Nanoparticle-based strategy in CAR-T cell immunotherapy: challenges, implications, and perspectives.

Introduction

Chimeric antigen receptor (CAR)-T cell therapy, an advanced form of adoptive cell immunotherapy, enhances the immune system’s ability to combat tumors by genetically modifying donor or patient T cells to express CARs [ 1 , 2 ]. The therapy generally consists of five critical steps: T cell isolation, activation, genetic engineering for CAR expression, CAR-T cell expansion, and reinfusion into patients to target cancer cells [ 3 ] ( Fig. 1 ) . CARs, expressed on the engineered CAR-T cells and designed to recognize specific cell surface structures, are mainly composed of four functional domains: the extracellular domain (ECD) with a single-chain variable fragment (scFv) for recognizing antigens and bypassing the limitations of the major histocompatibility complex (MHC), a transmembrane domain (TMD) for structural anchoring, a hinge domain (HD) for connecting ECD and TMD, and an intracellular domain for signal transduction [ 4 , 5 ]. After recognizing tumor antigens, these engineered CAR-T cells eliminate tumor cells through three mechanisms: (1) Through the perforin and granzyme pathway, perforin released by CD8 + T cells forms pores on the tumor cell membrane, allowing granzymes to enter and induce cell death [ 6 ]; (2) Through cytokine release, they regulate the tumor microenvironment (TME) and promote the polarization of macrophages to the M1 phenotype [ 7 ]; (3) Through the Fas/Fas ligand (FasL) pathway, the binding of Fas and FasL activates the adaptor protein Fas-associated death domain (FADD), triggering apoptosis [ 8 ] ( Fig. 1 ) . Notably, during CAR-T cell-mediated attack on target cells, granzyme B enters the tumor cell cytoplasm through perforin-formed pores in the plasma membrane, directly activating caspase-3. The activated caspase-3 subsequently cleaves gasdermin E (GSDME), thereby triggering pyroptosis, a proinflammatory form of programmed cell death, that ultimately leads to tumor cell membrane perforation, cellular swelling, and rupture [ 9 , 10 ]. This pyroptotic process promotes the massive release of damage-associated molecular patterns (DAMPs) from tumor cells [ 11 ]. These DAMPs specifically activate antigen-presenting cells (APCs, e.g., macrophages) infiltrating the TME, enhancing their antigen uptake and presentation capabilities. Consequently, this mechanism further amplifies the antitumor immune response mediated by CAR-T cells [ 12 ].

Fig. 1 The conventional CAR-T cell manufacturing process and the tumor-killing mechanisms. Blood is collected by venipuncture or apheresis, and T cells are isolated from either the patient or a healthy allogeneic donor. Subsequently, T cell activation is achieved using aAPCs or beads CD3/CD28. Genetic modification to generate CAR-T cells is then performed via either viral or electroporation gene delivery methods. During the patient’s lymphodepleting pre-treatment, the engineered CAR-T cells undergo ex vivo expansion in culture systems. Finally, the amplified CAR-T cell products are infused back into the patient, enabling specific recognition and elimination of tumor cells

Over the past 30 years, CAR-T cell technology has evolved to the fifth generation. The first-generation CAR-T cells achieve basic T cell activation by combining scFv with the CD3ζ signaling domain, but their efficacy and durability are limited [ 13 ]. The second generation introduces costimulatory domains (CSDs) such as CD28 and CD147 [ 14 ]. Incorporating an additional costimulatory domain into the third-generation framework significantly enhances the activity, proliferation, and durability of T cells [ 15 ]. The fourth generation, termed TRUCK (T-cells redirected for universal cytokine-mediated killing), integrates cytokine or immunomodulator genes to regulate the TME while simultaneously recruiting immune cells [ 15 ]. The latest fifth-generation CAR-T cells can respond to signals in the TME and provide a more controllable regulation method [ 16 ]. In particular, the emergence of Boolean logic-gated CAR-T cells has achieved precise control of activity through logical gates such as AND, OR, and NOT, improving specificity and efficacy while reducing side effects [ 17 , 18 ]. Currently, seven CAR-T cell therapies, including Kymriah™, Yescarta™, Tecartus™, Breyanzi ® , Abecma ® , Carvykti™, and Aucatzyl ® , have received the Food and Drug Administration (FDA) approval for hematologic malignancies [ 19 , 20 ]. Recent studies also explore CAR-T cell applications in non-cancer fields such as autoimmune diseases, infections, and transplantation, highlighting their transformative potential [ 21 – 24 ] ( Table 1 ) .

Table 1 Summary of clinical trials using CAR-T treated pathologies beyond tumor Disease type Condition Target Study Type Interventions Phase Status NCT Autoimmune diseases ITP BCMA Interventional Biological: autologous anti-BCMA chimeric antigen receptor T cells II Unknown NCT05315778 SLE CD19 Interventional Other: CAR T-cell therapy I Recruiting NCT06150651 SLE/SjS/SSc/DM/AAV CD19 Interventional Biological: CD19 targeted CAR-T cells I Recruiting NCT06056921 Scleroderma CD19/BCMA Interventional Biological: Assigned Interventions CD19/BCMA CAR T-cells Early I Recruiting NCT05085444 SjS CD19/BCMA Interventional Biological: Assigned Interventions CD19/BCMA CAR T-cells Early I Recruiting NCT05085431 Lupus nephritis CD19/BCMA Interventional Biological: Assigned Interventions CD19/BCMA CAR T-cells Early I Recruiting NCT05085418 SLE/SjS/SSc/IM/AAV CD19 Interventional Biological: Anti-CD19-CD3E-CAR-T cells NA Recruiting NCT06373081 Crohn’s disease/Ulcerative colitis/DM/Still diseases CD7 Interventional Biological: CD7 CAR T-cells Early I Recruiting NCT05239702 MS CD19 Interventional Biological: KYV-101 anti-CD19 CAR-T cell therapy Drug: Standard lymphodepletion regimen I Recruiting NCT06138132 AAV/IIM/Lupus nephritis/SjS/SLE/SSc CD19/BCMA Interventional Biological: CD19-BCMA CAR-T cells I/II Not yet recruiting NCT06350110 AAV/IM/SjS/SLE/SSc/APS CD19 Interventional Biological: BRL-301 NA Recruiting NCT05859997 MG CD19 Interventional Drug: CD19 CAR-T cells injection I Recruiting NCT05828225 Neuromyelitis optica CD19 Interventional Drug: CD19 CAR-T cells injection I Recruiting NCT05828212 AIHA CD19 Interventional Biological: ThisCART19A I Recruiting NCT06212154 IIM/MS/MG/NMOSD/MOGAD BCMA Interventional Biological: CT103A cells Drug: Cyclophosphamide and fludarabine Early I Recruiting NCT04561557 IIM/ITP/RA/SjS/SLE/ SSc CD19 Interventional Biological: T cell injection targeting CD19 chimeric antigen receptor Early I Not yet recruiting NCT06417398 Infectious diseases HIV bNAbs Interventional Biological: CAR-T cells I Unknown NCT04863066 HIV gp120 Interventional Biological: CAR-T cells I Recruiting NCT03240328 HIV gp120 Interventional Biological: CMV/HIV-CAR T Cells Early I Recruiting NCT06252402 HIV gp120 Interventional Drug: Cyclophosphamide Biological: LVgp120duoCAR-T cells, low dose | high dose 1 more I/II Recruiting NCT04648046 EBV LMP1 Interventional Biological: Cohort A: 3.0 × 10^6 CAR-T cells/kg Cohort B: 9.0 × 10^6 CAR-T cells/kg Cohort C:1.5 × 10^7 CAR-T cells/kg I Completed NCT05864924 Transplantation Kidney transplantation CD19/BCMA Interventional Drug: Cyclophosphamide Biological: CART-BCMA huCART19 I Recruiting NCT06056102 Haematopoietic cell transplantation CD19/CD20 Interventional Drug: Donor-derived CD19 CAR Therapy Bridged Allo-HSCT and Sequential Donor-derived CD22 CAR Therapy I Not yet recruiting NCT06326008 ITP immune thrombocytopenia, SLE systemic lupus erythematosus, SjS Sjogren’s syndrome, SSc systemic sclerosis, DM dermatomyositis, AAV anti-neutrophil cytoplasmic antibody‐associated vasculitis, IM inflammatory myopathy, MS multiple sclerosis, IIM idiopathic inflammatory myopathy, APS antiphospholipid syndrome, MG myasthenia gravis, AIHA autoimmune haemolytic anaemia, NMOSD neuromyelitis optica spectrum disorder, MOGAD myelin oligodendrocyte glycoprotein antibody‐associated disease, RA rheumatoid arthritis, BCMA B-cell maturation antigen

Despite its promise, challenges remain, such as high manufacturing costs, limited efficacy in solid tumors due to microenvironmental suppression, and severe adverse effects like cytokine release syndrome (CRS) [ 25 – 28 ]. In recent years, nanoparticles (NPs) have unlocked unprecedented opportunities in cancer immunotherapy due to their unique size effect, high specific surface area, precise targeting, and drug co-delivery capabilities, which have been successfully applied in tumor imaging, immunosuppressive microenvironment reprogramming, and vaccine delivery [ 29 – 32 ]. Through material selection and surface modification, the physical and chemical properties of NPs can be precisely regulated to optimize biocompatibility and improve efficiency, providing an innovative solution to break through the bottleneck of CAR-T cell therapy: NPs enhance CAR-T cell production by reducing genotoxicity and improving gene delivery efficiency, mimic artificial antigen-presenting cells (aAPCs) for T cell expansion, and enable in situ T cell engineering [ 33 – 35 ]. For solid tumors, NPs deliver chemokines, remodel the TME, enable multi-antigen targeting to improve CAR-T cell infiltration and persistence [ 36 ]. Additionally, NPs facilitate real-time monitoring and spatiotemporal control of CAR-T cell activity, mitigating CRS and neurotoxicity risks [ 37 , 38 ].

Diverse categories of NPs have been developed for cancer immunotherapy, including polymer-based NPs, lipid-based NPs, and inorganic NPs, each exhibiting unique advantages and distinct characteristics ( Table 2 ) . Herein, this review outlines the current obstacles faced by CAR-T cell therapy and systematically summarizes the three core application advancements of NPs in CAR-T cell therapy: optimizing manufacturing process, enhancing anti-tumor efficacy, enabling real-time monitoring and toxicity control. We also deeply discuss the technical challenges and future development directions of this combined therapy, exploring how artificial intelligence (AI) can aid the development of personalized immunotherapy to provide a theoretical basis for promoting clinical transformation.

Table 2 A summary of the pros and cons of representative nanoparticles Types of nanoparticles Examples Pros Cons References Polymer-based nanoparticles Polymer nanoparticles Dendrimers Polymeric micelles Excellent delivery carrier Tailored size and surface properties Sustained-release capability for controlled drug delivery Good stability Risk of particle aggregation Immunogenicity Potential hepatosplenic toxicity Limited clinical application [ 296 ] Lipid-based nanoparticles Liposomes Lipid nanoparticles Good biocompatibility and high bioavailability Extensive clinical adoption Payload flexibility Low immunogenicity Ease of surface modification Potential for drug leakage Immune perturbations in vivo Risk of hepatosplenic accumulation [ 297 , 298 ] Protein-based nanoparticles Ferritin, Albumin, Virus like particles Good biocompatibility and biodegradability Bioactivity modulation Low toxicity Potential immunogenicity Stability concerns Limited drug loading capacity [ 299 , 300 ] Inorganic nanoparticles Gold nanoparticles Iron oxide nanoparticles Silica nanoparticles Unique biological, chemical, electrical, magnetic properties Suitable for diagnostics, imaging, and photothermal therapy High stability Tunable size and shape Potential toxicity Limited solubility Clearance issues Regulatory hurdles [ 301 , 302 ] Peptide nanoparticles Stimuli-responsive peptide nanoparticles Versatile functionality Biodegradability Targeted delivery potential Potential immunogenicity Complex synthesis and stabilization requirements [ 303 ] Hybrid nanoparticles Peptide-polymer hybrid nanoparticles Polymer-lipid hybrid nanoparticles Large payload capacity High biocompatibility Good stability Low toxicity Technical challenges in design and manufacturing Potential immunogenicity [ 304 ] Biomimetic nanoparticles Membrane-coated nanoparticles Extracellular vesicles High biocompatibility Prolonged blood circulation time Low immunogenicity Targeting capability Complex fabrication Issues with non-specific accumulation Potential immunogenicity concerns [ 305 , 306 ]

Challenges in CAR-T cell therapy

CAR-T cell therapy encounters numerous hurdles, including inefficient manufacturing processes, suboptimal efficacy on solid tumors, poor CAR-T cell persistence, substantial antigen escape events, and serious side effects [ 39 – 41 ] ( Fig. 2 ) .

Fig. 2 Challenges in CAR-T Cell Therapy. (A) In conventional CAR-T manufacturing, viral vectors present challenges including immunogenicity, potential carcinogenicity, and limited cargo capacity, while electroporation may lead to cell membrane damage and cytotoxicity. (B) The intrinsic characteristics of solid tumors restrict CAR-T efficacy and persistence in multiple dimensions: physical barriers such as dense ECM and aberrant vasculature impede T-cell infiltration into the TME; metabolic features including hypoxia, acidic conditions, and nutrient depletion, combined with immunosuppressive components, accelerate CAR-T functional exhaustion; additionally, tumor antigen heterogeneity further compromises therapeutic efficacy. (C) Moreover, CAR-T cell therapy faces safety challenges such as CRS, on-target/off-target toxicity, and insufficient real-time monitoring capabilities

Manufacturing process

The clinical manufacturing procedure of CAR-T cells encompasses multiple in vitro steps, including T-cell isolation, CAR expression on T cells, and their amplification, demanding sophisticated clinical management, substantial infrastructure resources, and significant financial investment. Among these processes, the key step lies in the genetic modification of T cells for CAR expression via either viral or non-viral transduction [ 42 , 43 ]. To date, viral vectors, especially gamma-retrovirus and lentivirus, have been extensively used because of their high gene transduction efficiency and ability to integrate genes stably [ 42 ]. However, due to multiple problems of viral vectors, including immunogenicity, potential carcinogenic risks, complex production, high costs, and limited loading capacity, research has shifted to non-viral gene delivery technologies for alternative solutions [ 40 , 44 – 46 ].

Non-viral techniques, like electroporation, offer a safer and more adaptable means of gene transfer, free from the size limitations of viral vectors. Electroporation, which utilizes pulsed voltage to create transient pores in the cell membrane, can facilitate the entry of cargo into the cell and enable the expression of the transgene, possessing advantages like large cargo capacity and the ability to be used for mRNA modification [ 47 , 48 ]. Nevertheless, this technique, contingent upon electrical field strength and pulse duration, induces membrane disruption, cytoplasmic loss, and cytotoxicity, potentially altering gene expression patterns [ 49 , 50 ]. ( Fig. 2 A ) . Therefore, it is imperative to explore novel cargo delivery systems to address CAR-T cell manufacturing problems and drive its development.

Efficacy and persistence

Unlike hematologic malignancies, the treatment of solid tumors remains a Gordian knot. Although recent clinical trials have demonstrated that CAR-T cell therapy shows some efficacy in certain solid tumor types, such as sarcomas and neuroblastomas, the overall therapeutic effectiveness remains limited [ 51 , 52 ]. The inherent nature of solid tumors contributes to the constrained efficacy and persistence of CAR-T cells ( Fig. 2 B ) . As proof: (1) Upon injection into the bloodstream, CAR-T cells migrate to the tumor site via chemokine signals. Nevertheless, solid tumors suppress chemokine release, and the absence of matching receptors prevents their effective localization [ 53 ]. (2) After reaching the tumor site, physical barriers such as dense stroma and abnormal vasculature significantly hinder the migration, penetration, and extravasation of CAR-T cells, thereby limiting the attack on tumor cells [ 54 ]. (3) TME in solid tumors is typically characterized by hypoxia, acidity, oxidative stress, and nutrient depletion, along with immunosuppressive components such as soluble factors, cytokines like transforming growth factor-β (TGF-β), and immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) [ 54 , 55 ]. This hostile environment impairs CAR-T cell proliferation and accelerates functional decline, leading to non-responsiveness and tumor relapse. (4) Tumor antigen heterogeneity, another characteristic of solid tumors, also impacts CAR-T cell efficacy [ 56 ]. The absence or alteration of tumor cell antigens prevents single-target CAR-T cells from recognizing them. Although multi-target CARs can be designed to deal with this, it may lead to the risk of on-target/off-tumor toxicity [ 57 ].

Safety

Although CAR-T cell therapy exhibits a potent killing effect, the shortfall in control over efficacy, persistence, and localization after adoptive cell transfer results in an adverse immune response harmful to healthy tissues, such as CRS, immune effector cell associated neurotoxicity syndrome (ICANS), and immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) [ 58 ]. CRS is correlated with a substantial elevation in serum concentrations of pro-inflammatory cytokines induced by activated CAR-T cells, among which interleukin (IL)−6 functions as the key mediator [ 19 ]. Notably, this cytokine surge cascade arises not only from direct secretion by activated CAR-T cells but also from another key driver: pyroptosis, a gasdermin-mediated cell death pathway during CAR-T-mediated killing, that releases large amounts of DAMPs [ 12 ]. However, excessive DAMP release and subsequent hyperactivation of APCs further amplify proinflammatory cytokines (e.g., IL-1β, IL-6), ultimately triggering CRS [ 59 ]. Importantly, the level of GSDME correlates with CRS severity, underscoring the pivotal role of pyroptosis in CRS pathogenesis [ 9 ]. Clinically, CRS manifests as persistent fever, hypotension, hypoxia, or even multiple organ failure [ 19 , 60 ]. Fortunately, CRS responds to therapies directed at IL-6 and utilizing corticosteroids [ 61 ]. Recent studies show that pretreatment with the CD19-targeting monoclonal antibody tafasitamab reduces CRS severity in CD19-CAR-T therapy through CD19 antigen occupancy while enhancing antitumor efficacy and overall survival, offering a promising strategy to advance the achievement of a rational balance between CAR-T potency and CRS risk [ 62 ]. ICANS correlates with on-target/off-tumor recognition of cerebral antigens by CAR-T cells targeting CD19 and CD22, resulting in neurological symptoms including encephalopathy, tremor, and cerebral edema [ 63 , 64 ]. Other than supportive care and corticosteroids, IL-18 blockade and in situ conjugation of polyethylene glycol (PEG) to the surface of CAR-T cells (‘PEGylation’) have demonstrated potential in treating ICANS [ 65 – 67 ]. Like ICANS, IEC-HS frequently manifests in patients with serious CRS and is characterized by macrophage activation, leading to uncontrolled cytokine-driven inflammation. Studies have shown that corticosteroids +/- anakinra can be used for treating IEC-HS [ 68 , 69 ]. In addition, CAR-T cell therapy presents other safety concerns, such as graft-versus-host disease, off-target toxicity, anaphylactic reactions, potential viral insertional oncogenesis, and inadequate monitoring [ 70 ] ( Fig. 2 C ) . Thus, further research is required to address the aforementioned issues.

Integration of nanoparticles with CAR-T cell therapy

NPs, composed of different biomaterials such as lipids, polymers, and inorganic materials, exhibit beneficial properties, including reduced toxicity, low cost, and easy customization [ 8 ]. Over the past decade, NPs have presented feasible strategies in cancer immunotherapy by means of vaccine development and immunomodulatory drug delivery, indicating their potential to overcome the aforementioned challenges of CAR-T cell therapy.

Engineer CAR-T cell therapy

Due to their varied materials, decent stability, and expanded cargo-loading options, NPs are used to engineer CAR-T cells both in vitro and in vivo to improve availability, thereby becoming desirable alternatives to conventional gene delivery techniques and boosting production efficiency [ 38 , 71 ] ( Fig. 3 and Table 3 ) . Table 4 compares different gene delivery approaches in CAR-T cell therapy, highlighting key pros and cons.

Fig. 3 Use of nanoparticles for CAR-T cell generation and schematic diagram of the CAR-T-MNPs serving as the biomimetic drug delivery. (A) For in vitro production, T cells are first isolated from patient blood, activated, and then genetically modified via NP-mediated delivery of CAR-encoding genetic material. The engineered CAR-T cells undergo ex vivo expansion before reinfusion into the patient to exert antitumor effects. Functionalized NPs can serve as aAPC platforms to activate and proliferate CAR-T cells, significantly shortening preparation. Additionally, NPs act as cell-sorting tools to enhance CAR-T cell purity. (B) For in vivo generation, functionalized NPs carrying CAR-encoding genetic payloads are directly injected into the patient’s bloodstream. These NPs selectively transduce T cells, enabling in vivo expansion of CAR-T cells for targeted tumor eradication. This process can be optimized through antibody or SORT ligand modification strategies to achieve high-efficiency in vivo transfection. (C) The CAR-T-MNPs combine the target-specificity of CAR-T cells with the drug-loading capacity of NPs, thereby prolonging circulation time and enhancing tumor-targeting efficacy. SORT: selective organ targeting; CAR-T-MNPs: CAR-T-cell membrane-coated NPs

Table 3 Examples of nanoparticle-mediated transfection of CAR-T cells Carrier Cargo Target cells Transfection efficiency Disease References In vitro PBAE-based nanoparticles Leukemia-targeted 194-1BBz CAR plasmid DNA Primary human T cells 3.8% B-cell acute lymphoblastic leukemia. [ 90 ] Ionizable lipid Nanoparticle CD19-targeted CAR mRNA Jurkat cells Primary human T cells Equivalent to electroporation Acute lymphoblastic leukemia [ 33 ] Lipid Nanoparticle CD19-targeted CAR mRNA Human T cells 22.6% Leukemia [ 80 ] Ionizable lipid nanoparticle CD19-targeted CAR mRNA Jurkat cells Primary human T cells B10 LNPs are comparable to electroporation Acute lymphoblastic leukemia [ 79 ] Gold nanoparticles eGFP-mRNA Jurkat cells 45% NA [ 76 ] In vivo PBAE-based nanoparticles Leukemia-targeted 194-1BBz CAR plasmid DNA Primary human T cells 7.1% among CD3 + T cells on day 24 B-cell acute lymphoblastic leukemia [ 90 ] Cationic polymer nanoparticles Plasmid CAR with a T cell-specific CD2 promoter T cells in humanized NSG mice 14.3% Ovarian cancer [ 91 ] PBAE polymer nanoparticles Disease-specific CAR mRNA Circulating T cells 75 ± 11% of T cells on day 2 Leukemia Prostate cancer Hepatocellular carcinoma [ 307 ] Imidazole-based synthetic lipidoids Cre mRNA Primary mouse T cells 8.2% NA [ 308 ] SORT lipid nanoparticles CD19-targeted CAR mRNA Cre recombinase mRNA Splenic T cells 7% of total splenic T cells Lymphoreplete B cell lymphoma [ 98 ]

Table 4 Comparison between viral and non-viral vectors in CAR-T cell therapy [ 49 , 309 – 311 ] Types of vectors Viral vector Non-viral vector Electroporation Nanoparticle Advantages Easy to use Wide range of transfection mechanisms Large cargo capacity Low manufacturing costs Targeting potential Highly customizable Low cytotoxicity Suitable for in vivo and in vitro applications Disadvantages Size-limited cargo Immunogenicity Risk of genotoxicity High variability in small-scale production High production costs Limited large-scale manufacturing Risk of cytotoxicity Loss of cytoplasmic content Limited in vivo application Classically low transfection capacity Toxicity related to materials Potential hepatosplenic toxicity Efficiency Various Good Various Viability Good Poor Good

For in vitro transfection, Bozza and colleagues devised a non-integrating DNA nanovector that is free of viral components and capable of extrachromosomal replication, successfully generating CAR-T cells with enhanced anti-tumor efficacy in vitro. In addition, this nanovector is characterized by persistent transgene expression, non-immunogenicity, large cargo capacity, ease of manufacture, and affordability, underscoring its multifaceted advantages. Its in vitro validation, nevertheless, relies on healthy donor T cells rather than patient-derived ones and lacks TME simulation, meaning further research is still needed for its clinical translation [ 72 ]. mRNA features easy production, low cost, no concerns regarding long-term side effects, and flexible protein-encoding ability, thus serving as a potent tool for T cell gene engineering in vitro [ 73 ]. Moreover, NPs can encapsulate mRNA to protect it and facilitate T-cell uptake, thereby reprogramming tumor-associated genes in T cells via transient expression [ 33 ]. Researchers have employed miscellaneous types of NPs to boost mRNA delivery efficiency. For example, it was demonstrated that the structure of cationic polymers, the key components of polymer-based NPs, is pivotal in governing both gene transfection efficiency and cytotoxicity. In contrast to linear poly (2-dimethyl amino) ethyl methacrylate) (pDMAEMA), the comb- and Sunflower-shaped pDMAEMAs exhibited higher transfection efficiency and comparatively lower cytotoxicity, furnishing a valuable avenue for designing efficacious vectors to ensure successful in vitro gene delivery. However, their transfection efficiency remains lower than that of viral vectors, with transfection efficiency in primary Human T cells achieving only 25% (for mRNA) and 18% (for plasmid DNA), alongside limited in vivo validation and cell type specificity, hindering clinical translation due to unmet efficacy and generality demands [ 74 , 75 ]. Gold NP-mediated vapor nanobubble photoporation, which attained transfection rates of up to 45% and increased the number of transfected viable cells five times compared to electroporation in Jurkat T cells, is also a promising in vitro mRNA delivery technique. Nonetheless, it lacks validation in primary human T cells, has unclear long-term effects on cell functionality, and faces scalability challenges [ 76 ]. Recently, ionizable lipid nanoparticles (LNPs) have been applied for in vitro mRNA delivery to human T cells [ 77 , 78 ]. Through excipient optimization to enhance delivery efficiency and mitigate cytotoxicity, the B10 formulation emerged as the top performer, tripling the functional delivery compared to the standard one. Moreover, its generated CAR-T cells exhibited equivalent cancer cell-killing efficacy to those induced by lentivirus and electroporation. Even so, the mechanisms by which B10’s excipient composition enhances delivery remain unclear [ 79 ]. Furthermore, LNPs extend their applicability to other cell types, such as macrophages and natural killer (NK) cells. A study showed that by optimizing the LNP formulation and mRNA modification, in vitro mRNA transfection was achieved and anti-CD19 CAR macrophages were successfully produced, showing significant cytotoxic effects on B lymphoma [ 80 ]. Overall, among current mRNA nanocarriers for in vitro transfection, LNPs demonstrate the highest clinical potential, although each platform necessitates tailored optimization to overcome its particular limitations toward translational application.

The activation and expansion of CAR-T cells engineered in vitro is labour and time intensive, underlining the need for an efficient way to streamline production. T cell activation demands three signals: T cell receptor (TCR) signaling, costimulation, and cytokine signaling. In vivo, APCs deliver these signals, while in vitro, aAPCs are deployed to drive T cell activation [ 81 ]. Despite the capacity of prevalent commercial microbead aAPC systems like Dynabeads to restore T cell traits, issues including suboptimal T cell expansion rates, misregulated or limited functions of the generated T cells, and the necessity for extra steps to remove microbeads render the in vitro T cell activation and proliferation process cumbersome [ 82 – 84 ]. Fortunately, NPs can be modified and serve as aAPC platforms, enabling in vitro activation and proliferation of CAR-T cells and shortening the preparation time ( Fig. 3 A ) . Metzloff et al.. achieved one-step efficient production of mRNA CAR-T cells by directly conjugating fragments of CD3 and CD28 antibodies onto the surface of LNPs mimicking APCs, which subsequently alleviated tumor burden in murine xenograft models [ 85 ]. Notably, activating lipid nanoparticles (aLNPs) slashed the cell culture time for mRNA CAR-T cells by half, from 48 to 24 h. Simultaneously, dispensing with the need for activation magnetic beads substantially reduced both the preparation time and manufacturing costs, while the study is limited to preclinical murine models, long-term safety and scalability for clinical use remain unproven [ 85 ].

NPs could also serve as a pivotal tool in cell sorting, playing a significant role in CAR-T cell preparation ( Fig. 3 A ) [ 86 ]. NPs significantly enhanced magnetic labeling efficiency by utilizing magnetic amplification by sequentially connected antibodies and nanoparticles (MagSCAN) technology. This method increased the purity of positive fractions from 35 to 50% with conventional methods to 80%, thereby enabling the efficient isolation of high-purity CAR-T cells while maintaining cell viability above 90%. The sorted CAR-T cells demonstrated superior performance in anti-tumor therapy: when co-cultured with tumor cells, their killing efficiency was markedly enhanced, evidenced by increased tumor cell mortality and elevated release of pro-inflammatory molecules, such as tumor necrosis factors (TNFs) and interferon-gamma (IFN-γ). Additionally, the expression levels of cytokines, including IL-6 and IL-10, were significantly upregulated, indicating enhanced anti-tumor activity. Thus, the NP-based MagSCAN technology effectively improved the therapeutic efficacy of CAR-T cell therapy by enhancing both cell purity and functional activity. Notably, the linkers and MNPs involved may trigger immune responses, making direct clinical application unadvisable. Moreover, its effectiveness in whole blood without removing residual antibodies remains to be verified [ 87 ].

To circumvent the constraints of complexity, heavy cost, and high personalization associated with large-scale implementation of ex vivo-produced CAR-T cell products, in vivo T cell engineering holds promise ( Fig. 3 B ) [ 88 ]. Direct reprogramming of patient-derived T cells into CAR-T cells enables the generation of patient-specific cellular therapeutics. Polymer nanoparticles (PNPs) and LNPs are the most common and promising nanovectors employed in CAR-T cell engineering [ 89 ]. Utilizing PNPs as delivery vectors for in vivo generation of CAR-T cells constitutes a viable strategy. DNA-loaded PNPs delivered the transgene encoding leukemia-specific 194−1BBz CAR into circulating T-cell nuclei, leading to the regression of leukemia in mice, with an efficacy comparable to that of conventionally manufactured CAR-T cells. Yet, these studies are confined to murine models, and off-target transgene expression, though minimal, remains a concern [ 90 ]. Notably, in a recent study, Zhu and colleagues reported that they successfully generated CAR-T cells in vivo by injecting a supramolecular hydrogel system based on cationic PNPs and demonstrated its potent anti-tumor efficacy in NSG humanized xenograft mouse models. This method boasted strong local retention and low systemic toxicity, yet it relied on local tumor injection and had limited efficacy against metastases [ 91 ]. In comparison, PNPs enabled systemic administration, offering greater convenience in hematological tumors, but suffering from insufficient penetration in solid tumors. Both strategies avoided the risks associated with viral vectors, providing complementary optimization approaches for CAR-T therapy [ 90 , 91 ]. LNPs, another ideal carrier for promoting CAR-T cell therapy, enable in vivo generation of CAR-T cells by transporting therapeutic mRNA to lymphocytes [ 92 ]. Nonetheless, the current in vivo T cell production efficiency is suboptimal, and direct transfection of mRNA into in vivo T cells remains a technical hurdle [ 93 ]. The design and modification of NP surfaces may facilitate overcoming these hurdles by enhancing the in vivo- specific transfection of T cells ( Fig. 3 B ) . Zhou et al. constructed a CD3 antibody-modified LNP system loaded with a plasmid encoding IL-6 short hairpin RNA (shRNA) and CD19-CAR, which targeted and transfected T cells to generate IL-6 knockout CAR-T cells [ 92 ]. These engineered cells could kill leukemia tumor cells with high CD19 expression, reduce IL-6-induced CRS, and exhibit comparable anti-tumor effects to traditional CAR-T cells prepared ex vivo, greatly prolonging the survival of leukemia model mice. However, the transfection efficiency of this system remains lower than that of lentiviral methods, and off-target effects may still pose potential risks [ 92 ]. Furthermore, studies have demonstrated that anti-T cell marker selection (e.g., CD3/CD5/CD7) and antibody-conjugated lipid nanoparticles (Ab-LNPs) dosage critically regulate CAR expression levels in T cells. In vivo investigations revealed that CD3- and CD7-LNPs effectively delivered CAR mRNA, inducing robust CAR expression in circulating T cells, whereas CD5-LNPs failed to achieve efficient transfection or expression even at maximal doses. Remarkably, increasing the dosage of CD3- and CD7-LNPs further enhanced CAR expression [ 94 ].

Notably, targeted ligand modification isn’t essential for LNP-mediated in vivo T cell transfection. Certain LNPs with optimized formulations can circumvent the liver tropism of non-targeted carriers and accumulate in extrahepatic tissues [ 95 ]. Wang and co-authors developed a novel technique termed selective organ targeting (SORT), allowing for the controlled delivery of nucleic acids to target tissues without modifying the targeting ligands of NPs [ 96 ]. By incorporating a fifth component, the SORT molecule, into the traditional four-component LNP, they achieved precise regulation of tissue-specific delivery to the livers, lungs, and spleens of mice after intravenous injection [ 96 ]. Some researchers have identified the relevant mechanism: serum proteins can recognize exposed SORT molecules, adsorb to LNPs, and interact with target-organ cell receptors for targeted delivery [ 97 ]. Álvarez-Benedicto et al. have demonstrated that they achieved in situ production of CAR-T cells via intravenous injection of spleen-SORT LNPs unmodified by targeting ligands, reducing tumor metastasis, and improving the overall survival rate of B-cell lymphoma mice [ 98 ]. Notably, SORT LNPs have also been engineered for targeted delivery to other organs, including the lung, brain, pancreas, placenta, bone marrow, and lymph nodes [ 99 – 104 ]. To advance clinical translation, large animal model studies should be conducted to validate the safety and efficacy of SORT LNPs in a physiological environment more analogous to humans. Furthermore, optimizing LNP formulations and CAR designs can enhance targeting efficiency and therapeutic efficacy against highly aggressive tumors, thereby mitigating tumor escape.

Compared with conventional PNP and LNP-based gene delivery approaches, a recent study designed a novel in vivo CAR-T cell generation strategy through membrane fusion-mediated CAR protein delivery [ 105 ]. The researchers developed virus-mimetic fusogenic nanovesicles (FuNVs) as CAR protein carriers, which efficiently fused with T-cell membranes in vivo to deliver anti-CD19 CAR proteins directly to T cells, thereby generating functional anti-CD19 CAR-T cells. Although the strategy streamlined CAR-T production, avoided CRS, and showed potent anti-tumor effects, it might not have suited patients with impaired T-cell function, and fusogens with higher specificity were needed to reduce non-specific binding [ 105 ]. Interestingly, CAR-T cells can also serve as starting materials for the fabrication of NPs ( Fig. 3 C ) . Illustratively, the mesoporous silica NPs loaded with near-infrared (NIR) dye IR780 were coated with CAR-T cell membranes that specifically recognize Glypican-3⁺ hepatocellular carcinoma (HCC) cells, while poly (D, L-lactide-co-glycolic acid) NPs encapsulating the anticancer drug cisplatin were functionalized with anti-human epidermal growth factor receptor 2 (HER2)-specific CAR-T cell membranes. What’s more, 3-bromopyruvate-mixed Au NR@Cu₂₋ₓSe NPs were coated with CAR-T cell membranes capable of specifically recognizing CD19 antigens on the surface of NALM6 tumor cells, demonstrating potent anti-tumor efficacy both in vitro and in vivo [ 106 – 108 ]. Therefore, CAR-T cell membrane-coated NPs (CAR-T-MNPs) also represent a promising biomimetic delivery platform that combines CAR-T cell targeting specificity with NP drug-loading capacity, potentially augmenting the efficacy of various cancer treatments. For advancing the development of this field in the future, it is necessary to further improve the stability of loaded substances and verify the long-term in vivo metabolic safety of nanomaterials.

Enhance the anti-tumor efficacy of CAR-T cell therapy

Promote aggregation at tumor sites

Insufficient infiltration of T cells into tumor tissues impedes the efficacy of CAR-T cell therapy in treating solid tumors. To address this, NPs have been utilized to promote the aggregation of CAR-T cells to tumor sites ( Fig. 4 ) . IL-12 nano-stimulators (INSs), designed as redox-responsive human serum albumin NPs with IL-12, exemplify this approach [ 109 ] ( Fig. 4 A ) . Through bioorthogonal conjugation, INSs were conjugated with CAR-T cells to form INS-CAR-T biohybrids. Upon tumor antigen stimulation, the surface reducibility of CAR-T cells increased, prompting INS to release IL-12 in a responsive manner. This, in turn, drove the secretion of chemokine C-C motif ligand (CCL)−2, CCL5, and CXC chemokine ligand (CXCL)−10, selectively recruiting and expanding CD8 + CAR-T cells within the tumor and bolstering the anti-neoplastic potency. Although this design enables precise antigen-responsive activation, its effectiveness depends on specific antigen expression and may be limited in tumors with low antigen levels [ 109 ]. Beyond enhancing chemokine secretion, NPs can reshape the TME by removing physical barriers via photothermal therapy, thereby facilitating CAR-T cell therapy [ 110 ] ( Fig. 4 B ) . Tang et al. developed a hydroxyethyl starch-polycaprolactone (HES-PCL) nanodelivery system to co-load the TGF-β receptor inhibitor LY2157299 (LY) and photosensitizer indocyanine green (ICG), generating LY/ICG@HES-PCL NPs. These NPs were targeted to the lymphoma site via the enhanced permeability and retention (EPR) effect. The photothermal effect of ICG could destroy the extracellular matrix (ECM), dilate blood vessels, and loosen compact tissue, accelerating LY release. This release process could upregulate chemokines CXCL9/10/11 and the CAR-T cell CXC chemokine receptor (CXCR)−3 at the tumor site, facilitating CAR-T cell infiltration and enabling long-term anti-tumor effects. Despite its controllability, the utility of this system is limited by the shallow penetration depth of light [ 110 , 111 ]. Recent research has shown that chromium NP-based photoimmunotherapy harbors potential for enhancing CAR-T cell infiltration in solid tumors. The trivalent Cr3 + ions generated upon degradation of the nanocomposites could upregulate the expression of chemokines CCL3 and CXCL13, facilitating CAR-T cell infiltration. Even so, the long-term metabolic safety of Cr-based materials requires further validation [ 112 ]. Collectively, these technologies provide complementary strategies to promote the aggregation of CAR-T cells to tumor sites. The integration of precise targeting and microenvironment remodeling may offer promising directions for future research. Fig. 4 Strategies for nanoparticle-enhanced CAR-T Cell tumor infiltration. (A) Nano-stimulators enable redox-responsive IL-12 delivery, promoting secretion of chemokines to enhance CAR-T cell tumor recruitment and expansion. (B) NPs can be combined with PTT to co-deliver TGF-β inhibitor LY and photosensitizer ICG. This approach utilizes photothermal effects to disrupt ECM, dilate blood vessels, promote CAR-T cells infiltration, and upregulate chemokine expression. (C) Magneto-acoustic manipulation technology improves the transport efficiency of CAR-T cells conjugated with immunomagnetic NPs to tumor sites, enabling precise tumor localization and effective infiltration. PTT: photothermal therapy

Researchers could also employ magnetic-acoustic manipulation technology to enhance the transport efficiency of CAR-T cells to tumor sites ( Fig. 4 C ) . Immunomagnetic particles carrying anti-CD3/CD28 antibodies were conjugated to CAR-T cells via click chemistry, generating CAR-T-cell-based live microrobots (M-CAR-Ts) [ 113 ]. Under magnetic fields, M-CAR-Ts moved against blood flow, bypassed obstacles, and precisely targeted tumor periphery regions, while under acoustic fields, M-CAR-Ts further penetrated tumor tissues, promoting their infiltration into tumor sites. Additionally, the anti-CD3/CD28 antibodies also stimulated CAR-T cell proliferation and activation, significantly improving their tumor-suppressive effectiveness [ 113 ]. Nonetheless, it should be noted that this study was conducted solely in mouse models, which exhibit significant physiological differences from humans. The precision and stability of the magnetic-acoustic driving system may be compromised in deep tissues due to potential interference, potentially reducing its efficacy in larger animals or humans. Moreover, the immunomagnetic beads could elicit immune responses, and their long-term safety remains uncertain.

Modulate hostile TME

Upon infiltrating tumor sites, CAR-T cells encounter the hostile TME, which features hypoxia, acidity, and nutrient deprivation, along with various inhibitory cells and cytokines. These factors cause T cell dysfunction and induce treatment resistance [ 114 – 116 ]. NPs can reverse the immunosuppressive TME through multiple modalities, thereby enhancing the therapeutic efficacy of CAR-T cells in solid tumors ( Fig. 5 ) . Dong et al. developed catalase-encapsulated CaCO 3 nanoparticle-constructed colloidosomal microreactors (CCaP CSs), aiming to regulate tumor hypoxia and acidity within the TME [ 117 ]. By leveraging CaCO₃’s proton-scavenging capacity to neutralize acidic conditions and catalase’s ability to decompose H₂O₂, this dual-modality approach effectively alleviated hypoxia while reducing lactate accumulation. Notably, this TME remodeling strategy reversed immunosuppression and significantly potentiated the tumor suppression efficacy of epidermal growth factor receptor (EGFR)-expressing CAR-T cells in modeled human triple-negative breast cancer tumor xenografts. However, CCaP CSs rely on intratumoral injection, limiting their applicability to inaccessible tumors, and their oxygen generation depends on endogenous H₂O₂ levels, which may restrict efficacy in severely hypoxic environments [ 117 ]. Apart from directly modifying the hypoxic and acidic states of the TME, NPs can also indirectly remodel the immuno-hostile TME by regulating tumor cell metabolism [ 114 ]. The developed APHA@CM nanocatalyst, prepared by encapsulating horseradish peroxidase (HRP)-loaded Au/polydopamine nanoparticles (Au/PDA NPs) and Ag₂S quantum dots, targeted tumor cells by leveraging CAR-T cell membranes. This membrane-camouflaged design enhances tumor targeting and reduces immune clearance, a distinct advantage over CCaP CSs’ passive accumulation. Additionally, APHA@CM integrates multimodal imaging for precise therapeutic guidance, a feature absent in CCaP CSs. Au NPs mediated synergistic sonodynamic therapy (SDT) to induce immunogenic cell death (ICD) and reprogram the immunosuppressive TME. The PDA layer and HRP enhanced the efficiency of SDT, and HRP catalyzed the production of oxygen to alleviate hypoxia. Meanwhile, Au NPs exhibited glucose oxidase-like enzymatic activity, which inhibited glycolysis, reduced lactate efflux, improved the metabolic microenvironment, and promoted the activation and survival of CAR-T cells. Despite these advantages, APHA@CM’s reliance on external stimulation, such as ultrasound/laser for SDT/PTT, complicates clinical operation, and the long-term biocompatibility of Au-based materials remains to be verified [ 118 ] ( Fig. 5 A ) . Fig. 5 Strategies for nanoparticles reverse the immunosuppressive TME. (A) NPs can alleviate TME hypoxia and acidity: CaCO₃ NPs alleviate acidity through proton scavenging while catalase-mediated decomposition of H₂O₂ generates oxygen, reducing lactate accumulation. The APHA@CM nanocatalyst system employs HRP for oxygen production and utilizes the glucose oxidase-like activity of gold NPs to inhibit glycolysis and suppress lactate efflux, thereby improving metabolic conditions. (B) NPs deliver immunomodulators such as TLR7/8 agonists, PI3Kδ inhibitors, STING agonists, or anti-tumor cytokines in the TME, remodeling the immunosuppressive TME and augmenting the anti-tumor potency of CAR-T cells. (C) NPs can be co-encapsulated with hydrogels, enabling localized delivery of CAR-T cells, IL-15 cytokines, and anti-PD-L1 antibodies to effectively overcome immunosuppression in solid tumor microenvironments. HRP: horseradish peroxidase

NPs can also deliver diverse immunomodulatory drugs to suppress immunosuppressive cells and inhibitory molecules within the TME, thereby blocking immunosuppressive pathways ( Fig. 5 B ) . To illustrate, polymeric micellular nanoparticles (PMNPs) were engineered to co-deliver Toll-like receptor 7 and 8 (TLR7/8) agonists and phosphatidylinositol-3 kinase p110δ (PI3Kδ) inhibitors. This combination therapy promoted type 1 macrophage polarization while reducing MDSC accumulation and selectively depleting tissue-resident Tregs, thereby mitigating the immunosuppressive TME and paving the way for enhancing CAR-T cell therapy outcomes [ 119 ]. In another study, Zhu and colleagues developed nanovesicles expressing anti-programmed cell death Ligand 1 (PD-L1) and loaded with the interferon gene stimulator (STING) agonist cGAMP [ 120 ]. Upon inhalation, these nanovesicles swiftly accumulated in lung tissue and ferried cGAMP to PD-L1 highly-expressing cells in tumor tissue via the programmed cell death protein 1 (PD-1)/PD-L1 interaction. The internalized cGAMP triggered the release of pro-inflammatory cytokines, spurring CAR-T cell infiltration and depleting immunosuppressive cell populations such as Tregs and MDSCs. Meanwhile, by blocking PD-L1, the nanovesicles prevented CAR-T cell exhaustion, remodeling the immunosuppressive TME and augmenting the proliferation and anti-tumor potency of CAR-T cells in solid tumors [ 120 ]. Both strategies target immunosuppressive cells and amplify pro-inflammatory signals, yet differ in their delivery routes and primary mechanisms. Systemically administered PMNPs modulated myeloid cell polarization and Treg function, while inhalable lung-targeted nanovesicles integrated STING-mediated immune activation with checkpoint blockade to directly enhance CAR-T cell persistence. Together, these approaches demonstrate the versatility of NPs in remodeling the TME to improve immunotherapy, with their efficacy in solid tumors being influenced by route- and target-specific characteristics. Other than the aforementioned studies, immunomodulatory drugs like TGF-β inhibitors, A2a adenosine receptor antagonists, and α-GalCer agonists have also been documented [ 36 , 121 – 123 ].

Numerous studies have validated the role of pro-inflammatory cytokines, another type of immunomodulatory agent, in potentiating anti-tumor immunity [ 124 , 125 ]. Nevertheless, systemic administration of cytokines may lead to severe systemic toxicity [ 126 ]. Consequently, a NP-based “cellular backpack” strategy has been devised to accurately deliver cytokines at the opportune time and location, thereby synergistically enhancing T cell cytotoxicity and maximizing therapeutic outcomes while minimizing toxicity [ 127 ]. Tang et al. developed nanogels (NGs) loaded with IL-15 superagonist complex (IL-15Sa) [ 128 ]. Upon CAR-T cells recognizing tumor antigens, the NGs responded to the elevated reduction potential on the T cell surface, conjugated the IL-15Sa-loaded NPs with the CAR-T cell plasma membrane, and released IL-15Sa into the TME. This promoted the proliferation of intratumoral T cells while averting impacts on peripheral blood T cells. Compared with systemic administration of IL-15Sa, the NGs system boosted intratumoral T cell proliferation by a factor of 16, allowed for the safe use of up to 8 times the dose of IL-15Sa without eliciting toxic reactions, and markedly improved the tumor clearance rate [ 128 ]. Nevertheless, the “cellular backpack” strategy suffers from drawbacks such as complex manufacturing processes, lengthy production times, and potential impairment of T cell physiological functions. To address these and enhance CAR-T cell anti-tumor efficacy, Liu et al. labeled T cells by adding NPs containing unnatural azido sugars to the culture medium during cell expansion and employed click chemistry to directly attach anti-tumor cytokines to the cell surface. The approach augmented the in vivo effector function of T cells after adoptive transfer, activated the endogenous immune system to promote antigen spreading, and helped identify more tumor-specific antigens. It should be noted that metabolically labeled cytokines lack dynamic release regulation, potentially resulting in sustained systemic immune activation [ 129 ]. Besides the aforementioned strategies, CAR-T cells and auxiliary NPs can be co-encapsulated into hydrogels for local therapy ( Fig. 5 C ) . A biodegradable hydrogel reservoir for the localized delivery of CAR-T cells, IL-15 cytokine, and anti-PD-L1 antibody post-tumor resection was developed to prevent tumor recurrence, effectively overcoming immunosuppression in the solid TME and CAR-T cell depletion. Despite this promising approach, the necessity for surgical implantation of hydrogels may increase procedural complexity [ 130 ]. Overall, these findings indicate that localized delivery and stimulus-responsive release represent key strategies for enhancing T cell therapies. Technical pathways should be selected based on the characteristics of the TME: for instance, immunogenic tumors may benefit from nanogel-based systems, metabolic labeling offers advantages in manufacturing simplicity, while hydrogels are particularly suitable for surgically accessible tumors.

Restrict tumor antigen heterogeneity

Tumor antigen heterogeneity, characterized by antigenic differences and high mutation diversity among tumor cells, poses a major challenge to CAR-T cell therapy [ 131 ]. Researchers have carried out extensive investigations and devised multiple strategies to address tumor antigen heterogeneity, including multi-target CAR-T cells against diverse tumor-associated antigens (TAAs), BiTE (bi-specific T cell engager)-secreting CAR-T cells that enhance tumor recognition or universal CAR-T cells targeting switchable adaptors for flexible antigen engagement [ 132 – 134 ] ( Fig. 6 A ) . Fig. 6 Strategies to restrict tumor antigen heterogeneity. (A) Non-NP-based strategies to restrict tumor antigen heterogeneity. (B) The multifunctional NP (NbFGFR4-GrB-h-HFn) co-delivers nanobodies NbFGFR4 targeting tumor-specific antigen FGFR4 and broad-spectrum CD71 ligands (h-HFn). This design enables robust binding and GrB delivery across gastric cancer cells with variable FGFR4 expression, significantly enhancing the recognition of antigenically diverse tumors. (C) F-AgNPs utilize membrane fusion to modify exogenous antigens onto tumor cell surfaces. These in situ-modified antigens provide abundant recognition sites for CAR-T cells, enabling them to overcome limitations imposed by intrinsic tumor antigen heterogeneity and exert potent antitumor effects. (D) NP-vaccine combination strategy: Administered subcutaneously or intravenously, vaccine NPs are either internalized by peripheral DCs or directly transported to lymph nodes for DC interaction. Activated DCs migrate to lymph nodes to enhance CAR-T cells, thereby directing their tumor-specific killing. Subsequent tumor lysis releases diverse antigens (including non-vaccine-targeted neoantigens), which are captured by DCs and presented in draining lymph nodes. This process activates T-cell responses, broadening the immune system’s antigen recognition spectrum through antigen spreading. F-AgNPs: fusogenic antigen-loaded nanoparticle

NP-based CAR-T cell therapy also bears great prospects in restricting tumor antigen heterogeneity. The multitargeting NPs capable of binding both tumor-specific antigens (TSAs) and widely expressed antigens were designed to enhance the recognition of heterogeneous tumor cells [ 135 ]. Specifically, they fused the nanobodies (Nbs) targeting the TSA FGFR4 of gastric cancer with granzyme B (GrB) and conjugated the complex onto human ferritin (h-HFn), generating NbFGFR4-GrB-h-HFn NPs. These NPs utilize NbFGFR4 to target FGFR4 while leveraging h-HFn’s binding to CD71 (a receptor highly expressed on tumor cells) for dual targeting, enabling robust binding to gastric cancer cells across varying FGFR4 expression levels and delivering GrB. Notably, even in FGFR4-low-expressing MGC803 cells, CD71 ensures effective tumor engagement, thus overcoming antigen heterogeneity [ 135 ] ( Fig. 6 B ) . Similar strategies have been adopted in HCC characterized by high FGFR4 expression. FGFR4-ferritin NPs were administered to alpacas to construct a Nb phage library, from which FGFR4-targeting Nbs with high specificity and affinity were screened. The Nb-derived CAR-T cells, developed accordingly, overcame the antigen downregulation issues due to the inhibitory microenvironment and tumor heterogeneity, demonstrating remarkable anti-tumor efficacy both in vitro and in vivo. While Nb-derived CAR-T cells provide the benefit of sustained tumor surveillance via T cell expansion, their efficacy is limited by the dependence on FGFR4 expression, restricting their use in tumors with low or absent antigen levels [ 136 ]. In contrast, NbFGFR4-GrB-h-HFn NPs demonstrate broader applicability across a range of FGFR4 expression levels. However, NPs’ delivery efficiency is compromised by the structural barriers of solid tumors, which needs further study.

While these pioneering efforts have demonstrated the potential to enhance the efficacy of CAR-T cells in treating solid tumors, they remain confined to tumors possessing suitable intrinsic antigens, which underpin the functionality of CAR-T cells. In response, some researchers proposed leveraging exogenous antigens to perform in situ modification on solid tumors, thereby providing ample targets and achieving the redirection of immune recognition [ 137 ] ( Fig. 6 C ) . The developed fusogenic antigen-loaded nanoparticles (F-AgNPs) exploit the properties of tumor cell membranes after malignant transformation, namely negative charge and enhanced fluidity, facilitating their efficient fusion with F-AgNPs containing the cationic lipid DOTAP. Additionally, it employed tumor-derived extracellular vesicles (EVs) to propel antigen diffusion within tumor tissues. Through in situ antigen modification on the tumor cell membrane, this platform redirected CAR-T cells, regulated the expression level of exogenous antigens via dose adjustment, and averted target antigen loss through repeated administration. Consequently, a tumor-inherent antigen-independent target-redirected universal CAR-T (TRUE CAR-T) cell therapy was established, effectively overcoming tumor antigen heterogeneity. Nevertheless, the tumor selectivity of F-AgNPs relies on the membrane properties of tumor cells; off-target risks remain due to potential uptake by normal cells. Moreover, their tendency to accumulate in the liver and spleen necessitates further investigation into long-term safety [ 137 ].

Besides, coupling NPs with vaccine therapy to target diverse tumor antigenic epitopes and engage endogenous immunity also shines as a beacon of hope for overcoming tumor heterogeneity in CAR-T cell therapy [ 138 – 140 ] ( Fig. 6 D ) . The amph-vax vaccine developed by Ma et al. consists of amphiphilic ligands and adjuvants. Upon administration, it selectively migrates to lymph nodes and modifies the surface of APCs, significantly enhancing antigen presentation efficiency. This vaccine activates and expands potent tumor-killing CAR-T cells while overcoming tumor antigen heterogeneity through endogenous immune system activation and antigen spreading. Mechanistically, IFN-γ secreted by CAR-T cells not only sustains their cytotoxicity and effector factor expression but also recruits dendritic cells (DCs) and promotes antigen uptake. Under IFN-γ stimulation, DCs secrete IL-12, creating a positive feedback loop that amplifies CAR-T effector responses [ 138 , 141 ]. Furthermore, coupling this vaccine with NPs may enable more efficient lymph node-targeted delivery and APC anchoring, thereby potentiating CAR-T cell therapeutic efficacy. In another study, Zhang et al. fabricated a nanovaccine that encapsulated the TLR-9 agonist CpG ODN and was combined with tumor cell lysates (TCLs) and neoantigens [ 142 ]. Experimental results indicated that the NPs could efficiently transport CpG ODN and diverse tumor antigens to the draining lymph nodes. Therein, the CpG ODN spurred DC maturation and drove the secretion of proinflammatory cytokines like IL-6 and IFN-γ. Meanwhile, mature DCs manifested improved antigen-presenting capabilities, activating both T cells and B cells to elicit cellular and humoral immune responses. Furthermore, the vaccine-induced antibody generation synergized with CD16 CAR-T cells through antibody-dependent cellular cytotoxicity (ADCC) to impede tumor growth. Notably, the molecular mechanisms of ADCC and therapeutic synergy remain poorly defined. The use of TCLs and neoantigens in the nanovaccine introduces compositional heterogeneity, potentially resulting in batch variability and complicating standardized production. Future work should focus on validating findings in broader tumor models and species, clarifying the mode of action, and improving manufacturing consistency and safety evaluation [ 142 ].

In addition to the aforementioned aspects, NPs can augment the anti-tumor efficacy of CAR-T cell therapy via multiple mechanisms. Primarily, NPs mimic APCs to directly activate and stimulate T cells, exhibiting remarkable therapeutic potential in animal models [ 141 ]. Recent research has shown that NPs integrated CAR ligands into the immunological synapse (IS), actively recruiting APCs rather than merely mimicking them. This strategy leverages natural immune cell interactions to enhance targeting specificity, thereby optimizing CAR-T cell activation and bolstering anti-tumor efficacy. However, its effectiveness depends on CD169 expression by APCs, which may limit applicability in tumors with low CD169 levels [ 143 ]. Secondly, NPs can be combined with other immunotherapies to further amplify the anti-neoplastic activity of CAR-T cells. For instance, free delivery of immune checkpoint inhibitors such as blockers of PD-1, lymphocyte-activation gene 3 (LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), T cell immunoglobulin and mucin-domain containing 3 (TIM-3), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) bears great prospects in relieving T cell immunosuppression [ 144 – 147 ]. Alarmingly, extensive inhibition by free blockades may incite autoimmune reactions. To address this issue, researchers utilized LNPs to co-deliver CAR mRNA and small interfering RNA (siRNA) targeting PD-1 to T cells, temporarily disrupting endogenous PD-1 expression and reducing systemic immunosuppression risks. Despite these benefits, the efficacy of this approach relies on RNA stability and may require repeated administrations to sustain therapeutic effects [ 148 ]. Moreover, anchoring anti-PD-1 nanogels to CAR-T cell surfaces enhanced treatment efficacy through spatial-temporal co-localization and controlled drug release. Even so, the anchoring efficiency depends on cell surface modification steps, which may increase clinical complexity [ 149 ]. The combination of NPs-based CAR-T cell therapy with traditional therapies such as targeted therapy and radiotherapy unveils potential. For example, PMNPs concurrently delivered PI3Kδ inhibitors and TLR7/8 agonists, enhancing T cell adaptive immune responses and augmenting the anti-tumor effect of local radiotherapy [ 119 , 150 ]. NPs could also modify tumor cells by delivering the Fas gene to heighten the killing power of CAR-T cells against tumor cells. Yet, this strategy relies on the Fas-FasL pathway, which may limit its efficacy in tumors with low FasL expression [ 151 ]. In summary, each of the aforementioned technologies presents distinct advantages. Optimal strategy selection should be guided by specific tumor characteristics, target antigen expression profiles, and therapeutic context.

To address the bottlenecks of CAR-T cell therapy, such as immunosuppressive TME, cell exhaustion, and antigen heterogeneity, recent studies have focused on designing novel CAR variants incorporating innate immune domains. By activating innate immune signals and synergizing with adaptive immune effects, these variants significantly enhance the function and efficacy of CAR-T cells [ 152 – 156 ]. As core pattern recognition receptors (PRRs) of the innate immune system, the TLR family participates in the costimulation of CAR-T cells by regulating T cell activation, differentiation, and cytokine secretion [ 157 ]. Incorporating the Toll/interleukin-1 receptor (TIR) domain of TLR2 into CAR constructs targeting CD19 or mesothelin upregulated the secretion of proinflammatory cytokines (e.g., IL-2 and IFN-γ) and the expression of genes related to cell adhesion and synaptic transmission, significantly enhancing the in vitro and in vivo cytotoxic activity of CAR-T cells against leukemia and solid tumors [ 153 ]. In a clinical trial ( NCT02822326 ), 1928zT2 CAR-T cells harboring the TLR2 domain achieved complete remission in patients with relapse and refractory acute lymphoblastic leukemia (ALL), and mild CRS occurring in some treated patients was effectively managed with tocilizumab treatment [ 158 ]. The integration of the TLR4 domain also shows promise. Studies by Mikolič et al. demonstrated that second-generation anti-CD19 CAR-T cells containing the TLR4 domain exhibited stronger activation in both hematologic malignancies and solid tumor models. Although the response to solid tumors was slower, all tumor-bearing mice ultimately achieved complete remission [ 155 ].

The signaling module, composed of MyD88 (Myeloid Differentiation Primary Response gene) and CD40, represents another key optimization strategy. As a critical adaptor protein in the TLR pathway, MyD88 triggers the NF-κB (nuclear factor kappa B) and MAPK (mitogen-activated protein kinase) pathways by activating IRAK (IL-1 receptor-associated kinase) and TRAF6 (TNF receptor-associated factor 6) [ 159 ]. CD40, a costimulatory receptor, further amplifies these signals via TRAF proteins. Their synergy significantly enhances T cell proliferation, survival, and cytokine production [ 159 ]. Studies by Prinzing et al. confirmed that MyD88/CD40-containing CAR-T cells maintained a less differentiated phenotype (downregulated TBET and Blimp-1 expression) and upregulated the expression of cell cycle regulatory genes (MYB and FOXM1) after repeated stimulation, directly supporting their anti-exhaustion potential [ 160 ].

As a C-type lectin receptor (CLR), NKG2D (natural killer group 2-member D) recognizes stress-induced ligands (e.g., MICA, MICB, and ULBPs) that are upregulated on tumor cells due to DNA damage or oncogenic stress. Via adaptor proteins DAP10 (activating the PI3K-Akt pathway) or DAP12 (activating the Syk/ZAP70 pathway), NKG2D provides activation and signal integration for CAR-T cells without additional costimulatory domains, effectively addressing tumor antigen heterogeneity [ 161 , 162 ]. Currently, NKG2D-CAR-T products such as CYAD-01 have demonstrated significant tumor clearance potential in clinical studies for acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and multiple myeloma (MM) [ 163 ].

These findings confirm that the integration of innate immune signals with CAR-T cells synergizes innate and adaptive immune mechanisms, laying a foundation for next-generation therapies. Notably, NPs, with their excellent carrier properties, have enabled the precise delivery of innate immune ligands such as TLR agonists, effectively enhancing the targeted activation efficiency of immune cells and providing critical technical support for regulating the functions of innate immune components [ 119 , 142 , 164 ]. In the future, integrating NP delivery technology and AI-based design optimization is expected to further overcome the technical bottlenecks of CAR-T therapy, promote its application in solid tumors and long-term efficacy, and provide a more robust and durable solution for cancer immunotherapy.

Strengthen the monitoring and control of CAR-T cell therapy

In CAR-T cell therapy, severe adverse reactions like CRS partly result from inadequate monitoring of the location and duration of CAR-T cell-induced tumor cytotoxicity [ 165 ]. Consequently, it is imperative to develop CAR-T cell tracking methods. This will provide a comprehensive understanding of their in vivo biodistribution and behavioral patterns in patients, enabling timely troubleshooting to ensure safety and permitting prompt evaluation of treatment efficacy for therapy optimization. NPs present a significant opportunity for leveraging clinical imaging techniques to monitor the location, distribution, and activity of CAR-T cells. For instance, Kiru et al. devised a microfluidics-based iron oxide labeling system for CAR-T cells to enhance monitoring capabilities [ 166 ]. Through convective transfer and mechanical cell volume exchange, iron oxide NPs were incorporated into anti-B7-H3 CAR-T cells, obviating the need for transfection agents or genetic manipulation. This innovative approach allows for non-invasive detection of the labeled T cells using photoacoustic imaging (PAT), magnetic resonance imaging (MRI), and magnetic particle imaging (MPI), thereby providing a novel means to monitor CAR-T cells in solid tumors [ 166 ]. In comparison, one study employing iron oxide NPs achieved over 99% labeling efficiency by moderately activating T cells with IL-2 and CD3, without resorting to chemical or mechanical intervention [ 167 ]. By utilizing dextran-coated iron oxide NPs as an MRI contrast agent and the T2* relaxation measurement technique, researchers tracked CAR-T cells and monitored their distribution in intracranial gliomas. Experimental results demonstrated that the NPs could efficiently penetrate the subcellular cytoplasmic vesicles of T cells for labeling, without affecting the viability and effector functions of the T cells. Moreover, the distribution of T cells in the TME was found to be predictive of the anti-tumor treatment outcome [ 167 ]. However, the sole iron oxide NPs currently available for clinical application pose a risk of severe side effects in some patients, such as hypotension, hypersensitivity reactions, or allergic reactions [ 168 ]. Encouragingly, recent research has suggested that MegaPro-NPs may offer enhanced safety [ 169 ]. By labeling CAR-T cells with MegaPro-NPs and conducting in vitro cell iron uptake and activity function tests, along with in vivo MRI, MPI, and bioluminescence imaging (BLI) to monitor tumor growth, it has been confirmed that MegaPro-NPs can be effectively utilized for in vivo tracking of CAR-T cells in preclinical glioblastoma models [ 169 ]. Given that MegaPro-NPs have completed a Phase II clinical trial, the application in clinical cancer patients is expected shortly ( NCT03407495 ). In another study, an in vitro cell-labeling method based on heparin and protamine was employed to attach 89 Zr-labeled dual-modal positron emission tomography (PET)/near-infrared fluorescent (NIRF) silica NPs to CAR-T cells [ 170 ]. This approach enabled non-invasive tracking of infused CAR-T cells via whole-body PET and NIRF imaging for over one week in tumor-bearing mice, providing valuable insights into their spatial distribution and fate. Nevertheless, a key limitation of this technique lies in the gradual dissociation of NPs from CAR-T cells, followed by their uptake by tumor cells, which may compromise the accuracy of cell tracking and fail to reflect the true distribution of viable CAR-T cells. Furthermore, the radiological safety profile of 89 Zr requires further evaluation before clinical translation [ 170 ].

Conventional CAR-T cells, lacking artificial control in vivo, can elicit rapid and durable clinical responses that may lead to severe, even fatal adverse events, particularly on-target/off-tumor toxicity effects and CRS. On-target/off-tumor toxicity effects stem from the expression of CAR-T cell targets in normal tissues, whereas CRS results from the intense interplay between CAR-T cells, tumor cells, and host immune cells, which triggers the swift activation and expansion of CAR-T cells, followed by massive cytokine release [ 39 ]. Consequently, beyond using NPs to monitor CAR-T cell distribution and activity, it is essential to employ them for regulating CAR-T cell function and quantity ( Fig. 7 ) . This will help avert excessive activation and proliferation, thereby curtailing side effects and safeguarding treatment safety. In response, researchers have developed “switch” mechanisms, such as a gelatinase Matrix metalloproteinase 2/9-based nano-modified switch, to modulate CAR-T cell activity, leveraging the high expression of gelatinase in the TME and its minimal presence in normal tissues [ 171 ]. By embedding a gelatinase-responsive peptide within methoxy poly(ethylene glycol)-polycaprolactone (PEG-PCL) copolymers, researchers obtained a PEG-PCL conjugate that was cleaved upon secretion of gelatinase into the TME. This cleavage caused NPs to deform and release drugs into the tumor tissue. Thus, NPs with switch molecules allowed precise control and continuous, stable activation of CAR-T cells, eliminating adverse reactions from sudden cytokine surges [ 171 ]. Similarly, by combining light-switchable CAR-T cells (LiCAR-Ts) with an upconversion nanoplate (UCNP) system, researchers utilized UCNPs to convert NIR light into blue light to activate LiCAR-T cells. The anti-tumor immune response was precisely triggered only when tumor antigens and light coexist, realizing a spatio-temporally regulated immune response [ 172 ]. This combined system has been validated in melanoma and lymphoma mouse tumor models, demonstrating enhanced CAR-T cell therapy safety and marked suppression of tumor growth, thereby providing a promising strategy for precision immunotherapy [ 172 , 173 ]. Employing an analogous remote-control mechanism, another team utilized gold nanorods to convert NIR light into heat, enabling remote control of CAR-T cell activity through photothermal signals. Coupled with thermal-sensitive genetic switches like 7 H-YB, this system induced localized expression of IL-15 Superagonists at 40–42 °C, improving the precision, safety, and antitumor potency of the treatment [ 174 ] ( Fig. 7 A ) . Despite their therapeutic benefits, these technologies present distinct limitations. Nano-modified switch relies on endogenous matrix metalloproteinase (MMP) expression, which restricts its applicability in tumors with low MMP levels. LiCAR-T depends on external light activation, and its efficacy diminishes in deep tumors due to significant attenuation of light intensity. Although the photothermal approach, mediated by gold nanorods, can trigger transgene expression via heat shock response elements, its precision is comparatively lower due to thermal diffusion, which may cause off-target activation in surrounding normal tissues. Fig. 7 Nanoparticles designed to modulate the function and quantity of CAR-T cells. (A) UCNPs and gold nanorods can convert NIR light into blue light and heat, respectively, to activate CAR-T cells, enabling antitumor responses triggered only in the coexistence of tumor antigens and light, thus enhancing treatment safety. (B) NP-based cytokine regulation strategy. (C) NPs switch tumor cell death phenotype. UCNPs: Upconversion nanoplatelets; NIR light: near-infrared light

Besides the “switch” mechanism, NPs can improve CAR-T cell therapy safety by suppressing cytokine release. Zhou et al. used LNPs, which outperform cationic polymers, to encapsulate IL-6 shRNA and CAR gene plasmids, then modified them with CD3 antibody [ 92 ]. They created IL-6 knockdown CAR-T cells in vivo via intravenous injection, maintaining anti-tumor effects while reducing IL-6 release and CRS incidence, bypassing the complexity and cost of traditional in vitro methods. However, this strategy offers limited control over the broader cytokine network, as secretion of other proinflammatory cytokines such as IL-2 and TNF-α persists. Moreover, its clinical applicability may be constrained by variable transfection efficiency and potential off-target effects, warranting further investigation into the consistency and safety of cytokine modulation [ 92 ] ( Fig. 7 B ) . Another study functionalized melanoma-specific CAR-T cells with superparamagnetic iron oxide nanoparticles (SPIONs) [ 175 ]. SPIONs serve as contrast agents for X-ray tomography, MRI, micro-computed tomography, and magnetic guidance carriers [ 176 ]. Following SPION internalization, T cells acquired magnetic controllability, enabling magnetic field-guided precise tumor targeting while significantly reducing off-target distribution and associated damage. Importantly, targeting pyroptosis or DAMPs release is a well-recognized strategy to mitigate CAR-T-associated inflammatory complications, and the SPIONs-based modification strategy aligns with this approach: it preserves overall CAR-T cytotoxicity while suppressing secretion of cytokines (e.g., TNFα, IFNγ), but also shifts target cell death from inflammatory pyroptosis to non-inflammatory apoptosis. This occurs via a mechanism wherein reduced cytokine and granzyme B secretion inhibits the expression and cleavage of gasdermin proteins, thereby markedly lowering systemic inflammatory risks and ultimately enhancing both safety and targeting precision in melanoma treatment. Nevertheless, these findings are limited to in vitro models and require further validation [ 175 ] ( Fig. 7 C ) .

Recently, CAR⁺ EVs derived from CAR-T cells have emerged as a novel tactic to mitigate immunotherapy side effects, attributed to their natural nanostructure, low immunogenicity, and high biocompatibility. The biomolecular constituents and functions within them closely correlate with the source cells and can mediate intercellular crosstalk, participating in biological processes such as immunity and inflammation [ 177 ]. A previous report has shown that CAR-T cells could release EVs, primarily in the form of exosomes bearing CAR constructs, which expressed antibodies against EGFR and HER2, exhibiting potent anti-tumor effects with low toxicity in breast cancer [ 178 ]. Building on this, Haque and colleagues innovatively produced “off-the-shelf” targeted CAR exosomes using in vitro parental cells and CAR plasmids, simplifying the cumbersome traditional CAR-T cell preparation [ 179 ]. They demonstrated that CD19 CAR exosomes, instead of whole CD19 CAR-T cells, could induce cytotoxicity in CD19-positive leukemia B cells while sparing negative cells and curtail the CRS risk, thereby charting a novel course for cancer nano-immunotherapy [ 179 ]. Apart from their robust anti-tumor potential, CAR⁺ EVs serve as both CAR-T cell activation markers and predictors of therapy-related adverse reactions. In vitro assays revealed that following co-culture of CD19 CAR-T cells with CD19⁺ target cells, the release of CAR⁺ EVs spiked within 1 to 4 h, signifying a close link between CAR⁺ EV release and target cell binding, thereby representing an immediate response to CD19 CAR-T cell activation. In vivo, CAR⁺ EVs could be detected in plasma just 1 h after patients received CD19 CAR-T cells, and their plasma presence paralleled the disappearance of circulating CAR-T cells, further attesting to their capacity to reflect CAR-T cell activation in vivo [ 180 ]. Furthermore, the clinical trial highlighted plasma CAR⁺ EVs as early predictors of ICANS. The data revealed that ICANS patients exhibited significantly higher plasma CAR⁺ EV levels in the early phase (e.g., 1 h and day 1 post-infusion) compared to non-ICANS patients. Specifically, a plasma CAR⁺ EV concentration above 132.8 CAR⁺ EVs/µL at 1 h post-infusion predicted ICANS with 89.29% sensitivity and 74.58% specificity, while a level above 224.5 CAR⁺ EVs/µL on day 1 achieved 96.55% sensitivity and 80.36% specificity. These results suggested that CAR⁺ EVs could predict ICANS up to 4 days before symptom onset (median onset: day 5) and correlate with ICANS severity [ 180 ]. Taken together, the applications of NPs offer novel pathways to improve CAR-T cell therapy safety and propel the advancement of cancer immunotherapy.

Limitations and solution strategies: no garden is without weeds

Over the past three decades, nanotechnology has witnessed rapid advancements, leading to the creation of numerous NPs, such as the most representative LNPs and PNPs, which are specifically designed for the targeted delivery of anti-tumor drugs. To date, at least 15 cancer nanomedicines have been approved worldwide, concurrently with over 80 novel drugs under clinical trial evaluation [ 181 ]. Currently, FDA-approved or clinically investigated nanomedicines are primarily based on organic nanomaterials, such as NP liposomes. Representative examples include Vyxeos ® (liposomal daunorubicin and cytarabine for injection) and Marqibo ® (liposomal vincristine sulfate injection), approved for treating leukemia after successful clinical trials [ 182 ] ( Table 5 ) . Extensive preclinical research has explored NP-based strategies, showing promising potential to boost tumor-antagonizing immunity ( Table 6 ) . However, NP-based CAR-T cell immunotherapy remains in its early stages, with only one ongoing clinical trial employing 64Cu SPIONs and PET-MRI to study cilta-cel’s in vivo transport in extramedullary myeloma, while recruitment for this trial continues ( NCT05666700 ). Before the large-scale clinical translation of NP-based CAR-T cell therapy, improvements should be considered in the following aspects. Table 5 FDA-approved nanomedicines for anti-cancer therapy Drug name Company Nanoparticle material Active ingredients Indications Date of first approval Oncaspar Sigma TAU Polymer protein conjugate Pegaspargase Acute lymphoblastic leukemia 1994 Doxil Baxter Healthcare Corp Liposome Doxorubicin hydrochloride Ovarian cancer AIDS-related Kaposi’s sarcoma Multiple myeloma 1995 DaunoXome Galen (UK) Liposome Daunorubicin citrate Kaposi’s sarcoma 1996 DepoCyt IPacira Pharms Inc Liposome Cytarabine Lymphomatous meningitis 1999 Abraxane Bristol-Myers Nanoparticle-bound albumin Paclitaxel Lung cancer Metastatic breast cancer Metastatic pancreatic cancer 2005 Marqibo Acrotech Liposome Vincristine sulfate Acute lymphoblastic leukemia 2012 Onivyde Ipsen Liposome Irinotecan hydrochloride Metastatic pancreatic cancer 2015 Vyxeos Celator Pharms Liposome Cytarabine Daunorubicin Acute myeloid leukemia 2017 Fyarro Aadi Sub Albumin-bound nanoparticles Sirolimus Malignant perivascular epithelioid cell tumor 2021 Table 6 Representative nanoparticle-based strategies for enhancing the CAR-T cell therapy Strategies Nanoparticles Cargos Administration Functions References CAR-T cell engineering Spleen SORT lipid nanoparticles CD19-targeted CAR mRNA Intravenous injection Produce CAR-T cells in situ [ 98 ] Ionizable lipid nanoparticles CAR mRNA and siRNA targeting PD-1 Co-incubation Program CAR-T cells and downregulate PD-1 expression [ 148 ] Activating lipid nanoparticles CD19-targeted CAR mRNA Co-incubation Enable one-step activation and transfection of primary human T cells [ 85 ] Magnetic nanoparticles NA Co-incubation Enable high-performance magnetic cell sorting [ 87 ] Promote aggregation at tumor sites Human serum albumin nanoparticles IL-12 Intravenous injection Release IL-12, recruit and expand CAR-T cells through chemokines at tumor sites [ 109 ] LY/ICG@HES-PCL nanoparticles LY ICG Intravenous injection Reshape the TME via photothermal therapy to facilitate CAR-T cell therapy [ 111 ] Immunomagnetic particles NA Intravenous injection Decorate CAR-T cells to enable magnetic-acoustic actuation for precise tumor targeting and in situ immune activation [ 113 ] Modulate hostile TME CaCO3 nanoparticles Catalase Intratumoral injection Neutralize tumor acidity, attenuate tumor hypoxia, and suppress lactate production [ 117 ] Au/polydopamine nanoparticles Horseradish peroxidase Intravenous injection Inhibit the glycolytic metabolism of tumor cells, reducing lactate efflux, reprogramming tumor immunosuppression [ 118 ] Polymeric micellular nanoparticles TLR 7/8 agonist and PI3k delta inhibitor Intravenous injection Intraperitoneal injection Diminish the immunosuppressive TME [ 119 ] Protein nanogels IL-15 Sa Intravenous injection Boost intratumoral T cell proliferation and limit toxic reactions [ 128 ] Restrict tumor antigen heterogeneity Human ferritin nanoparticles Granzyme B Intravenous injection Enable robust binding to gastric cancer cells and demonstrate superior cytotoxicity [ 135 ] Fusogenic nanoparticles Exogenous antigens Intravenous injection Intraperitoneal injection Leverage exogenous antigens to perform in situ modification on solid tumors [ 137 ] Strengthen the monitoring of CAR-T cells Iron oxide nanoparticles NA Mechanoporation Intravenous injection Achieve PAT, MRI and MPI multimodal imaging [ 166 ] MegaPro nanoparticles NA Co-incubation Intracardiac injections Realize MRI, MPI, and BLI multi-modality imaging [ 169 ] 89 Zr-labeled dual-modal PET/NIRF silica NPs NA Intravenous injection Intraperitoneal injection Accomplish PET/NIRF dual-modal imaging [ 170 ] Enhance the control of CAR-T cells Gelatinase-responsive nanoparticles Switch molecules Intraperitoneal injection Allow precise control and continuous, stable activation of CAR-T cells [ 171 ] Upconversion nanoplates NA Intravenous injection Intratumoral injection Convert NIR light into blue light to activate light-switchable CAR-T cells [ 172 ] Lipid nanoparticles Plasmid containing IL-6 shRNA and CD19-CAR Intravenous injection Engineer T cells to generate CAR-T cells with IL-6 gene suppression [ 92 ] Superparamagnetic iron oxide nanoparticles NA Co-incubation Enable magnetic control of CAR-T cells, suppress inflammatory cytokine release, and switch tumor cell death mechanism [ 175 ]

Production of NPs

NP production faces challenges in sterility control, quality control, surface modification, and scalable manufacturing, all of which are critical for successful biomedical applications. Sterility and quality control ensure safety and batch consistency, while surface modification enhances stability, targeting, and therapeutic efficacy. Additionally, scalable production, a key bottleneck in transitioning NPs from the laboratory to clinical applications, requires solutions to environmental safety and regulatory issues [ 183 ].

Sterility and quality control

Like other medical devices or drugs, NPs for in vivo use must remain sterile to prevent treatment failure and toxic reactions [ 184 ]. Various sterilization techniques, including non-ionizing radiation, ionizing radiation, autoclaving, sterile filtration, and chemical agents such as formaldehyde and ethylene oxide, have been developed [ 185 – 187 ]. However, just as the saying “every coin has two sides” goes, each sterilization method possesses both merits and demerits, potentially adversely affecting the properties of NPs. Therefore, it is essential to consider factors such as NP formulation, batch size, and terminal sterilization limits to select the most suitable sterilization method for maximizing benefits [ 187 ]. It has been shown that purification strategies can remove residual production components, thereby avoiding toxicity [ 188 ]. Besides sterility, strict quality control of NP size, composition, and shape ensures batch consistency. During production, analytical methods such as dynamic light scattering, microscopy, and zeta potential measurements play crucial roles in characterizing NPs, maintaining quality, and achieving reproducible manufacturing [ 189 , 190 ].

Surface modification and stability

Extensive research has been conducted on NP surface modification to prolong their circulation time, enable precise drug delivery, and provide targeted payload release [ 191 ]. As the most representative approach, PEGylation involves coating NPs with PEG to prevent aggregation, opsonization, and phagocytosis, thereby extending blood circulation duration and promoting accumulation at pathological sites via the enhanced EPR effect [ 192 , 193 ]. However, the immunogenicity of PEGylated NPs can trigger anti-PEG antibody generation, thereby accelerating blood clearance and reducing therapeutic efficacy [ 194 , 195 ]. To alleviate this concern, various strategies have been explored, including designing cleavable PEG structures, reducing PEG chain number and length to minimize antigenicity, and incorporating tumor-targeting ligands to enhance therapeutic efficacy and targeting [ 193 , 196 ]. Alternative modification strategies, such as employing hyaluronic acid, polyglycerol, surfactants, or small targeting peptides (e.g., cyclic peptide Arg-Gly-Asp) and blood proteins like albumin, have also been investigated [ 197 – 200 ].

Scaling up production

Scalable production remains a critical bottleneck in clinical translation. Compared to small-scale laboratory preparation, the industrial-scale production of NPs requires stringent control of sterility and quality to ensure safety and product consistency, making the process more complex and costly [ 183 ]. Beyond selecting suitable sterilization methods, employing advanced software and analytical tools for process optimization, along with clarifying NP physicochemical properties, understanding key component interactions, and exploring characteristic-performance relationships, enhances nanomedicine manufacturing reproducibility [ 201 ].

Large-scale NP production poses significant environmental safety challenges. When handling dry nanomaterials, their small size allows them to disperse like dust in the air, potentially being inhaled and deposited in the lungs, causing tissue damage [ 202 ]. Even worse, some NPs can penetrate the skin barrier, making dermal exposure a potential risk. Therefore, personal protective measures must be strengthened during nanomedicine manufacturing to minimize the potential harm to personal health [ 203 ]. To predict immune interactions, extensive toxicity testing should be performed [ 201 ]. Pleasingly, manufacturing NPs entirely in liquid environments significantly reduces potential environmental impact, making closed production systems an effective strategy. Meanwhile, to guide clinical research and accelerate clinical translation of nanomedicines, the DELIVER framework has been introduced, encompassing critical elements including design, experimental, manufacturing, preclinical, clinical, regulatory, and business considerations [ 204 ].

Cargo delivery and expression of CAR

NPs face multiple barriers in mediating CAR cargo delivery, including target distribution barriers, extracellular barriers, and intracellular barriers. To enhance T cell transfection efficiency, researchers have optimized NP design through various strategies. This section will highlight the barriers and corresponding solutions encountered at each major step from NP administration to final CAR expression in chronological order ( Fig. 8 ) .

Fig. 8 Barriers of nanoparticle as a carrier for CAR cargo. (A) Administration routes for NPs. (B) Intravenous injection of NPs into the bloodstream faces multiple challenges: First, plasma proteins adsorb onto the NP surface to form a protein corona, which alters their surface properties and affects in vivo distribution and stability. Second, blood shear stress may disrupt NP structure, interfering with their distribution and delivery functions. Additionally, endovascular nucleases, proteases, and other enzymes can degrade nucleic acids encapsulated within NPs, while interactions between NPs and blood cells further reduce their stability and delivery efficiency. (C) Throughout this process, NPs may also be cleared from the systemic circulation via the MPS, hepatobiliary elimination (by feces), or renal excretion (by urine). (D) The sequential steps and potential barriers in cargo delivery to target cells: After reaching target cells, NPs bind to the cell membrane and undergo cellular uptake through different endocytic routes. Upon uptake by T cells, NPs are typically encapsulated within endosomes, making effective endosomal escape critical. Following endosomal escape, NPs release their cargo to generate CAR-T cells, where DNA cargo must be transported to the nucleus for transcription, while mRNA cargo can directly bind to ribosomes in the cytoplasm to initiate translation. MPS: mononuclear phagocyte system

Reaching target cells

NPs face multiple barriers before reaching target cells, with these challenges varying based on the administration method. They can be administered via various routes, including intravenous injection, local injection (e.g., heart, brain, eye, tumor, or utero), intranasal administration, inhalation, and oral delivery [ 205 ] ( Fig. 8 A ) . Each administration route requires crossing specific barriers to enter the bloodstream. For example, oral delivery must overcome intestinal barriers, including mucus, immune components, and microbiota, while non-vascular methods like subcutaneous injections require overcoming barriers like the vascular endothelium [ 206 ]. Taking the commonly used intravenous injection in clinical practice as an example, NPs encounter multiple barriers upon entering the bloodstream ( Fig. 8 B ) . Following contact with blood, NPs rapidly adsorb plasma proteins and biomolecules, forming a protein corona, which often leads to opsonization, marking them for recognition and degradation by the mononuclear phagocyte system (MPS), including Kupffer cells, splenic macrophages, and bone marrow monocytes [ 196 , 207 ]. NPs not phagocytosed by MPS may be eliminated via hepatobiliary or renal systems based on size [ 208 , 209 ] ( Fig. 8 C ) . Meanwhile, the protein corona alters the surface properties of NPs, influencing their distribution and stability [ 210 ]. Furthermore, shear stress in the bloodstream may disrupt NP structures, further interfering with their distribution and delivery [ 211 ]. NPs may also interact with blood cells, reducing stability and delivery efficiency [ 212 ]. Worse still, intravascular enzymes such as nucleases and proteases degrade the nucleic acids encapsulated by NPs [ 213 ]. Due to these barriers, only a limited number of NPs reach their targets and effectively deliver the cargo [ 214 ].

To address these challenges, researchers have developed various targeting strategies. Passive targeting leverages NPs’ size and surface properties, whereas active targeting involves surface modifications with ligands like small molecules, aptamers, antibodies, peptides, or cells [ 214 , 215 ]. Endogenous targeting exploits NPs’ composition to bind specific plasma proteins, while stimuli-responsive targeting utilizes materials that change under specific conditions [ 97 , 99 , 216 ]. In practical applications, multiple strategies are often combined to achieve the best results.

Binding and endocytic uptake

After reaching target cells, NPs need to bind to the cell membrane and cross this barrier to achieve intracellular delivery. The interaction between NPs and cell surfaces is influenced by multiple factors, including their charge, shape, and size [ 217 ]. For instance, anionic NPs may exhibit poor cellular uptake due to electrostatic repulsion with the negatively charged cell membrane, while excessive positive charge on cationic NPs can cause membrane damage [ 217 ]. Following the binding, NPs enter cells primarily through endocytosis or direct membrane fusion [ 218 ]. Nonetheless, T cells are notoriously difficult to transfect, significantly hindering NP-cell surface binding and subsequent cellular internalization [ 219 ]. Ligands such as anti-CD8 antibodies, transferrin, and integrin can be added to enhance binding and uptake [ 220 , 221 ]. Particle size also critically influences endocytic pathways, including clathrin-mediated, caveolin-mediated, macropinocytotic, and clathrin/caveolin-independent mechanisms, making size optimization crucial for T cell uptake [ 218 ].

Escaping from the endosomes

Upon uptake by T cells, NPs and their cargo are typically entrapped in endosomes, which gradually mature, acidify, and fuse with lysosomes, ultimately leading to cargo degradation [ 222 ]. Efficient endosomal escape is thus essential for successful delivery. Strategies include modifying NP composition (e.g., employing ionizable lipids, proton sponge molecules, or calcium ions) or designing specific structures like cuboplex nanostructures [ 223 – 226 ]. Further research into endosomal escape mechanisms and novel NP modifications is needed to advance clinical applications.

Expressing the cargo as CAR

Following endosomal escape, NPs need to release their cargo to generate CAR-T cells. For DNA cargo, NPs must deliver it to the nucleus for transcription, requiring T cells to be in an active mitotic state. Therefore, it is crucial to activate primary T cells, which can be achieved through aAPCs or CD3/CD28 magnetic beads [ 227 ]. In addition, incorporating nuclear localization signal (NLS) peptides or modifying DNA can enhance therapeutic transgene expression in T lymphocytes [ 90 , 228 ]. For mRNA cargo, once released into the cytoplasm, it can directly bind to ribosomes to initiate translation, bypassing the need to enter the nucleus. However, mRNA offers short-term expression and requires frequent dosing to maintain efficacy, which may lead to cumulative toxicity and systemic inflammatory responses [ 140 ]. Potential solutions incorporate developing sustained-release delivery systems, optimizing mRNA sequences, investigating self-amplifying and trans-amplifying RNA systems, and exploring the application of circular RNA [ 229 – 233 ].

T cells have cell type-specific barriers and present additional gene delivery challenges. Autophagosome formation reduces polymer-mediated transfection efficiency, potentially addressed by manipulating autophagy via vacuolar protein sorting 34 and UNC-51-like kinase 1 complexes [ 234 ]. Slow endosomal acidification in T cells can be countered by pH-sensitive monomers with high pKa values to promote endosomal escape [ 235 ]. Overall, T cell transfection is a multi-step process, and the capability of NPs to overcome the aforementioned barriers is crucial for their successful delivery.

Efficacy and security in combined application

Combining NPs with CAR-T cell therapy confronts a gauntlet of obstacles that jeopardize anti-neoplastic effectiveness. Immunogenicity is a major concern; specifically, PEGylated LNPs can induce anti-PEG antibodies, leading to rapid clearance [ 236 ]. Moreover, immune cell membrane-coated NPs may also exhibit immunogenicity due to MHCs, affecting therapeutic outcomes [ 237 ]. Thus, a thorough investigation of the physicochemical properties of NPs and modification strategies, as well as their impact on pharmacokinetics and targeted delivery, is essential before NP clinical application. As previously mentioned, opsonization, MPS clearance, hepatobiliary, and renal system excretion will further reduce the concentration of NPs at target sites while increasing non-target tissue aggregation [ 207 – 209 ]. Some NPs could also suppress the functions of monocytes, macrophages, and NK cells, as well as pro-inflammatory cytokine production, leading to immune suppression [ 238 ]. Optimizing NP size, shape, charge, and surface modifications can enhance targeting, reduce immune suppression, and improve anti-tumor potency.

The potential hazards of NPs cannot be overlooked, as evidenced by the significant cytotoxicity demonstrated by magnetite and copper oxide NPs [ 239 , 240 ]. LNPs may also induce adverse effects through multiple mechanisms, including IgM-mediated pseudoallergy, IgE-mediated allergic reactions, and autoimmune responses [ 241 , 242 ]. In addition, the limited biodegradability of some NPs presents risks of long-term accumulation and chronic toxicity, but surface modifications and biodegradable materials can mitigate these risks [ 143 , 243 ]. Moreover, many countries have enacted relevant laws and regulations to standardize the research and application of nanotechnology, thereby ensuring safety [ 244 ].

Regulatory frameworks

The clinical translation of NP-based strategy in CAR-T cell therapy also faces unique regulatory challenges: such combination products are subject to dual regulatory frameworks for both cell therapy products and NP-based therapy, but current guidelines lack integrated standards for such combined products. Understanding the existing regulatory landscape and addressing its inherent contradictions are crucial for advancing these therapies from laboratory research to clinical application.

CAR-T cell therapy

Globally, the United States, the European Union (EU), and Japan have established comprehensive regulatory frameworks for CAR-T cell therapy to facilitate innovation while ensuring patient safety. In the United States, the FDA classifies CAR-T products as human gene therapy products, overseen by the Center for Biologics Evaluation and Research (CBER) [ 245 ]. To accelerate approval, the FDA offers expedited pathways such as the Regenerative Medicine Advanced Therapy (RMAT) designation, which provides priority review and early regulatory communication for products addressing unmet medical needs without requiring demonstrated superiority over existing treatments [ 246 ]. The Breakthrough Therapy designation further shortens approval timelines for products showing substantial improvement in early clinical studies [ 247 ]. In the product development and manufacturing phases, manufacturers must submit detailed Investigational New Drug (IND) applications, including protocols for production and quality control. Manufacturing processes must strictly comply with current Good Manufacturing Practices (cGMP), with rigorous controls across the entire production process, from vector sourcing and T-cell handling to cryopreservation and transport, ensuring consistency and product stability [ 248 ].

In the EU, CAR-T cell therapy is classified as Advanced Therapy Medicinal Products (ATMPs), and their review system is jointly established by the Committee for Advanced Therapies (CAT) and the Committee for Medicinal Products for Human Use (CHMP) under the European Medicines Agency (EMA). The EU has established the Priority Medicine (PRIME) scheme, which targets innovative medicines for the treatment of serious diseases with unmet medical needs. Eligible CAR-T products can apply for this scheme and gain support, such as early communication with the EMA and accelerated evaluation [ 249 ]. Additionally, the conditional marketing authorization pathway allows for the approval of a product that can demonstrate significant benefits and controllable risks based on limited data, on the condition that further studies are conducted post-authorization to confirm its efficacy and safety [ 250 ]. Notably, ATMPs must submit marketing authorization applications through a centralized approval procedure, and the manufacturing process must comply with the GMP specifically developed by the EMA. Due to genetic modification, some member states also require environmental risk assessments to ensure public health and ecological safety [ 251 ].

Under the joint oversight of the Pharmaceuticals and Medical Devices Agency (PMDA) and the Ministry of Health, Labour and Welfare (MHLW), Japan classifies and regulates CAR-T products as regenerative medicine products. Japan has established the SAKIGAKE designation (Forerunner designation or pioneer drug), which is granted to innovative products first applied for in Japan and with potential to address unmet medical needs. Recipients enjoy preferences like priority review and shortened review cycles to accelerate market access [ 252 ]. Additionally, the Time-limited conditional approval pathway permits marketing approval based on non-confirmatory clinical trial data, though enterprises must supplement confirmatory data within a post-marketing specified period to retain marketing authorization [ 253 ]. During product development and clinical research, enterprises must comply with relevant regulations and guidelines. For example, the Act on the Safety of Regenerative Medicine implements risk-classified management for regenerative medicine technologies, with products of different risk levels subject to distinct review processes and requirements. Strict quality control standards also apply to manufacturing to ensure product quality and safety [ 251 ]. In summary, although the regulatory frameworks in the United States, EU, and Japan differ in classification and approval mechanisms, they share the core objective of ensuring safety, efficacy, and quality control. Through structured regulatory systems, expedited pathways, and lifecycle management, these regions support CAR-T innovation while safeguarding patient access to reliable therapies.

NP-based therapy

In the global regulatory landscape for NP-based therapy, the United States, Europe, and Japan have established tailored regulatory frameworks that align with their respective pharmaceutical oversight systems. These frameworks not only focus on the unique physicochemical properties and potential risks of nanomaterials but also provide guidance for their clinical translation [ 254 ]. In the United States, the FDA employs a multidimensional and adaptive regulatory strategy centered on risk assessment and product specificity. The agency has issued several key guidelines: the 2014 guidance Considering Whether an FDA-Regulated Product Involves the Application of Nanotechnology outlines criteria for nanomaterials, incorporating both size (1–100 nm) and size-dependent properties [ 255 ]; the 2018 guidance Liposome Drug Products specifies requirements for chemistry, manufacturing, controls (CMC), pharmacokinetics, and labeling [ 256 ]; and the 2022 guidance Drug Products , Including Biological Products , that Contain Nanomaterials refines the risk assessment framework, emphasizing evaluation of physicochemical properties, biological interactions, and long-term stability, along with the control of critical quality attributes (CQAs) [ 257 ]. The FDA also encourages early industry engagement and adopts “product-specific premarket reviews” to accommodate technological diversity [ 255 ].

In Europe, the EMA leads a regulatory approach focused on comparability studies and detailed control. Through scientific reflection papers, the EMA emphasizes the consistency and safety of nanomedicines and their reference products [ 258 ]. For intravenous liposomal formulations, the EMA requires demonstration of comparability in pharmaceutical quality, nonclinical data, and clinical performance [ 259 ]. For coated nanomedicines, it focuses on the stability of coatings, metabolic pathways, heterogeneity of surface coverage, process validation, and risk assessments of coating detachment [ 260 ]. For block copolymer micelles, early regulatory communication is advised to define CQAs, along with comprehensive documentation of synthesis, purification, and raw materials [ 261 ]. The European Nanomedicine Characterization Laboratory (EU-NCL) is advancing method standardization, though this effort remains under public consultation [ 262 ].

Japan’s regulatory system, overseen by the MHLW and the PMDA, follows a case-by-case methodology within existing pharmaceutical frameworks, supplemented by specialized provisions. Though no standalone nanomedicine regulation exists, the MHLW and PMDA issue joint reflection papers to elaborate on regulatory requirements: for example, block copolymer micelle products require data on synthesis, formation mechanisms, and stability [ 263 ]; a 2016 reflection paper on siRNA-loaded nanotherapeutics clearly outlined CMC standards, nonclinical toxicology, and clinical trial design [ 264 ]. Japan has established a Nanomedicine Initiative Working Group to discuss regulatory needs, but its regulatory progress lags behind that of the United States and Europe, with guidelines for emerging nanomedicine formulations still under development. Moreover, the approval process relies heavily on sponsors proactively submitting supporting data [ 254 ]. Overall, while the United States, European, and Japanese regulatory systems all prioritize safety and efficacy, they differ in emphasis: the United States system is risk-based and flexible, Europe emphasizes comparability and detailed control, and Japan adopts an incremental, case-based approach. These divergent frameworks necessitate compliance with multiple standards, underscoring the need for greater international harmonization to facilitate the global development of NP-based therapeutics.

NP-CAR-T combined therapy

When NPs are used in combination with CAR-T cell therapy, their integration of multiple technical dimensions, including nanomaterials, cell therapy, and gene editing, poses significant challenges to existing regulatory frameworks designed for single therapy. Due to the absence of specific guidelines for the approval and commercialization of such combined products, the regulatory landscape for NP-enhanced CAR-T cell therapy remains poorly defined. Multiple agencies may be involved in oversight. For example, in the United States, oversight may involve the FDA’s CBER, its Center for Drug Evaluation and Research (CDER), and the National Institutes of Health (NIH), which regulates gene therapies. However, the unclear demarcation of responsibilities among these agencies often leads to prolonged approval processes [ 265 ]. Unlike viral vectors with well-established regulatory pathways, non-viral vectors such as LNPs may reduce the risk of insertional mutagenesis but could necessitate the reconstruction of regulatory standards. For instance, NP-mediated CAR expression via mRNA is transient and may require repeated administrations, yet existing regulatory systems lack risk assessment indicators for such dosing strategies. Moreover, quality control indicators for viral vectors (e.g., viral titer, integration efficiency) are inapplicable to NPs, demanding the development of NP-specific parameters such as cargo encapsulation efficiency and transfection uniformity [ 266 ]. The synergistic interaction between NPs and CAR-T cells also introduces complex risk considerations: NPs carry the risk of accumulation, while CAR-T cells pose the risk of CRS; their combination may amplify these risks. However, current regulations do not mandate quantitative analysis of synergistic risks, making it difficult to balance therapeutic benefits against potential toxicities. Furthermore, as NP-based CAR-T cell therapy are still largely in the preclinical stage, the absence of long-term follow-up data fails to meet regulatory requirements for combination products [ 267 ].

To address these challenges, the following potential solutions can be adopted: First, promote interdisciplinary regulatory coordination by establishing a regulatory working group for NP-based cell therapy. This group should integrate expert resources from the fields of nanomaterials, cell therapy, and gene editing, and explore a “modular review” approach to evaluate the safety of NPs and CAR-T cells separately, thereby streamlining the approval process. Second, develop a dedicated regulatory framework. Drawing on the successful application of LNPs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, which accelerated the clinical translation of NP delivery systems, specialized guidelines for NP-based CAR-T cell therapy should be developed [ 268 ]. These guidelines should clarify key quality attributes of NPs (e.g., encapsulation efficiency, drug loading, stability) and safety evaluation indicators (e.g., maximum safe dose, organ accumulation threshold). Finally, optimize the risk-benefit assessment model by requiring the submission of quantitative data on the synergistic effects of NPs and CAR-T cells, for instance, by comparing the fold increase in efficacy with the incidence of additional toxicities. A dynamic risk monitoring system should also be established, including post-marketing continuous monitoring of NP biodistribution and long-term toxicity. Overcoming these regulatory hurdles requires collaboration among academia, industry, and agencies. Stakeholders should work within existing frameworks while advancing innovation through international standards, improved preclinical models, and transparent risk-benefit communication [ 269 ]. By establishing efficient and safety-conscious pathways, we can accelerate the clinical translation of these innovative combined therapies.

Artificial intelligence-powered nanoparticle-based CAR-T cell therapy

AI, a branch of computer science, aims to enable computers or computer-controlled machines to perform complex tasks requiring “human intelligence”. It is broadly categorized into three types: artificial general intelligence, artificial narrow intelligence, and artificial superintelligence. Key subfields include machine learning (ML), artificial neural networks, and deep learning [ 270 ]. In recent years, the rapid advancement of AI has ushered in a transformative era in medical and biological research [ 271 – 274 ]. The deep integration of AI and NPs is transforming CAR-T cell therapy from a standalone cellular treatment into a multi-technology-integrated precision medicine system. Through AI-driven target screening, product design optimization, delivery efficiency enhancement, efficacy monitoring, and toxicity prediction, this approach may establish a comprehensive “manufacturing optimization, efficacy maximization, and safety management” therapeutic framework.

Manufacturing optimization

To address the time-consuming and high complexity of CAR-T cell manufacturing, ML algorithms integrate multi-omics and functional data to accurately predict CQAs (e.g., CD4/CD8 ratios, memory cell subsets) for identifying optimal resource allocation and experimental conditions [ 275 ]. This approach also enables real-time quality prediction through early-phase parameter monitoring, facilitating scalable and standardized production of therapeutic cells [ 275 ]. Notably, large-scale collaborative initiatives are now underway to harness AI for advancing clinical applications of cell therapies. The European community-funded project on AI-driven, Decentralized Production for Advanced Therapies in the Hospital exemplifies such efforts [ 276 ].

Integrating ML and high-throughput NP synthesis, characterization, and functional testing enables data-driven candidate selection. Yamankurt et al. investigated Spherical nucleic acid candidates for cancer vaccines. They first identified 11 Spherical nucleic acid characteristics, generating 3,072 potential NP formulations, of which 960 were synthesized for high-throughput immunostimulation screening. Subsequently, they developed ML models using the experimental data to elucidate structure-immunoactivity relationships. This study demonstrates how combining ML with high-throughput NP screening can significantly reduce the number of designs requiring experimental testing, thereby shortening the research and development cycle and improving manufacturing efficiency [ 277 ]. While the work lays a methodological foundation for AI-aided NP design, it requires further improvement in model universality, multi-objective optimization, and production scale adaptation.

Efficacy maximization

A key objective in CAR-T cell research is identifying clinically safe and effective target antigens. Ideal targets are surface markers strongly and exclusively expressed on tumor cells but absent from vital non-malignant cells. AI tools facilitate this process by analyzing single-cell RNA and protein datasets to compare tumor and normal cell profiles.

In one study, an AI-powered deep learning model integrated multiple single-cell datasets and identified myeloid markers CD86 and colony-stimulating factor 1 receptor (CSF1R) as promising targets for AML [ 278 ]. Following tumor-target antigen identification, designing stable and high-affinity CAR proteins is crucial to trigger precise antitumor responses, with scFv structural optimization being particularly critical for enhancing therapeutic efficacy. Martarelli et al. employed an AI-powered computational platform to perform molecular docking and targeted molecular dynamics analysis on anti-CD30 monoclonal antibodies, enabling systematic screening of optimal scFv candidates for CAR construction. This study demonstrates the potential of computational modeling platforms to advance novel CAR engineering while reducing costs and resource consumption. However, the study relies on known antibody sequences, making it difficult to generate novel single-chain variable fragments [ 279 ]. Moreover, AI has been employed to predict optimal CAR signaling motifs by training on a library of 2,300 synthetic costimulatory domains, enabling optimized CAR-T cell phenotype, proliferation, and persistence. While the study features a rich library of motif combinations, the library construction depends on known signaling motifs and fails to cover motifs with unknown functions [ 280 ]. In the future, it is necessary to combine AI-generated design and multi-dimensional experimental validation to advance CAR design.

AI algorithms can also optimize NP delivery through multiple pathways, enhancing the efficacy of cancer therapy. Firstly, by employing ML and computational modeling, AI enables multidimensional optimization of NP characteristics, including size, surface charge, biointerface interactions, drug encapsulation efficiency, and controlled release kinetics [ 281 ]. Secondly, AI accelerates the development and optimization of novel NP formulations while enabling precise prediction of their in vivo behaviors. In a landmark study, drug-loaded ferritin NPs were combined with image-segmentation-based ML to analyze over 67,000 tumor vasculatures, successfully predicting NP permeability. This approach facilitated the design of genetically engineered protein NPs that significantly enhance transendothelial transport in low-permeability tumors, promoting NP accumulation within solid tumors [ 282 ]. Besides, AI algorithms facilitate the discovery of NPs targeting specific tissues and cells, thereby accelerating the realization of intelligent targeted delivery within the TME [ 283 ]. Some researchers developed a high-throughput DNA barcoding system, in which nucleic acid-barcoded LNPs were tracked via deep sequencing to quantify biodistribution. This approach precisely monitors LNP dynamics in both normal and tumor tissues, providing a powerful platform for screening highly tumor-specific nanocarriers [ 283 ]. By integrating nanosystems with sensors and imaging agents, combined with AI algorithms, this platform empowers real-time monitoring and feedback of tumor response, providing critical insights for treatment evaluation and adaptive strategy development. Such closed-loop feedback systems optimize drug delivery, dynamically adjust dosing, and even switch therapeutic modalities to maximize efficacy and overcome drug resistance [ 284 ].

Safety management

CRS and ICANS represent the primary side effects of CAR-T cell therapy. While studies on CRS and ICANS are limited by insufficient datasets and incomplete mechanistic understanding, AI has demonstrated promising potential in toxicity prediction. One group employed a ML approach with 16 models based on regression models and decision tree models to predict grade 4–5 CRS in anti-CD19 CAR-T-treated B-cell ALL patients. By measuring serum cytokine profiles, such as IFN-γ and sgp130 (soluble glycoprotein 130) before the onset of CRS, they achieved over 85% sensitivity and specificity [ 285 ]. Another study developed a ML-based logistic regression model to predict ICANS in 204 Axicabtagene Ciloleucel (axi-cel)-treated follicular lymphoma or diffuse large B-cell lymphoma patients, achieving 77% accuracy and 82% sensitivity [ 286 ]. Current predictive models, such as supervised multivariate models, generally fall short of the high-performance threshold (typically over 85%). However, expanding datasets and advanced AI models may enhance predictive accuracy, enabling proactive toxicity management and safer CAR-T cell therapy.

In addition to the aforementioned aspects, AI demonstrates significant potential to enhance NP-based CAR-T cell therapy through multifaceted approaches, particularly by leveraging large-scale data analytics to optimize patient selection, predict therapeutic responses, and improve early cancer screening and diagnosis [ 287 – 289 ]. It is noteworthy that current AI applications in cancer immunotherapy are still in their infancy, confronting substantial challenges including insufficient high-quality datasets, inherent complexities of biological systems and patient information, regulatory concerns, and the imperative for clinical validation through rigorous trials. Nevertheless, the development of integrated AI platforms incorporating multi-omics data such as genomics, proteomics, and imaging holds considerable promise to revolutionize the therapeutic workflow, from target antigen selection to clinical implementation, thereby ushering in a new era of precision-designed, patient-specific cellular immunotherapies with optimized efficacy and safety profiles [ 290 , 291 ].

Conclusion and future perspectives

The combined application of CAR-T cell therapy and NPs offers an innovative solution for cancer treatment, with its core values manifested in the following three aspects: technological innovation, therapeutic enhancement, and safety control. Firstly, nanocarriers such as PNPs and LNPs enable efficient CAR delivery via mRNA transient expression or DNA site-specific integration, mitigating risks of insertional mutagenesis and cytotoxicity while reducing manufacturing timelines from weeks to days. Additionally, NPs enhance therapeutic efficacy by delivering chemokine modulators, degrading ECM barriers, and reprogramming immunosuppressive microenvironments, thereby improving CAR-T cell infiltration and functional persistence in solid tumors. Thirdly, safety control is ensured through iron oxide NPs enabling in vivo dynamic imaging of CAR-T cells, while “molecular switch” nanosystems precisely regulate CAR-T cell activity to minimize risks of CRS and neurotoxicity. Looking forward, the integration of AI is expected to substantially optimize this therapeutic strategy through AI-driven target screening, product design optimization, delivery efficiency enhancement, efficacy monitoring, and toxicity prediction, which will establish novel paradigms for developing more efficacious and safer immunotherapeutic approaches.

Despite significant strides in the application of NPs in CAR-T cell therapy over the past five years, its clinical translation still faces numerous challenges: uncertainty in NP mechanisms and physicochemical properties; scalability challenges in large-scale production; limitations in in vivo transduction targeting; concerns regarding long-term biocompatibility; potential immunogenic reactions; and translation challenges in experimental models. In this regard, future research should focus on:

Elucidating the pharmacokinetics, biodistribution, metabolism, clearance, and toxicity of NPs, along with their toxicity and immune system interaction mechanisms. Simplifying the design of biomaterial systems, optimizing manufacturing processes, meeting regulatory requirements, and overcoming industrialization barriers. Developing multifunctional integrated nanosystems by optimizing NP and nucleic acid designs or creating new materials to achieve efficient transfection, enhance therapeutic efficacy, and ensure safety. Conducting in-depth studies in immunocompetent mice, immunodeficient and humanized mouse models, as well as non-human primates to promote clinical translation. Integrating AI and high-throughput technologies to optimize nanomaterial design, or utilizing organoid and chip models to construct TME biomimetic systems for more accurate prediction of NPs’ behavior in humans [ 292 , 293 ]. Further expanding research on targeting NK cells and other effector cell subsets, exploring their therapeutic potential beyond tumors, and advancing the field of adoptive cell therapy [ 294 , 295 ].

With the deep integration of materials science, biomedicine, and clinical oncology, NP-based CAR-T cell therapy is poised to overcome existing hurdles in cancer therapy, ushering in a novel era of personalized and controllable “living drugs” in precision medicine. The academic community and the industry need to collaborate on innovation, improve international regulatory frameworks, accelerate the translation from laboratory to clinic, and provide safer and more effective treatment options for cancer patients.

Abbreviations

Chimeric antigen receptor

Extracellular domain

Single-chain variable fragment

Major histocompatibility complex

Transmembrane domain

Hinge domain

Tumor microenvironment

Fas ligand

Fas-associated death domain

Gasdermin E

Damage-associated molecular patterns

Costimulatory domain

T-cells redirected for universal cytokine-mediated killing

Food and Drug Administration

Cytokine release syndrome

Nanoparticle

Artificial antigen-presenting cell

Artificial intelligence

Transforming growth factor-β

Regulatory T cell

Myeloid-derived suppressor cell

Immune effector cell associated neurotoxicity syndrome

Immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome

Interleukin

Polyethylene glycol

Lipid nanoparticle

Natural killer

T cell receptor

Activating lipid nanoparticle

Magnetic amplification by sequentially connected antibodies and nanoparticle

Tumor necrosis factor

Interferon gamma

Polymer nanoparticle

Short hairpin RNA

Selective organ targeting

Fusogenic nanovesicles

Near-infrared

Hepatocellular carcinoma

Human epidermal growth factor receptor 2

IL-12 nano-stimulator

Chemokine C-C motif ligand

CXC chemokine ligand

Hydroxyethyl starch-polycaprolactone

LY2157299

Indocyanine green

Enhanced permeability and retention

Extracellular matrix

CXC chemokine receptor

CAR-T microrobot

Epidermal growth factor receptor

Catalase-encapsulated CaCO3 nanoparticle-constructed colloidosomal microreactors

Horseradish peroxidase

Polydopamine

Synergistic sonodynamic therapy

Immunogenic cell death

Polymeric micellular nanoparticle

Toll-like receptor

Programmed cell death ligand 1

Interferon gene stimulator

Programmed cell death protein 1

Nanogel

IL-15 superagonist complex

Tumor-specific antigen

Human ferritin

Nanobodies

Granzyme B

Fusogenic antigen loaded nanoparticle

Extracellular vesicle

Target-redirected universal CAR-T

Immunological synapse

Lymphocyte-activation gene 3

T cell immunoreceptor with Ig and ITIM domain

T cell immunoglobulin and mucin-domain containing 3

Cytotoxic T-lymphocyte-associated protein 4

Small interfering RNA

Pattern recognition receptors

Toll/interleukin-1 receptor

Acute lymphoblastic leukemia

Myeloid Differentiation Primary Response gene 88

Nuclear factor kappa B

Mitogen-activated protein kinase

IL-1 receptor-associated kinase

TNF receptor-associated factor 6

C-type lectin receptor

Natural killer group 2-member D

Acute myeloid leukemia

Myelodysplastic syndromes

Multiple myeloma

Dendritic cell

Antibody-dependent cell cytotoxicity

Phosphatidylinositol-3 kinase p110δ

Photoacoustic imaging

Magnetic resonance imaging

Magnetic particle imaging

Bioluminescence imaging

Positron emission tomography

Near infrared fluorescent

Polycaprolactone

Light-switchable CAR-T cell

Upconversion nanoplate

Superparamagnetic iron oxide nanoparticle

Matrix metalloproteinase

Mononuclear phagocyte system

Nuclear localization signal

European Union

Center for Biologics Evaluation and Research

Regenerative Medicine Advanced Therapy

Investigational New Drug

Current Good Manufacturing Practices

Advanced Therapy Medicinal Products

Committee for Advanced Therapies

Committee for Medicinal Products for Human Use

European Medicines Agency

Priority Medicine

Pharmaceuticals and Medical Devices Agency

Ministry of Health, Labour and Welfare

Chemistry, manufacturing, controls

Critical quality attributes

European Nanomedicine Characterization Laboratory

Center for Drug Evaluation and Research

National Institutes of Health

Severe acute respiratory syndrome coronavirus 2

Machine learning

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Dong Shang, Zhaokai Zhou, Run Shi, Zhan Wang and Pengpeng Zhang contributed equally to this work and share the first authorship.

Acknowledgements

Not applicable.

Authors’ contributions

LC, QL, and RX provided direction and guidance throughout the preparation of this manuscript. DS, ZKZ, RS, ZW, and PPZ wrote and edited the manuscript. ZKZ reviewed and made significant revisions to the manuscript. FP, HBL, GYC, HZQ, YDX, XZ, WJC, YJC, and SY collected and prepared the related papers. All authors read and approved the final manuscript.

Funding

This work was supported by the Natural Science Foundation of Hunan Province (2024JJ5494), Natural Science Foundation of Changsha City (Kq2403094), Hunan Provincial Development and Reform Commission of Innovative Research Program (2021-212-23), Hunan Innovative Province Construction Special Project (2021ZK4025), and the Hunan Provincial Department of Finance Gra (2024-31, 45, 2022 − 151, 2021 − 139 and 2020-83).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

References

1. Singh AK McGuirk JP CAR T cells: continuation in a revolution of immunotherapy Lancet Oncol 2020 21 3 e168 78 10.1016/S1470-2045(19)30823-X 32135120 Singh AK, McGuirk JP. CAR T cells: continuation in a revolution of immunotherapy. Lancet Oncol. 2020;21(3):e168–78. 32135120 10.1016/S1470-2045(19)30823-X

2. Qian K CAR-T-cell products in solid tumors: progress, challenges, and strategies Interdiscip Med 2024 2 2 e20230047 10.1002/INMD.20230047 Qian K, et al. CAR-T-cell products in solid tumors: progress, challenges, and strategies. Interdiscip Med. 2024;2(2):e20230047.

3. Ellis GI Sheppard NC Riley JL Genetic engineering of T cells for immunotherapy Nat Rev Genet 2021 22 7 427 47 10.1038/s41576-021-00329-9 33603158 PMC8217325 Ellis GI, Sheppard NC, Riley JL. Genetic engineering of T cells for immunotherapy. Nat Rev Genet. 2021;22(7):427–47. 33603158 10.1038/s41576-021-00329-9 PMC8217325

4. Rafiq S Hackett CS Brentjens RJ Engineering strategies to overcome the current roadblocks in CAR T cell therapy Nat Rev Clin Oncol 2020 17 3 147 67 10.1038/s41571-019-0297-y 31848460 PMC7223338 Rafiq S, Hackett CS, Brentjens RJ. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol. 2020;17(3):147–67. 31848460 10.1038/s41571-019-0297-y PMC7223338

5. Alabanza L Function of novel anti-CD19 chimeric antigen receptors with human variable regions is affected by hinge and transmembrane domains Mol Ther 2017 25 11 2452 65 10.1016/j.ymthe.2017.07.013 28807568 PMC5675490 Alabanza L, et al. Function of novel anti-CD19 chimeric antigen receptors with human variable regions is affected by hinge and transmembrane domains. Mol Ther. 2017;25(11):2452–65. 28807568 10.1016/j.ymthe.2017.07.013 PMC5675490

6. Cullen SP Martin SJ Mechanisms of granule-dependent killing Cell Death Differ 2008 15 2 251 62 10.1038/sj.cdd.4402244 17975553 Cullen SP, Martin SJ. Mechanisms of granule-dependent killing. Cell Death Differ. 2008;15(2):251–62. 17975553 10.1038/sj.cdd.4402244

7. Morrissey MA Chimeric antigen receptors that trigger phagocytosis Elife 2018 10.7554/eLife.36688 29862966 PMC6008046 Morrissey MA, et al. Chimeric antigen receptors that trigger phagocytosis. Elife. 2018. 10.7554/eLife.36688. 29862966 10.7554/eLife.36688 PMC6008046

8. Wang L Activation of cancer immunotherapy by nanomedicine Front Pharmacol 2022 13 1041073 10.3389/fphar.2022.1041073 36618938 PMC9814015 Wang L, et al. Activation of cancer immunotherapy by nanomedicine. Front Pharmacol. 2022;13:1041073. 36618938 10.3389/fphar.2022.1041073 PMC9814015

9. Liu Y Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome Sci Immunol 2020 10.1126/sciimmunol.aax7969 31953257 Liu Y, et al. Gasdermin E-mediated target cell pyroptosis by CAR T cells triggers cytokine release syndrome. Sci Immunol. 2020. 10.1126/sciimmunol.aax7969. 31953257 10.1126/sciimmunol.aax7969

10. Shi J Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death Nature 2015 526 7575 660 5 10.1038/nature15514 26375003 Shi J, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015;526(7575):660–5. 26375003 10.1038/nature15514

11. Gong T Damp-sensing receptors in sterile inflammation and inflammatory diseases Nat Rev Immunol 2020 20 2 95 112 10.1038/s41577-019-0215-7 31558839 Gong T, et al. Damp-sensing receptors in sterile inflammation and inflammatory diseases. Nat Rev Immunol. 2020;20(2):95–112. 31558839 10.1038/s41577-019-0215-7

12. Zhang Z Zhang Y Lieberman J Lighting a fire: can we harness pyroptosis to ignite antitumor immunity?? Cancer Immunol Res 2021 9 1 2 7 10.1158/2326-6066.CIR-20-0525 33397791 PMC7789047 Zhang Z, Zhang Y, Lieberman J. Lighting a fire: can we harness pyroptosis to ignite antitumor immunity?? Cancer Immunol Res. 2021;9(1):2–7. 33397791 10.1158/2326-6066.CIR-20-0525 PMC7789047

13. Kravets VG Zhang Y Sun H Chimeric-antigen-receptor (CAR) T cells and the factors influencing their therapeutic efficacy J Immunol Res Ther 2017 2 1 100 13 30443604 PMC6233887 Kravets VG, Zhang Y, Sun H. Chimeric-antigen-receptor (CAR) T cells and the factors influencing their therapeutic efficacy. J Immunol Res Ther. 2017;2(1):100–13. 30443604 PMC6233887

14. Weinkove R Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations Clin Transl Immunol 2019 8 5 e1049 10.1002/cti2.1049 PMC6511336 31110702 Weinkove R, et al. Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations. Clin Transl Immunol. 2019;8(5):e1049. 10.1002/cti2.1049 PMC6511336 31110702

15. Harrer DC CAR triggered release of type-1 interferon limits CAR T-cell activities by an artificial negative autocrine loop Cells 2022 10.3390/cells11233839 36497099 PMC9737386 Harrer DC, et al. CAR triggered release of type-1 interferon limits CAR T-cell activities by an artificial negative autocrine loop. Cells. 2022. 10.3390/cells11233839. 36497099 10.3390/cells11233839 PMC9737386

16. Smirnov S Recent advances on CAR-T signaling pave the way for prolonged persistence and new modalities in clinic Front Immunol 2024 15 1335424 10.3389/fimmu.2024.1335424 38455066 PMC10918004 Smirnov S, et al. Recent advances on CAR-T signaling pave the way for prolonged persistence and new modalities in clinic. Front Immunol. 2024;15:1335424. 38455066 10.3389/fimmu.2024.1335424 PMC10918004

17. Moreno C Alternative CAR therapies: recent approaches in engineering chimeric antigen receptor immune cells to combat cancer Biomedicines 2022 10.3390/biomedicines10071493 35884798 PMC9313317 Moreno C, et al. Alternative CAR therapies: recent approaches in engineering chimeric antigen receptor immune cells to combat cancer. Biomedicines. 2022. 10.3390/biomedicines10071493. 35884798 10.3390/biomedicines10071493 PMC9313317

18. Asmamaw Dejenie T Current updates on generations, approvals, and clinical trials of CAR T-cell therapy Hum Vaccin Immunother 2022 18 6 2114254 10.1080/21645515.2022.2114254 36094837 PMC9746433 Asmamaw Dejenie T, et al. Current updates on generations, approvals, and clinical trials of CAR T-cell therapy. Hum Vaccin Immunother. 2022;18(6):2114254. 36094837 10.1080/21645515.2022.2114254 PMC9746433

19. Zhu C Rationally designed approaches to augment CAR-T therapy for solid tumor treatment Bioact Mater 2024 33 377 95 38059121 10.1016/j.bioactmat.2023.11.002 PMC10696433 Zhu C, et al. Rationally designed approaches to augment CAR-T therapy for solid tumor treatment. Bioact Mater. 2024;33:377–95. 38059121 10.1016/j.bioactmat.2023.11.002 PMC10696433

20. Lee A. Obecabtagene autoleucel: first approval. Mol Diagn Ther. 2025;29(3): 419–23. 10.1007/s40291-025-00771-z 39888581

21. Rurik JG CAR T cells produced in vivo to treat cardiac injury Science 2022 375 6576 91 6 10.1126/science.abm0594 34990237 PMC9983611 Rurik JG, et al. CAR T cells produced in vivo to treat cardiac injury. Science. 2022;375(6576):91–6. 34990237 10.1126/science.abm0594 PMC9983611

22. Mackensen A Anti-CD19 CAR t cell therapy for refractory systemic lupus erythematosus Nat Med 2022 28 10 2124 32 10.1038/s41591-022-02017-5 36109639 Mackensen A, et al. Anti-CD19 CAR t cell therapy for refractory systemic lupus erythematosus. Nat Med. 2022;28(10):2124–32. 36109639 10.1038/s41591-022-02017-5

23. Qin H Insights into next-generation immunotherapy designs and tools: molecular mechanisms and therapeutic prospects J Hematol Oncol 2025 18 1 62 10.1186/s13045-025-01701-6 40483473 PMC12145627 Qin H, et al. Insights into next-generation immunotherapy designs and tools: molecular mechanisms and therapeutic prospects. J Hematol Oncol. 2025;18(1):62. 40483473 10.1186/s13045-025-01701-6 PMC12145627

24. Zhang W BCMA-CD19 bispecific CAR-T therapy in refractory chronic inflammatory demyelinating polyneuropathy hLife 2024 2 8 434 8 10.1016/j.hlife.2024.05.005 Zhang W, et al. BCMA-CD19 bispecific CAR-T therapy in refractory chronic inflammatory demyelinating polyneuropathy. hLife. 2024;2(8):434–8.

25. Elsallab M Maus MV Expanding access to CAR t cell therapies through local manufacturing Nat Biotechnol 2023 41 12 1698 708 10.1038/s41587-023-01981-8 37884746 Elsallab M, Maus MV. Expanding access to CAR t cell therapies through local manufacturing. Nat Biotechnol. 2023;41(12):1698–708. 37884746 10.1038/s41587-023-01981-8

26. Dana H CAR-T cells: early successes in blood cancer and challenges in solid tumors Acta Pharm Sin B 2021 11 5 1129 47 10.1016/j.apsb.2020.10.020 34094824 PMC8144892 Dana H, et al. CAR-T cells: early successes in blood cancer and challenges in solid tumors. Acta Pharm Sin B. 2021;11(5):1129–47. 34094824 10.1016/j.apsb.2020.10.020 PMC8144892

27. Xiao X Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies J Exp Clin Cancer Res 2021 40 1 367 10.1186/s13046-021-02148-6 34794490 PMC8600921 Xiao X, et al. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J Exp Clin Cancer Res. 2021;40(1):367. 34794490 10.1186/s13046-021-02148-6 PMC8600921

28. Liu Z Immunosuppression in tumor immune microenvironment and its optimization from CAR-T cell therapy Theranostics 2022 12 14 6273 90 10.7150/thno.76854 36168626 PMC9475465 Liu Z, et al. Immunosuppression in tumor immune microenvironment and its optimization from CAR-T cell therapy. Theranostics. 2022;12(14):6273–90. 36168626 10.7150/thno.76854 PMC9475465

29. Saeed M Engineering nanoparticles to reprogram the tumor immune microenvironment for improved cancer immunotherapy Theranostics 2019 9 26 7981 8000 10.7150/thno.37568 31754376 PMC6857062 Saeed M, et al. Engineering nanoparticles to reprogram the tumor immune microenvironment for improved cancer immunotherapy. Theranostics. 2019;9(26):7981–8000. 31754376 10.7150/thno.37568 PMC6857062

30. Dhas N Nanoengineered platform-based microenvironment-triggered immunotherapy in cancer treatment Front Biosci (Landmark Ed) 2024 29 10 349 10.31083/j.fbl2910349 39473401 Dhas N, et al. Nanoengineered platform-based microenvironment-triggered immunotherapy in cancer treatment. Front Biosci (Landmark Ed). 2024;29(10):349. 39473401 10.31083/j.fbl2910349

31. Zhao Y Cui Y Sun Y Brain-targeting nanoparticle drug delivery systems for brain tumors Front Biosci (Landmark Ed) 2025 30 1 27934 10.31083/FBL27934 39862100 Zhao Y, Cui Y, Sun Y. Brain-targeting nanoparticle drug delivery systems for brain tumors. Front Biosci (Landmark Ed). 2025;30(1):27934. 39862100 10.31083/FBL27934

32. Kalayil N, Budar AA, Dave RK. Nanofibers for drug delivery: design and fabrication strategies. BIOI' 2024;5(1):1–18.

33. Billingsley MM Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering Nano Lett 2020 20 3 1578 89 10.1021/acs.nanolett.9b04246 31951421 PMC7313236 Billingsley MM, et al. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Lett. 2020;20(3):1578–89. 31951421 10.1021/acs.nanolett.9b04246 PMC7313236

34. Meyer RA Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation Small 2015 11 13 1519 25 10.1002/smll.201402369 25641795 PMC4529071 Meyer RA, et al. Biodegradable nanoellipsoidal artificial antigen presenting cells for antigen specific T-cell activation. Small. 2015;11(13):1519–25. 25641795 10.1002/smll.201402369 PMC4529071

35. Pinto IS Cordeiro RA Faneca H Polymer- and lipid-based gene delivery technology for CAR T cell therapy J Control Release 2023 353 196 215 10.1016/j.jconrel.2022.11.038 36423871 Pinto IS, Cordeiro RA, Faneca H. Polymer- and lipid-based gene delivery technology for CAR T cell therapy. J Control Release. 2023;353:196–215. 36423871 10.1016/j.jconrel.2022.11.038

36. Yang M The application of nanoparticles in cancer immunotherapy: targeting tumor microenvironment Bioact Mater 2021 6 7 1973 87 33426371 10.1016/j.bioactmat.2020.12.010 PMC7773537 Yang M, et al. The application of nanoparticles in cancer immunotherapy: targeting tumor microenvironment. Bioact Mater. 2021;6(7):1973–87. 33426371 10.1016/j.bioactmat.2020.12.010 PMC7773537

37. Bogart LK Nanoparticles for imaging, sensing, and therapeutic intervention ACS Nano 2014 8 4 3107 22 10.1021/nn500962q 24641589 PMC4123720 Bogart LK, et al. Nanoparticles for imaging, sensing, and therapeutic intervention. ACS Nano. 2014;8(4):3107–22. 24641589 10.1021/nn500962q PMC4123720

38. Gong N Nanomaterials for T-cell cancer immunotherapy Nat Nanotechnol 2021 16 1 25 36 10.1038/s41565-020-00822-y 33437036 Gong N, et al. Nanomaterials for T-cell cancer immunotherapy. Nat Nanotechnol. 2021;16(1):25–36. 33437036 10.1038/s41565-020-00822-y

39. Sterner RC Sterner RM CAR-T cell therapy: current limitations and potential strategies Blood Cancer J 2021 11 4 69 10.1038/s41408-021-00459-7 33824268 PMC8024391 Sterner RC, Sterner RM. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 2021;11(4):69. 33824268 10.1038/s41408-021-00459-7 PMC8024391

40. Zhou Z The underlying mechanism of chimeric antigen receptor (CAR)-T cell therapy triggering secondary T-cell cancers: mystery of the sphinx?? Cancer Lett 2024 597 217083 10.1016/j.canlet.2024.217083 38925363 Zhou Z, et al. The underlying mechanism of chimeric antigen receptor (CAR)-T cell therapy triggering secondary T-cell cancers: mystery of the sphinx? Cancer Lett. 2024;597:217083. 38925363 10.1016/j.canlet.2024.217083

41. Zhou S Present status and advances in chimeric antigen receptor T cell therapy for glioblastoma Front Biosci (Landmark Ed) 2023 28 9 206 10.31083/j.fbl2809206 37796692 Zhou S, et al. Present status and advances in chimeric antigen receptor T cell therapy for glioblastoma. Front Biosci (Landmark Ed). 2023;28(9):206. 37796692 10.31083/j.fbl2809206

42. Yu G Recent advancements in biomaterials for chimeric antigen receptor T cell immunotherapy Biomater Res 2024 28 0045 10.34133/bmr.0045 39011521 PMC11246982 Yu G, et al. Recent advancements in biomaterials for chimeric antigen receptor T cell immunotherapy. Biomater Res. 2024;28:0045. 39011521 10.34133/bmr.0045 PMC11246982

43. Yan L Boosting CAR-T cell therapy with CRISPR technology hLife 2024 2 8 380 96 10.1016/j.hlife.2024.06.002 Yan L, et al. Boosting CAR-T cell therapy with CRISPR technology. hLife. 2024;2(8):380–96.

44. Khan AN Immunogenicity of CAR-T cell therapeutics: evidence, mechanism and mitigation Front Immunol 2022 13 886546 10.3389/fimmu.2022.886546 35677038 PMC9169153 Khan AN, et al. Immunogenicity of CAR-T cell therapeutics: evidence, mechanism and mitigation. Front Immunol. 2022;13:886546. 35677038 10.3389/fimmu.2022.886546 PMC9169153

45. Zhang J Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL Nature 2022 609 7926 369 74 10.1038/s41586-022-05140-y 36045296 PMC9452296 Zhang J, et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature. 2022;609(7926):369–74. 36045296 10.1038/s41586-022-05140-y PMC9452296

46. Prazeres P Delivery of plasmid DNA by ionizable lipid nanoparticles to induce CAR expression in T cells Int J Nanomed 2023 18 5891 904 10.2147/IJN.S424723 PMC10590593 37873551 Prazeres P, et al. Delivery of plasmid DNA by ionizable lipid nanoparticles to induce CAR expression in T cells. Int J Nanomed. 2023;18:5891–904. 10.2147/IJN.S424723 PMC10590593 37873551

47. Hu T Automating CAR-T transfection with micro and Nano-Technologies Small Methods 2024 8 8 e2301300 10.1002/smtd.202301300 38054597 Hu T, et al. Automating CAR-T transfection with micro and Nano-Technologies. Small Methods. 2024;8(8):e2301300. 38054597 10.1002/smtd.202301300

48. Zhou Z Revolutionising cancer immunotherapy: advancements and prospects in non-viral CAR-NK cell engineering Cell Prolif 2025 58 4 e13791 10.1111/cpr.13791 39731215 PMC11969250 Zhou Z, et al. Revolutionising cancer immunotherapy: advancements and prospects in non-viral CAR-NK cell engineering. Cell Prolif. 2025;58(4):e13791. 39731215 10.1111/cpr.13791 PMC11969250

49. Harris E Elmer JJ Optimization of electroporation and other non-viral gene delivery strategies for T cells Biotechnol Prog 2021 37 1 e3066 10.1002/btpr.3066 32808434 Harris E, Elmer JJ. Optimization of electroporation and other non-viral gene delivery strategies for T cells. Biotechnol Prog. 2021;37(1):e3066. 32808434 10.1002/btpr.3066

50. Kavanagh H A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications Cytotherapy 2021 23 9 852 60 10.1016/j.jcyt.2021.03.002 33941482 PMC8386197 Kavanagh H, et al. A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications. Cytotherapy. 2021;23(9):852–60. 33941482 10.1016/j.jcyt.2021.03.002 PMC8386197

51. Hegde M Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial Nat Cancer 2024 5 6 880 94 10.1038/s43018-024-00749-6 38658775 PMC11588040 Hegde M, et al. Autologous HER2-specific CAR T cells after lymphodepletion for advanced sarcoma: a phase 1 trial. Nat Cancer. 2024;5(6):880–94. 38658775 10.1038/s43018-024-00749-6 PMC11588040

52. Del Bufalo F GD2-CART01 for relapsed or refractory high-risk neuroblastoma N Engl J Med 2023 388 14 1284 95 10.1056/NEJMoa2210859 37018492 Del Bufalo F, et al. GD2-CART01 for relapsed or refractory high-risk neuroblastoma. N Engl J Med. 2023;388(14):1284–95. 37018492 10.1056/NEJMoa2210859

53. Mi J Ye Q Min Y Advances in nanotechnology development to overcome current roadblocks in CAR-T therapy for solid tumors Front Immunol 2022 13 849759 10.3389/fimmu.2022.849759 35401561 PMC8983935 Mi J, Ye Q, Min Y. Advances in nanotechnology development to overcome current roadblocks in CAR-T therapy for solid tumors. Front Immunol. 2022;13:849759. 35401561 10.3389/fimmu.2022.849759 PMC8983935

54. Kankeu Fonkoua LA CAR t cell therapy and the tumor microenvironment: current challenges and opportunities Mol Ther 2022 25 69 77 10.1016/j.omto.2022.03.009 PMC8980704 35434273 Kankeu Fonkoua LA, et al. CAR t cell therapy and the tumor microenvironment: current challenges and opportunities. Mol Ther. 2022;25:69–77. 10.1016/j.omto.2022.03.009 PMC8980704 35434273

55. Guo Z, Saw PE, Jon S. Non-Invasive physical stimulation to modulate the tumor microenvironment: unveiling a new frontier in cancer therapy. BIOI. 2024;5(1):1–14.

56. Hou AJ Chen LC Chen YY Navigating CAR-T cells through the solid-tumour microenvironment Nat Rev Drug Discov 2021 20 7 531 50 10.1038/s41573-021-00189-2 33972771 Hou AJ, Chen LC, Chen YY. Navigating CAR-T cells through the solid-tumour microenvironment. Nat Rev Drug Discov. 2021;20(7):531–50. 33972771 10.1038/s41573-021-00189-2

57. Hirabayashi K Dual targeting CAR-T cells with optimal costimulation and metabolic fitness enhance antitumor activity and prevent escape in solid tumors Nat Cancer 2021 2 9 904 18 10.1038/s43018-021-00244-2 34746799 PMC8570569 Hirabayashi K, et al. Dual targeting CAR-T cells with optimal costimulation and metabolic fitness enhance antitumor activity and prevent escape in solid tumors. Nat Cancer. 2021;2(9):904–18. 34746799 10.1038/s43018-021-00244-2 PMC8570569

58. Morris EC Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy Nat Rev Immunol 2022 22 2 85 96 10.1038/s41577-021-00547-6 34002066 PMC8127450 Morris EC, et al. Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy. Nat Rev Immunol. 2022;22(2):85–96. 34002066 10.1038/s41577-021-00547-6 PMC8127450

59. Giavridis T Car T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade Nat Med 2018 24 6 731 8 10.1038/s41591-018-0041-7 29808005 PMC6410714 Giavridis T, et al. Car T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med. 2018;24(6):731–8. 29808005 10.1038/s41591-018-0041-7 PMC6410714

60. Mahdi J Tumor inflammation-associated neurotoxicity Nat Med 2023 29 4 803 10 10.1038/s41591-023-02276-w 37024595 PMC10166099 Mahdi J, et al. Tumor inflammation-associated neurotoxicity. Nat Med. 2023;29(4):803–10. 37024595 10.1038/s41591-023-02276-w PMC10166099

61. Li X Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel Nat Biomed Eng 2023 7 9 1129 41 10.1038/s41551-023-01084-4 37696984 Li X, et al. Suppression of cytokine release syndrome during CAR-T-cell therapy via a subcutaneously injected interleukin-6-adsorbing hydrogel. Nat Biomed Eng. 2023;7(9):1129–41. 37696984 10.1038/s41551-023-01084-4

62. Sakemura RL, Manriquez Roman C, Horvei P et al. , . CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS. Blood. 2024;143(3):258–271. Blood, 2024. 144(14): p. 1544. 10.1182/blood.2022018905 PMC10808250 37879074

63. Parker KR Single-Cell analyses identify brain mural cells expressing CD19 as potential Off-Tumor targets for CAR-T immunotherapies Cell 2020 183 1 126 e14217 10.1016/j.cell.2020.08.022 32961131 PMC7640763 Parker KR, et al. Single-Cell analyses identify brain mural cells expressing CD19 as potential Off-Tumor targets for CAR-T immunotherapies. Cell. 2020;183(1):126–e14217. 32961131 10.1016/j.cell.2020.08.022 PMC7640763

64. Jess J, et al. CD22 CAR T-cell associated hematologic toxicities, endothelial activation and relationship to neurotoxicity. J Immunother Cancer. 2023;11(6):e005898. 10.1136/jitc-2022-005898 PMC10277551 37295816

65. Brown BD Immune effector cell associated neurotoxicity (ICANS) in pediatric and young adult patients following chimeric antigen receptor (CAR) T-cell therapy: can we optimize early diagnosis?? Front Oncol 2021 11 634445 10.3389/fonc.2021.634445 33763368 PMC7982581 Brown BD, et al. Immune effector cell associated neurotoxicity (ICANS) in pediatric and young adult patients following chimeric antigen receptor (CAR) T-cell therapy: can we optimize early diagnosis?? Front Oncol. 2021;11:634445. 33763368 10.3389/fonc.2021.634445 PMC7982581

66. Diorio C Comprehensive serum proteome profiling of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome patients with B-cell ALL receiving CAR T19 Clin Cancer Res 2022 28 17 3804 13 10.1158/1078-0432.CCR-22-0822 35705524 PMC9444956 Diorio C, et al. Comprehensive serum proteome profiling of cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome patients with B-cell ALL receiving CAR T19. Clin Cancer Res. 2022;28(17):3804–13. 35705524 10.1158/1078-0432.CCR-22-0822 PMC9444956

67. Gong N In situ pegylation of CAR T cells alleviates cytokine release syndrome and neurotoxicity Nat Mater 2023 22 12 1571 80 10.1038/s41563-023-01646-6 37696939 Gong N, et al. In situ pegylation of CAR T cells alleviates cytokine release syndrome and neurotoxicity. Nat Mater. 2023;22(12):1571–80. 37696939 10.1038/s41563-023-01646-6

68. Hines MR Immune effector Cell-Associated hemophagocytic Lymphohistiocytosis-Like syndrome Transpl Cell Ther 2023 29 7 e4381 43816 10.1016/j.jtct.2023.03.006 PMC10330221 36906275 Hines MR, et al. Immune effector Cell-Associated hemophagocytic Lymphohistiocytosis-Like syndrome. Transpl Cell Ther. 2023;29(7):e4381–43816. 10.1016/j.jtct.2023.03.006 PMC10330221 36906275

69. Posey AD Young RM June CH Future perspectives on engineered T cells for cancer Trends Cancer 2024 10 8 687 95 10.1016/j.trecan.2024.05.007 38853073 Posey AD Jr., Young RM, June CH. Future perspectives on engineered T cells for cancer. Trends Cancer. 2024;10(8):687–95. 38853073 10.1016/j.trecan.2024.05.007

70. De Marco RC Monzo HJ Ojala PM CAR T cell therapy: a versatile living drug Int J Mol Sci 2023 10.3390/ijms24076300 37047272 PMC10094630 De Marco RC, Monzo HJ, Ojala PM. CAR T cell therapy: a versatile living drug. Int J Mol Sci. 2023. 10.3390/ijms24076300. 37047272 10.3390/ijms24076300 PMC10094630

71. Moretti A The past, present, and future of non-viral CAR T cells Front Immunol 2022 13 867013 10.3389/fimmu.2022.867013 35757746 PMC9218214 Moretti A, et al. The past, present, and future of non-viral CAR T cells. Front Immunol. 2022;13:867013. 35757746 10.3389/fimmu.2022.867013 PMC9218214

72. Bozza M A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells Sci Adv 2021 10.1126/sciadv.abf1333 33853779 PMC8046366 Bozza M, et al. A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells. Sci Adv. 2021. 10.1126/sciadv.abf1333. 33853779 10.1126/sciadv.abf1333 PMC8046366

73. Foster JB Barrett DM Karikó K The emerging role of in vitro-transcribed mRNA in adoptive T cell immunotherapy Mol Ther 2019 27 4 747 56 10.1016/j.ymthe.2019.01.018 30819612 PMC6453504 Foster JB, Barrett DM, Karikó K. The emerging role of in vitro-transcribed mRNA in adoptive T cell immunotherapy. Mol Ther. 2019;27(4):747–56. 30819612 10.1016/j.ymthe.2019.01.018 PMC6453504

74. Cheng Y Nano-Sized sunflower polycations as effective gene transfer vehicles Small 2016 12 20 2750 8 10.1002/smll.201502930 27061622 PMC5052141 Cheng Y, et al. Nano-Sized sunflower polycations as effective gene transfer vehicles. Small. 2016;12(20):2750–8. 27061622 10.1002/smll.201502930 PMC5052141

75. Olden BR Cationic polymers for non-viral gene delivery to human T cells J Control Release 2018 282 140 7 10.1016/j.jconrel.2018.02.043 29518467 PMC6008197 Olden BR, et al. Cationic polymers for non-viral gene delivery to human T cells. J Control Release. 2018;282:140–7. 29518467 10.1016/j.jconrel.2018.02.043 PMC6008197

76. Raes L Intracellular delivery of mRNA in adherent and suspension cells by vapor nanobubble photoporation Nano-Micro Lett 2020 12 1 185 10.1007/s40820-020-00523-0 PMC7770675 34138203 Raes L, et al. Intracellular delivery of mRNA in adherent and suspension cells by vapor nanobubble photoporation. Nano-Micro Lett. 2020;12(1):185. 10.1007/s40820-020-00523-0 PMC7770675 34138203

77. Chen Z mRNA-laden lipid nanoparticle-enabled humanized CD19 CAR-T-cell engineering for the eradication of leukaemic cells Br J Haematol 2025 206 2 628 43 10.1111/bjh.19988 39761676 PMC11829146 Chen Z, et al. mRNA-laden lipid nanoparticle-enabled humanized CD19 CAR-T-cell engineering for the eradication of leukaemic cells. Br J Haematol. 2025;206(2):628–43. 39761676 10.1111/bjh.19988 PMC11829146

78. Xiao K mRNA-based chimeric antigen receptor T cell therapy: basic principles, recent advances and future directions Interdiscip Med 2024 2 1 e20230036 10.1002/INMD.20230036 Xiao K, et al. mRNA-based chimeric antigen receptor T cell therapy: basic principles, recent advances and future directions. Interdiscip Med. 2024;2(1):e20230036.

79. Billingsley MM Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR T cells Nano Lett 2022 22 1 533 42 10.1021/acs.nanolett.1c02503 34669421 PMC9335860 Billingsley MM, et al. Orthogonal design of experiments for optimization of lipid nanoparticles for mRNA engineering of CAR T cells. Nano Lett. 2022;22(1):533–42. 34669421 10.1021/acs.nanolett.1c02503 PMC9335860

80. Ye Z Vitro engineering chimeric antigen receptor macrophages and T cells by lipid Nanoparticle-Mediated mRNA delivery ACS Biomater Sci Eng 2022 8 2 722 33 10.1021/acsbiomaterials.1c01532 35104103 Ye Z, et al. Vitro engineering chimeric antigen receptor macrophages and T cells by lipid Nanoparticle-Mediated mRNA delivery. ACS Biomater Sci Eng. 2022;8(2):722–33. 35104103 10.1021/acsbiomaterials.1c01532

81. Wang C Bioengineering of artificial antigen presenting cells and lymphoid organs Theranostics 2017 7 14 3504 16 10.7150/thno.19017 28912891 PMC5596439 Wang C, et al. Bioengineering of artificial antigen presenting cells and lymphoid organs. Theranostics. 2017;7(14):3504–16. 28912891 10.7150/thno.19017 PMC5596439

82. Hou F Particle-based artificial antigen-presenting cell systems for T cell activation in adoptive T cell therapy ACS Nano 2024 18 12 8571 99 10.1021/acsnano.3c10180 38483840 Hou F, et al. Particle-based artificial antigen-presenting cell systems for T cell activation in adoptive T cell therapy. ACS Nano. 2024;18(12):8571–99. 38483840 10.1021/acsnano.3c10180

83. Fadel TR A carbon nanotube-polymer composite for T-cell therapy Nat Nanotechnol 2014 9 8 639 47 10.1038/nnano.2014.154 25086604 Fadel TR, et al. A carbon nanotube-polymer composite for T-cell therapy. Nat Nanotechnol. 2014;9(8):639–47. 25086604 10.1038/nnano.2014.154

84. Li Y Kurlander RJ Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation J Transl Med 2010 8 104 10.1186/1479-5876-8-104 20977748 PMC2987859 Li Y, Kurlander RJ. Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: differing impact on CD8 T cell phenotype and responsiveness to restimulation. J Transl Med. 2010;8:104. 20977748 10.1186/1479-5876-8-104 PMC2987859

85. Metzloff AE Antigen presenting cell mimetic lipid nanoparticles for rapid mRNA CAR T cell cancer immunotherapy Adv Mater 2024 36 26 e2313226 10.1002/adma.202313226 38419362 PMC11209815 Metzloff AE, et al. Antigen presenting cell mimetic lipid nanoparticles for rapid mRNA CAR T cell cancer immunotherapy. Adv Mater. 2024;36(26):e2313226. 38419362 10.1002/adma.202313226 PMC11209815

86. Moradi N Bio-conjugation of anti-human CD3 monoclonal antibodies to magnetic nanoparticles by using cyanogen bromide: a potential for cell sorting and noninvasive diagnosis Int J Biol Macromol 2021 192 72 81 10.1016/j.ijbiomac.2021.09.129 34606792 Moradi N, et al. Bio-conjugation of anti-human CD3 monoclonal antibodies to magnetic nanoparticles by using cyanogen bromide: a potential for cell sorting and noninvasive diagnosis. Int J Biol Macromol. 2021;192:72–81. 34606792 10.1016/j.ijbiomac.2021.09.129

87. Wang Z Nanoparticle amplification labeling for high-performance magnetic cell sorting Nano Lett 2022 22 12 4774 83 10.1021/acs.nanolett.2c01018 35639489 Wang Z, et al. Nanoparticle amplification labeling for high-performance magnetic cell sorting. Nano Lett. 2022;22(12):4774–83. 35639489 10.1021/acs.nanolett.2c01018

88. Garbayo E RNA-loaded nanoparticles for the treatment of hematological cancers Adv Drug Deliv Rev 2024 214 115448 10.1016/j.addr.2024.115448 39303823 Garbayo E, et al. RNA-loaded nanoparticles for the treatment of hematological cancers. Adv Drug Deliv Rev. 2024;214:115448. 39303823 10.1016/j.addr.2024.115448

89. Hajj KA Whitehead KA Tools for translation: non-viral materials for therapeutic mRNA delivery Nat Rev Mater 2017 10.1038/natrevmats.2017.56 Hajj KA, Whitehead KA. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat Rev Mater. 2017. 10.1038/natrevmats.2017.56.

90. Smith TT In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers Nat Nanotechnol 2017 12 8 813 20 10.1038/nnano.2017.57 28416815 PMC5646367 Smith TT, et al. In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers. Nat Nanotechnol. 2017;12(8):813–20. 28416815 10.1038/nnano.2017.57 PMC5646367

91. Zhu C Injectable supramolecular hydrogels for in situ programming of Car-T cells toward solid tumor immunotherapy Adv Mater 2024 36 5 e2310078 10.1002/adma.202310078 37947048 Zhu C, et al. Injectable supramolecular hydrogels for in situ programming of Car-T cells toward solid tumor immunotherapy. Adv Mater. 2024;36(5):e2310078. 37947048 10.1002/adma.202310078

92. Zhou JE Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo J Control Release 2022 350 298 307 10.1016/j.jconrel.2022.08.033 36002054 Zhou JE, et al. Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo. J Control Release. 2022;350:298–307. 36002054 10.1016/j.jconrel.2022.08.033

93. Riley RS Delivery technologies for cancer immunotherapy Nat Rev Drug Discov 2019 18 3 175 96 10.1038/s41573-018-0006-z 30622344 PMC6410566 Riley RS, et al. Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov. 2019;18(3):175–96. 30622344 10.1038/s41573-018-0006-z PMC6410566

94. Billingsley MM Vivo mRNA CAR T cell engineering via targeted ionizable lipid nanoparticles with extrahepatic tropism Small 2024 20 11 e2304378 10.1002/smll.202304378 38072809 Billingsley MM, et al. Vivo mRNA CAR T cell engineering via targeted ionizable lipid nanoparticles with extrahepatic tropism. Small. 2024;20(11):e2304378. 38072809 10.1002/smll.202304378

95. Cheng Q Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing Nat Nanotechnol 2020 15 4 313 20 10.1038/s41565-020-0669-6 32251383 PMC7735425 Cheng Q, et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol. 2020;15(4):313–20. 32251383 10.1038/s41565-020-0669-6 PMC7735425

96. Wang X Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery Nat Protoc 2023 18 1 265 91 10.1038/s41596-022-00755-x 36316378 PMC9888002 Wang X, et al. Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat Protoc. 2023;18(1):265–91. 36316378 10.1038/s41596-022-00755-x PMC9888002

97. Dilliard SA Cheng Q Siegwart DJ On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles Proc Natl Acad Sci U S A 2021 10.1073/pnas.2109256118 34933999 PMC8719871 Dilliard SA, Cheng Q, Siegwart DJ. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc Natl Acad Sci U S A. 2021. 10.1073/pnas.2109256118. 34933999 10.1073/pnas.2109256118 PMC8719871

98. Álvarez-Benedicto E Spleen SORT LNP generated in situ CAR T cells extend survival in a mouse model of lymphoreplete B cell lymphoma Angew Chem Int Ed Engl 2023 62 44 e202310395 10.1002/anie.202310395 37651468 PMC10826899 Álvarez-Benedicto E, et al. Spleen SORT LNP generated in situ CAR T cells extend survival in a mouse model of lymphoreplete B cell lymphoma. Angew Chem Int Ed Engl. 2023;62(44):e202310395. 37651468 10.1002/anie.202310395 PMC10826899

99. Hughes KA Use of stimulatory responsive soft nanoparticles for intracellular drug delivery Nano Res 2023 16 5 6974 90 10.1007/s12274-022-5267-5 36685637 PMC9840428 Hughes KA, et al. Use of stimulatory responsive soft nanoparticles for intracellular drug delivery. Nano Res. 2023;16(5):6974–90. 36685637 10.1007/s12274-022-5267-5 PMC9840428

100. Khare P Lipid nanoparticle-mediated drug delivery to the brain Adv Drug Deliv Rev 2023 197 114861 10.1016/j.addr.2023.114861 37150326 Khare P, et al. Lipid nanoparticle-mediated drug delivery to the brain. Adv Drug Deliv Rev. 2023;197:114861. 37150326 10.1016/j.addr.2023.114861

101. Melamed JR Ionizable lipid nanoparticles deliver mRNA to pancreatic β cells via macrophage-mediated gene transfer Sci Adv 2023 9 4 eade1444 10.1126/sciadv.ade1444 36706177 PMC9882987 Melamed JR, et al. Ionizable lipid nanoparticles deliver mRNA to pancreatic β cells via macrophage-mediated gene transfer. Sci Adv. 2023;9(4):eade1444. 36706177 10.1126/sciadv.ade1444 PMC9882987

102. Swingle KL Ionizable lipid nanoparticles for in vivo mRNA delivery to the placenta during pregnancy J Am Chem Soc 2023 145 8 4691 706 10.1021/jacs.2c12893 36789893 PMC9992266 Swingle KL, et al. Ionizable lipid nanoparticles for in vivo mRNA delivery to the placenta during pregnancy. J Am Chem Soc. 2023;145(8):4691–706. 36789893 10.1021/jacs.2c12893 PMC9992266

103. Xue L Rational design of bisphosphonate lipid-like materials for mRNA delivery to the bone microenvironment J Am Chem Soc 2022 144 22 9926 37 10.1021/jacs.2c02706 35616998 Xue L, et al. Rational design of bisphosphonate lipid-like materials for mRNA delivery to the bone microenvironment. J Am Chem Soc. 2022;144(22):9926–37. 35616998 10.1021/jacs.2c02706

104. Chen J Lipid nanoparticle-mediated lymph node-targeting delivery of mRNA cancer vaccine elicits robust CD8(+) T cell response Proc Natl Acad Sci U S A 2022 119 34 e2207841119 10.1073/pnas.2207841119 35969778 PMC9407666 Chen J, et al. Lipid nanoparticle-mediated lymph node-targeting delivery of mRNA cancer vaccine elicits robust CD8(+) T cell response. Proc Natl Acad Sci U S A. 2022;119(34):e2207841119. 35969778 10.1073/pnas.2207841119 PMC9407666

105. Zhao G In vivo production of CAR-T cells using virus-mimetic fusogenic nanovesicles Sci Bull 2024 69 3 354 66 10.1016/j.scib.2023.11.055 38072706 Zhao G, et al. In vivo production of CAR-T cells using virus-mimetic fusogenic nanovesicles. Sci Bull. 2024;69(3):354–66. 10.1016/j.scib.2023.11.055 38072706

106. Ma W Coating biomimetic nanoparticles with chimeric antigen receptor T cell-membrane provides high specificity for hepatocellular carcinoma photothermal therapy treatment Theranostics 2020 10 3 1281 95 10.7150/thno.40291 31938065 PMC6956810 Ma W, et al. Coating biomimetic nanoparticles with chimeric antigen receptor T cell-membrane provides high specificity for hepatocellular carcinoma photothermal therapy treatment. Theranostics. 2020;10(3):1281–95. 31938065 10.7150/thno.40291 PMC6956810

107. Wu W CAR T cell membrane camouflaged nanocatalyst augments CAR T cell therapy efficacy against solid tumor Small 2024 20 37 e2401299 10.1002/smll.202401299 38746996 Wu W, et al. CAR T cell membrane camouflaged nanocatalyst augments CAR T cell therapy efficacy against solid tumor. Small. 2024;20(37):e2401299. 38746996 10.1002/smll.202401299

108. Yaman S Targeted chemotherapy via HER2-based chimeric antigen receptor (CAR) engineered T-cell membrane coated polymeric nanoparticles Bioact Mater 2024 34 422 35 38282968 10.1016/j.bioactmat.2023.12.027 PMC10821609 Yaman S, et al. Targeted chemotherapy via HER2-based chimeric antigen receptor (CAR) engineered T-cell membrane coated polymeric nanoparticles. Bioact Mater. 2024;34:422–35. 38282968 10.1016/j.bioactmat.2023.12.027 PMC10821609

109. Luo Y IL-12 nanochaperone-engineered CAR T cell for robust tumor-immunotherapy Biomaterials 2022 281 121341 10.1016/j.biomaterials.2021.121341 34995901 Luo Y, et al. IL-12 nanochaperone-engineered CAR T cell for robust tumor-immunotherapy. Biomaterials. 2022;281:121341. 34995901 10.1016/j.biomaterials.2021.121341

110. Chen Z Nanoengineered CAR-T biohybrids for solid tumor immunotherapy with microenvironment Photothermal-Remodeling strategy Small 2021 17 14 e2007494 10.1002/smll.202007494 33711191 Chen Z, et al. Nanoengineered CAR-T biohybrids for solid tumor immunotherapy with microenvironment Photothermal-Remodeling strategy. Small. 2021;17(14):e2007494. 33711191 10.1002/smll.202007494

111. Tang Y TGF-β blocking combined with photothermal therapy promote tumor targeted migration and long-term antitumor activity of CAR-T cells Mater Today Bio 2023 20 100615 10.1016/j.mtbio.2023.100615 37063775 PMC10090704 Tang Y, et al. TGF-β blocking combined with photothermal therapy promote tumor targeted migration and long-term antitumor activity of CAR-T cells. Mater Today Bio. 2023;20:100615. 37063775 10.1016/j.mtbio.2023.100615 PMC10090704

112. Zou Q Photo-metallo-immunotherapy: fabricating Chromium-Based nanocomposites to enhance CAR-T cell infiltration and cytotoxicity against solid tumors Adv Mater 2025 37 2 e2407425 10.1002/adma.202407425 38899741 PMC11733712 Zou Q, et al. Photo-metallo-immunotherapy: fabricating Chromium-Based nanocomposites to enhance CAR-T cell infiltration and cytotoxicity against solid tumors. Adv Mater. 2025;37(2):e2407425. 38899741 10.1002/adma.202407425 PMC11733712

113. Tang X Magnetic-Acoustic sequentially actuated CAR T cell microrobots for precision navigation and in situ antitumor immunoactivation Adv Mater 2023 35 18 e2211509 10.1002/adma.202211509 36807373 Tang X, et al. Magnetic-Acoustic sequentially actuated CAR T cell microrobots for precision navigation and in situ antitumor immunoactivation. Adv Mater. 2023;35(18):e2211509. 36807373 10.1002/adma.202211509

114. Chen Y Intratumoral lactate depletion based on injectable nanoparticles-hydrogel composite system synergizes with immunotherapy against postablative hepatocellular carcinoma recurrence Adv Healthc Mater 2024 13 6 e2303031 10.1002/adhm.202303031 37848188 Chen Y, et al. Intratumoral lactate depletion based on injectable nanoparticles-hydrogel composite system synergizes with immunotherapy against postablative hepatocellular carcinoma recurrence. Adv Healthc Mater. 2024;13(6):e2303031. 37848188 10.1002/adhm.202303031

115. Singleton DC Macann A Wilson WR Therapeutic targeting of the hypoxic tumour microenvironment Nat Rev Clin Oncol 2021 18 12 751 72 10.1038/s41571-021-00539-4 34326502 Singleton DC, Macann A, Wilson WR. Therapeutic targeting of the hypoxic tumour microenvironment. Nat Rev Clin Oncol. 2021;18(12):751–72. 34326502 10.1038/s41571-021-00539-4

116. Kopecka J Hypoxia as a driver of resistance to immunotherapy Drug Resist Updat 2021 59 100787 10.1016/j.drup.2021.100787 34840068 Kopecka J, et al. Hypoxia as a driver of resistance to immunotherapy. Drug Resist Updat. 2021;59:100787. 34840068 10.1016/j.drup.2021.100787

117. Dong Z Tumor microenvironment modulating CaCO(3) -Based colloidosomal microreactors can generally reinforce cancer immunotherapy Adv Mater 2024 36 9 e2308254 10.1002/adma.202308254 37918820 Dong Z, et al. Tumor microenvironment modulating CaCO(3) -Based colloidosomal microreactors can generally reinforce cancer immunotherapy. Adv Mater. 2024;36(9):e2308254. 37918820 10.1002/adma.202308254

118. Li H A near-infrared-II fluorescent nanocatalyst for enhanced CAR T cell therapy against solid tumor by immune reprogramming ACS Nano 2023 17 12 11749 63 10.1021/acsnano.3c02592 37319120 Li H, et al. A near-infrared-II fluorescent nanocatalyst for enhanced CAR T cell therapy against solid tumor by immune reprogramming. ACS Nano. 2023;17(12):11749–63. 37319120 10.1021/acsnano.3c02592

119. Yazdimamaghani M Tumor microenvironment immunomodulation by nanoformulated TLR 7/8 agonist and PI3k delta inhibitor enhances therapeutic benefits of radiotherapy Biomaterials 2025 312 122750 10.1016/j.biomaterials.2024.122750 39126779 PMC11401478 Yazdimamaghani M, et al. Tumor microenvironment immunomodulation by nanoformulated TLR 7/8 agonist and PI3k delta inhibitor enhances therapeutic benefits of radiotherapy. Biomaterials. 2025;312:122750. 39126779 10.1016/j.biomaterials.2024.122750 PMC11401478

120. Zhu T Inhalable nanovesicles loaded with a STING agonist enhance CAR-T cell activity against solid tumors in the lung Nat Commun 2025 16 1 262 10.1038/s41467-024-55751-4 39747173 PMC11695690 Zhu T, et al. Inhalable nanovesicles loaded with a STING agonist enhance CAR-T cell activity against solid tumors in the lung. Nat Commun. 2025;16(1):262. 39747173 10.1038/s41467-024-55751-4 PMC11695690

121. Zhang F Nanoparticles that reshape the tumor milieu create a therapeutic window for effective T-cell therapy in solid malignancies Cancer Res 2018 78 13 3718 30 10.1158/0008-5472.CAN-18-0306 29760047 PMC6030470 Zhang F, et al. Nanoparticles that reshape the tumor milieu create a therapeutic window for effective T-cell therapy in solid malignancies. Cancer Res. 2018;78(13):3718–30. 29760047 10.1158/0008-5472.CAN-18-0306 PMC6030470

122. Zheng Y Enhancing adoptive cell therapy of cancer through targeted delivery of small-molecule immunomodulators to internalizing or noninternalizing receptors ACS Nano 2017 11 3 3089 100 10.1021/acsnano.7b00078 28231431 PMC5647839 Zheng Y, et al. Enhancing adoptive cell therapy of cancer through targeted delivery of small-molecule immunomodulators to internalizing or noninternalizing receptors. ACS Nano. 2017;11(3):3089–100. 28231431 10.1021/acsnano.7b00078 PMC5647839

123. Siriwon N Car-t cells surface-engineered with drug-encapsulated nanoparticles can ameliorate intratumoral t-cell hypofunction Cancer Immunol Res 2018 6 7 812 24 10.1158/2326-6066.CIR-17-0502 29720380 Siriwon N, et al. Car-t cells surface-engineered with drug-encapsulated nanoparticles can ameliorate intratumoral t-cell hypofunction. Cancer Immunol Res. 2018;6(7):812–24. 29720380 10.1158/2326-6066.CIR-17-0502

124. Yi M Targeting cytokine and chemokine signaling pathways for cancer therapy Signal Transduct Target Ther 2024 9 1 176 10.1038/s41392-024-01868-3 39034318 PMC11275440 Yi M, et al. Targeting cytokine and chemokine signaling pathways for cancer therapy. Signal Transduct Target Ther. 2024;9(1):176. 39034318 10.1038/s41392-024-01868-3 PMC11275440

125. Leonard WJ Lin JX Strategies to therapeutically modulate cytokine action Nat Rev Drug Discov 2023 22 10 827 54 10.1038/s41573-023-00746-x 37542128 Leonard WJ, Lin JX. Strategies to therapeutically modulate cytokine action. Nat Rev Drug Discov. 2023;22(10):827–54. 37542128 10.1038/s41573-023-00746-x

126. Propper DJ Balkwill FR Harnessing cytokines and chemokines for cancer therapy Nat Rev Clin Oncol 2022 19 4 237 53 10.1038/s41571-021-00588-9 34997230 Propper DJ, Balkwill FR. Harnessing cytokines and chemokines for cancer therapy. Nat Rev Clin Oncol. 2022;19(4):237–53. 34997230 10.1038/s41571-021-00588-9

127. Shields CWt Cellular backpacks for macrophage immunotherapy Sci Adv 2020 6 18 eaaz6579 10.1126/sciadv.aaz6579 32494680 PMC7190308 Shields CWt, et al. Cellular backpacks for macrophage immunotherapy. Sci Adv. 2020;6(18):eaaz6579. 32494680 10.1126/sciadv.aaz6579 PMC7190308

128. Tang L Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery Nat Biotechnol 2018 36 8 707 16 10.1038/nbt.4181 29985479 PMC6078803 Tang L, et al. Enhancing T cell therapy through TCR-signaling-responsive nanoparticle drug delivery. Nat Biotechnol. 2018;36(8):707–16. 29985479 10.1038/nbt.4181 PMC6078803

129. Liu Y Cytokine conjugation to enhance T cell therapy Proc Natl Acad Sci U S A 2023 120 1 e2213222120 10.1073/pnas.2213222120 36577059 PMC9910457 Liu Y, et al. Cytokine conjugation to enhance T cell therapy. Proc Natl Acad Sci U S A. 2023;120(1):e2213222120. 36577059 10.1073/pnas.2213222120 PMC9910457

130. Hu Q Inhibition of post-surgery tumour recurrence via a hydrogel releasing CAR-T cells and anti-PDL1-conjugated platelets Nat Biomed Eng 2021 5 9 1038 47 10.1038/s41551-021-00712-1 33903744 PMC9102991 Hu Q, et al. Inhibition of post-surgery tumour recurrence via a hydrogel releasing CAR-T cells and anti-PDL1-conjugated platelets. Nat Biomed Eng. 2021;5(9):1038–47. 33903744 10.1038/s41551-021-00712-1 PMC9102991

131. Tang L Arming CAR-T cells with cytokines and more: innovations in the fourth-generation CAR-T development Mol Ther 2023 31 11 3146 62 10.1016/j.ymthe.2023.09.021 37803832 PMC10638038 Tang L, et al. Arming CAR-T cells with cytokines and more: innovations in the fourth-generation CAR-T development. Mol Ther. 2023;31(11):3146–62. 37803832 10.1016/j.ymthe.2023.09.021 PMC10638038

132. Maalej KM CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances Mol Cancer 2023 22 1 20 10.1186/s12943-023-01723-z 36717905 PMC9885707 Maalej KM, et al. CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances. Mol Cancer. 2023;22(1):20. 36717905 10.1186/s12943-023-01723-z PMC9885707

133. Gao H Unlocking the potential of chimeric antigen receptor T cell engineering immunotherapy: long road to achieve precise targeted therapy for hepatobiliary pancreatic cancers Int J Biol Macromol 2025 297 139829 10.1016/j.ijbiomac.2025.139829 39814310 Gao H, et al. Unlocking the potential of chimeric antigen receptor T cell engineering immunotherapy: long road to achieve precise targeted therapy for hepatobiliary pancreatic cancers. Int J Biol Macromol. 2025;297:139829. 39814310 10.1016/j.ijbiomac.2025.139829

134. Wu Z Universal CAR cell therapy: challenges and expanding applications Transl Oncol 2025 51 102147 10.1016/j.tranon.2024.102147 39413693 PMC11525228 Wu Z, et al. Universal CAR cell therapy: challenges and expanding applications. Transl Oncol. 2025;51:102147. 39413693 10.1016/j.tranon.2024.102147 PMC11525228

135. Xia B Development of chimeric nanobody-granzyme B functionalized ferritin nanoparticles for precise tumor therapy Pharmacol Res 2025 213 107628 10.1016/j.phrs.2025.107628 39880067 Xia B, et al. Development of chimeric nanobody-granzyme B functionalized ferritin nanoparticles for precise tumor therapy. Pharmacol Res. 2025;213:107628. 39880067 10.1016/j.phrs.2025.107628

136. Lin K Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology J Transl Med 2024 22 1 349 10.1186/s12967-024-05159-x 38610029 PMC11015683 Lin K, et al. Development of nanobodies targeting hepatocellular carcinoma and application of nanobody-based CAR-T technology. J Transl Med. 2024;22(1):349. 38610029 10.1186/s12967-024-05159-x PMC11015683

137. Sun Z In situ antigen modification-based target-redirected universal chimeric antigen receptor T (TRUE CAR-T) cell therapy in solid tumors J Hematol Oncol 2022 15 1 29 10.1186/s13045-022-01246-y 35303900 PMC8932190 Sun Z, et al. In situ antigen modification-based target-redirected universal chimeric antigen receptor T (TRUE CAR-T) cell therapy in solid tumors. J Hematol Oncol. 2022;15(1):29. 35303900 10.1186/s13045-022-01246-y PMC8932190

138. Ma L Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity Cell 2023 186 15 3148 e316520 10.1016/j.cell.2023.06.002 37413990 PMC10372881 Ma L, et al. Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity. Cell. 2023;186(15):3148–e316520. 37413990 10.1016/j.cell.2023.06.002 PMC10372881

139. Sellars MC Wu CJ Fritsch EF Cancer vaccines: building a bridge over troubled waters Cell 2022 185 15 2770 88 10.1016/j.cell.2022.06.035 35835100 PMC9555301 Sellars MC, Wu CJ, Fritsch EF. Cancer vaccines: building a bridge over troubled waters. Cell. 2022;185(15):2770–88. 35835100 10.1016/j.cell.2022.06.035 PMC9555301

140. Chen Z Synergistic integration of mRNA-LNP with CAR-engineered immune cells: pioneering progress in immunotherapy Mol Ther 2024 32 11 3772 92 10.1016/j.ymthe.2024.09.019 39295145 PMC11573621 Chen Z, et al. Synergistic integration of mRNA-LNP with CAR-engineered immune cells: pioneering progress in immunotherapy. Mol Ther. 2024;32(11):3772–92. 39295145 10.1016/j.ymthe.2024.09.019 PMC11573621

141. Isser A Livingston NK Schneck JP Biomaterials to enhance antigen-specific T cell expansion for cancer immunotherapy Biomaterials 2021 268 120584 10.1016/j.biomaterials.2020.120584 33338931 PMC7856270 Isser A, Livingston NK, Schneck JP. Biomaterials to enhance antigen-specific T cell expansion for cancer immunotherapy. Biomaterials. 2021;268:120584. 33338931 10.1016/j.biomaterials.2020.120584 PMC7856270

142. Zhang X CD16 CAR-T cells enhance antitumor activity of CpG ODN-loaded nanoparticle-adjuvanted tumor antigen-derived vaccinevia ADCC approach J Nanobiotechnol 2023 21 1 159 10.1186/s12951-023-01900-8 PMC10199637 37208748 Zhang X, et al. CD16 CAR-T cells enhance antitumor activity of CpG ODN-loaded nanoparticle-adjuvanted tumor antigen-derived vaccinevia ADCC approach. J Nanobiotechnol. 2023;21(1):159. 10.1186/s12951-023-01900-8 PMC10199637 37208748

143. Zang H Ganglioside-functionalized nanoparticles for chimeric antigen receptor T-cell activation at the immunological synapse ACS Nano 2022 16 11 18408 20 10.1021/acsnano.2c06516 36282488 PMC9815837 Zang H, et al. Ganglioside-functionalized nanoparticles for chimeric antigen receptor T-cell activation at the immunological synapse. ACS Nano. 2022;16(11):18408–20. 36282488 10.1021/acsnano.2c06516 PMC9815837

144. Kuchroo JR The double-edged sword: harnessing PD-1 blockade in tumor and autoimmunity Sci Immunol 2021 6 65 eabf4034 10.1126/sciimmunol.abf4034 34739340 PMC13541067 Kuchroo JR, et al. The double-edged sword: harnessing PD-1 blockade in tumor and autoimmunity. Sci Immunol. 2021;6(65):eabf4034. 34739340 10.1126/sciimmunol.abf4034 PMC13541067

145. Wang H Immune checkpoint blockade and CAR-T cell therapy in hematologic malignancies J Hematol Oncol 2019 12 1 59 10.1186/s13045-019-0746-1 31186046 PMC6558778 Wang H, et al. Immune checkpoint blockade and CAR-T cell therapy in hematologic malignancies. J Hematol Oncol. 2019;12(1):59. 31186046 10.1186/s13045-019-0746-1 PMC6558778

146. Lee YH PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR T cells Mol Ther 2022 30 2 579 92 10.1016/j.ymthe.2021.10.004 34628052 PMC8821960 Lee YH, et al. PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR T cells. Mol Ther. 2022;30(2):579–92. 34628052 10.1016/j.ymthe.2021.10.004 PMC8821960

147. Ma S Identification of a small-molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical mouse models Sci Transl Med 2023 15 722 eadg6752 10.1126/scitranslmed.adg6752 37967204 Ma S, et al. Identification of a small-molecule Tim-3 inhibitor to potentiate T cell-mediated antitumor immunotherapy in preclinical mouse models. Sci Transl Med. 2023;15(722):eadg6752. 37967204 10.1126/scitranslmed.adg6752

148. Hamilton AG Ionizable lipid nanoparticles with integrated immune checkpoint inhibition for mRNA CAR T cell engineering Adv Healthc Mater 2023 12 30 e2301515 10.1002/adhm.202301515 37602495 Hamilton AG, et al. Ionizable lipid nanoparticles with integrated immune checkpoint inhibition for mRNA CAR T cell engineering. Adv Healthc Mater. 2023;12(30):e2301515. 37602495 10.1002/adhm.202301515

149. Chen X Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels Nanomedicine 2022 45 102591 10.1016/j.nano.2022.102591 35907618 Chen X, et al. Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels. Nanomedicine. 2022;45:102591. 35907618 10.1016/j.nano.2022.102591

150. Hao M, et al. Combination of metabolic intervention and T cell therapy enhances solid tumor immunotherapy. Sci Transl Med. 2020;12(571):eaaz6667. 10.1126/scitranslmed.aaz6667 33239389

151. Al Subeh ZY Lipid nanoparticle delivery of Fas plasmid restores Fas expression to suppress melanoma growth in vivo ACS Nano 2022 16 8 12695 710 10.1021/acsnano.2c04420 35939651 PMC9721370 Al Subeh ZY, et al. Lipid nanoparticle delivery of Fas plasmid restores Fas expression to suppress melanoma growth in vivo. ACS Nano. 2022;16(8):12695–710. 35939651 10.1021/acsnano.2c04420 PMC9721370

152. Collinson-Pautz MR Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies Leukemia 2019 33 9 2195 207 10.1038/s41375-019-0417-9 30816327 PMC6756044 Collinson-Pautz MR, et al. Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies. Leukemia. 2019;33(9):2195–207. 30816327 10.1038/s41375-019-0417-9 PMC6756044

153. Lai Y Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T cells Leukemia 2018 32 3 801 8 10.1038/leu.2017.249 28841215 Lai Y, et al. Toll-like receptor 2 costimulation potentiates the antitumor efficacy of CAR T cells. Leukemia. 2018;32(3):801–8. 28841215 10.1038/leu.2017.249

154. Mata M Inducible activation of MyD88 and CD40 in CAR T cells results in controllable and potent antitumor activity in preclinical solid tumor models Cancer Discov 2017 7 11 1306 19 10.1158/2159-8290.CD-17-0263 28801306 PMC5780189 Mata M, et al. Inducible activation of MyD88 and CD40 in CAR T cells results in controllable and potent antitumor activity in preclinical solid tumor models. Cancer Discov. 2017;7(11):1306–19. 28801306 10.1158/2159-8290.CD-17-0263 PMC5780189

155. Mikolič V Toll-like receptor 4 signaling activation domains promote CAR T cell function against solid tumors Mol Ther Oncol 2024 32 2 200815 10.1016/j.omton.2024.200815 38840781 PMC11152746 Mikolič V, et al. Toll-like receptor 4 signaling activation domains promote CAR T cell function against solid tumors. Mol Ther Oncol. 2024;32(2):200815. 38840781 10.1016/j.omton.2024.200815 PMC11152746

156. Yang D T cells expressing NKG2D chimeric antigen receptors efficiently eliminate glioblastoma and cancer stem cells J Immunother Cancer 2019 7 1 171 10.1186/s40425-019-0642-9 31288857 PMC6617951 Yang D, et al. T cells expressing NKG2D chimeric antigen receptors efficiently eliminate glioblastoma and cancer stem cells. J Immunother Cancer. 2019;7(1):171. 31288857 10.1186/s40425-019-0642-9 PMC6617951

157. Komai-Koma M TLR2 is expressed on activated T cells as a costimulatory receptor Proc Natl Acad Sci U S A 2004 101 9 3029 34 10.1073/pnas.0400171101 14981245 PMC365739 Komai-Koma M, et al. TLR2 is expressed on activated T cells as a costimulatory receptor. Proc Natl Acad Sci U S A. 2004;101(9):3029–34. 14981245 10.1073/pnas.0400171101 PMC365739

158. Weng J A novel generation 1928zt2 car t cells induce remission in extramedullary relapse of acute lymphoblastic leukemia J Hematol Oncol 2019 12 1 117 10.1186/s13045-019-0816-4 31747962 PMC6865043 Weng J, et al. A novel generation 1928zt2 car t cells induce remission in extramedullary relapse of acute lymphoblastic leukemia. J Hematol Oncol. 2019;12(1):117. 31747962 10.1186/s13045-019-0816-4 PMC6865043

159. Foster AE Regulated expansion and survival of chimeric antigen Receptor-Modified T cells using small molecule-dependent inducible MyD88/CD40 Mol Ther 2017 25 9 2176 88 10.1016/j.ymthe.2017.06.014 28697888 PMC5589084 Foster AE, et al. Regulated expansion and survival of chimeric antigen Receptor-Modified T cells using small molecule-dependent inducible MyD88/CD40. Mol Ther. 2017;25(9):2176–88. 28697888 10.1016/j.ymthe.2017.06.014 PMC5589084

160. Prinzing B MyD88/CD40 signaling retains CAR T cells in a less differentiated state JCI Insight 2020 10.1172/jci.insight.136093 33148882 PMC7710311 Prinzing B, et al. MyD88/CD40 signaling retains CAR T cells in a less differentiated state. JCI Insight. 2020. 10.1172/jci.insight.136093. 33148882 10.1172/jci.insight.136093 PMC7710311

161. Wensveen FM Jelenčić V Polić B NKG2D: a master regulator of immune cell responsiveness Front Immunol 2018 9 441 10.3389/fimmu.2018.00441 29568297 PMC5852076 Wensveen FM, Jelenčić V, Polić B. NKG2D: a master regulator of immune cell responsiveness. Front Immunol. 2018;9:441. 29568297 10.3389/fimmu.2018.00441 PMC5852076

162. Ng YY Cells expressing NKG2D CAR with a DAP12 signaling domain stimulate lower cytokine production while effective in tumor eradication Mol Ther 2021 29 1 75 85 10.1016/j.ymthe.2020.08.016 32956627 PMC7790889 Ng YY, et al. Cells expressing NKG2D CAR with a DAP12 signaling domain stimulate lower cytokine production while effective in tumor eradication. Mol Ther. 2021;29(1):75–85. 32956627 10.1016/j.ymthe.2020.08.016 PMC7790889

163. Sallman DA CYAD-01, an autologous NKG2D-based CAR T-cell therapy, in relapsed or refractory acute myeloid leukaemia and myelodysplastic syndromes or multiple myeloma (THINK): haematological cohorts of the dose escalation segment of a phase 1 trial Lancet Haematol 2023 10 3 e191 202 10.1016/S2352-3026(22)00378-7 36764323 Sallman DA, et al. CYAD-01, an autologous NKG2D-based CAR T-cell therapy, in relapsed or refractory acute myeloid leukaemia and myelodysplastic syndromes or multiple myeloma (THINK): haematological cohorts of the dose escalation segment of a phase 1 trial. Lancet Haematol. 2023;10(3):e191–202. 36764323 10.1016/S2352-3026(22)00378-7

164. Kim H Polymeric nanoparticles encapsulating novel TLR7/8 agonists as immunostimulatory adjuvants for enhanced cancer immunotherapy Biomaterials 2018 164 38 53 10.1016/j.biomaterials.2018.02.034 29482062 Kim H, et al. Polymeric nanoparticles encapsulating novel TLR7/8 agonists as immunostimulatory adjuvants for enhanced cancer immunotherapy. Biomaterials. 2018;164:38–53. 29482062 10.1016/j.biomaterials.2018.02.034

165. Liu L Cellular and molecular imaging of CAR-T cell-based immunotherapy Adv Drug Deliv Rev 2023 203 115135 10.1016/j.addr.2023.115135 37931847 PMC11052581 Liu L, et al. Cellular and molecular imaging of CAR-T cell-based immunotherapy. Adv Drug Deliv Rev. 2023;203:115135. 37931847 10.1016/j.addr.2023.115135 PMC11052581

166. Kiru L In vivo imaging of nanoparticle-labeled CAR T cells Proc Natl Acad Sci U S A 2022 10.1073/pnas.2102363119 35101971 PMC8832996 Kiru L, et al. In vivo imaging of nanoparticle-labeled CAR T cells. Proc Natl Acad Sci U S A. 2022. 10.1073/pnas.2102363119. 35101971 10.1073/pnas.2102363119 PMC8832996

167. Hunger J In vivo nanoparticle-based T cell imaging can predict therapy response towards adoptive T cell therapy in experimental glioma Theranostics 2023 13 15 5170 82 10.7150/thno.87248 37908732 PMC10614679 Hunger J, et al. In vivo nanoparticle-based T cell imaging can predict therapy response towards adoptive T cell therapy in experimental glioma. Theranostics. 2023;13(15):5170–82. 37908732 10.7150/thno.87248 PMC10614679

168. Lu M FDA report: ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease Am J Hematol 2010 85 5 315 9 10.1002/ajh.21656 20201089 Lu M, et al. FDA report: ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease. Am J Hematol. 2010;85(5):315–9. 20201089 10.1002/ajh.21656

169. Wu WE Multimodal in vivo tracking of chimeric antigen receptor T cells in preclinical glioblastoma models Invest Radiol 2023 58 6 388 95 10.1097/RLI.0000000000000946 36729074 PMC10164035 Wu WE, et al. Multimodal in vivo tracking of chimeric antigen receptor T cells in preclinical glioblastoma models. Invest Radiol. 2023;58(6):388–95. 36729074 10.1097/RLI.0000000000000946 PMC10164035

170. Harmsen S A dual-modal pet/near infrared fluorescent nanotag for long-term immune cell tracking Biomaterials 2021 269 120630 10.1016/j.biomaterials.2020.120630 33395580 PMC7870530 Harmsen S, et al. A dual-modal pet/near infrared fluorescent nanotag for long-term immune cell tracking. Biomaterials. 2021;269:120630. 33395580 10.1016/j.biomaterials.2020.120630 PMC7870530

171. Wang X Nanomodified switch induced precise and moderate activation of CAR-T cells for solid tumors Adv Sci 2023 10 12 e2205044 10.1002/advs.202205044 PMC10131841 36755195 Wang X, et al. Nanomodified switch induced precise and moderate activation of CAR-T cells for solid tumors. Adv Sci. 2023;10(12):e2205044. 10.1002/advs.202205044 PMC10131841 36755195

172. Nguyen NT Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety Nat Nanotechnol 2021 16 12 1424 34 10.1038/s41565-021-00982-5 34697491 PMC8678207 Nguyen NT, et al. Nano-optogenetic engineering of CAR T cells for precision immunotherapy with enhanced safety. Nat Nanotechnol. 2021;16(12):1424–34. 34697491 10.1038/s41565-021-00982-5 PMC8678207

173. Nguyen NT Nano-optogenetic CAR-T cell immunotherapy Methods Mol Biol 2024 2748 267 78 10.1007/978-1-0716-3593-3_17 38070119 Nguyen NT, et al. Nano-optogenetic CAR-T cell immunotherapy. Methods Mol Biol. 2024;2748:267–78. 38070119 10.1007/978-1-0716-3593-3_17

174. Miller IC Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control Nat Biomed Eng 2021 5 11 1348 59 10.1038/s41551-021-00781-2 34385695 PMC8791016 Miller IC, et al. Enhanced intratumoural activity of CAR T cells engineered to produce immunomodulators under photothermal control. Nat Biomed Eng. 2021;5(11):1348–59. 34385695 10.1038/s41551-021-00781-2 PMC8791016

175. Pfister F et al. Loading of CAR-T cells with magnetic nanoparticles for controlled targeting suppresses inflammatory cytokine release and switches tumor cell death mechanism. MedComm (2020), 2025. 6(1): p. e70039. 10.1002/mco2.70039 PMC11702464 39764559

176. Unterweger H Comparative in vitro and in vivo evaluation of different iron Oxide-Based contrast agents to promote clinical translation in compliance with patient safety Int J Nanomed 2023 18 2071 86 10.2147/IJN.S402320 PMC10128873 37113796 Unterweger H, et al. Comparative in vitro and in vivo evaluation of different iron Oxide-Based contrast agents to promote clinical translation in compliance with patient safety. Int J Nanomed. 2023;18:2071–86. 10.2147/IJN.S402320 PMC10128873 37113796

177. Buzas EI The roles of extracellular vesicles in the immune system Nat Rev Immunol 2023 23 4 236 50 10.1038/s41577-022-00763-8 35927511 PMC9361922 Buzas EI. The roles of extracellular vesicles in the immune system. Nat Rev Immunol. 2023;23(4):236–50. 35927511 10.1038/s41577-022-00763-8 PMC9361922

178. Fu W CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity Nat Commun 2019 10 1 4355 10.1038/s41467-019-12321-3 31554797 PMC6761190 Fu W, et al. CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity. Nat Commun. 2019;10(1):4355. 31554797 10.1038/s41467-019-12321-3 PMC6761190

179. Haque S Vaiselbuh SR CD19 chimeric antigen receptor-exosome targets CD19 positive B-lineage acute lymphocytic leukemia and induces cytotoxicity Cancers (Basel) 2021 10.3390/cancers13061401 33808645 PMC8003442 Haque S, Vaiselbuh SR. CD19 chimeric antigen receptor-exosome targets CD19 positive B-lineage acute lymphocytic leukemia and induces cytotoxicity. Cancers (Basel). 2021. 10.3390/cancers13061401. 33808645 10.3390/cancers13061401 PMC8003442

180. Storci G, et al. CAR + extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells. J Clin Invest. 2024;134(14):e173096. 10.1172/JCI173096 PMC11245152 38833312

181. Fan D Nanomedicine in cancer therapy Signal Transduct Target Ther 2023 8 1 293 10.1038/s41392-023-01536-y 37544972 PMC10404590 Fan D, et al. Nanomedicine in cancer therapy. Signal Transduct Target Ther. 2023;8(1):293. 37544972 10.1038/s41392-023-01536-y PMC10404590

182. Li J Development and application of nanomaterials, nanotechnology and nanomedicine for treating hematological malignancies J Hematol Oncol 2023 16 1 65 10.1186/s13045-023-01460-2 37353849 PMC10290401 Li J, et al. Development and application of nanomaterials, nanotechnology and nanomedicine for treating hematological malignancies. J Hematol Oncol. 2023;16(1):65. 37353849 10.1186/s13045-023-01460-2 PMC10290401

183. Halwani AA Development of pharmaceutical nanomedicines: from the bench to the market Pharmaceutics 2022 14 1 106 10.3390/pharmaceutics14010106 35057002 PMC8777701 Halwani AA. Development of pharmaceutical nanomedicines: from the bench to the market. Pharmaceutics. 2022;14(1):106. 35057002 10.3390/pharmaceutics14010106 PMC8777701

184. França A Sterilization matters: consequences of different sterilization techniques on gold nanoparticles Small 2010 6 1 89 95 10.1002/smll.200901006 19937607 França A, et al. Sterilization matters: consequences of different sterilization techniques on gold nanoparticles. Small. 2010;6(1):89–95. 19937607 10.1002/smll.200901006

185. Masson V Influence of sterilization processes on poly(epsilon-caprolactone) nanospheres Biomaterials 1997 18 4 327 35 10.1016/S0142-9612(96)00144-5 9068895 Masson V, et al. Influence of sterilization processes on poly(epsilon-caprolactone) nanospheres. Biomaterials. 1997;18(4):327–35. 9068895 10.1016/s0142-9612(96)00144-5

186. Geh KJ Hubert M Winter G Progress in formulation development and sterilisation of freeze-dried oligodeoxynucleotide-loaded gelatine nanoparticles Eur J Pharm Biopharm 2018 129 10 20 10.1016/j.ejpb.2018.05.016 29777773 Geh KJ, Hubert M, Winter G. Progress in formulation development and sterilisation of freeze-dried oligodeoxynucleotide-loaded gelatine nanoparticles. Eur J Pharm Biopharm. 2018;129:10–20. 29777773 10.1016/j.ejpb.2018.05.016

187. Bernal-Chávez SA Insights into terminal sterilization processes of nanoparticles for biomedical applications Molecules 2021 10.3390/molecules26072068 33916823 PMC8038324 Bernal-Chávez SA, et al. Insights into terminal sterilization processes of nanoparticles for biomedical applications. Molecules. 2021. 10.3390/molecules26072068. 33916823 10.3390/molecules26072068 PMC8038324

188. Tehrani SF Purification processes of polymeric nanoparticles: how to improve their clinical translation? J Control Release 2023 360 591 612 10.1016/j.jconrel.2023.06.038 37422123 Tehrani SF, et al. Purification processes of polymeric nanoparticles: how to improve their clinical translation? J Control Release. 2023;360:591–612. 37422123 10.1016/j.jconrel.2023.06.038

189. Filippov SK Dynamic light scattering and transmission electron microscopy in drug delivery: a roadmap for correct characterization of nanoparticles and interpretation of results Mater Horiz 2023 10 12 5354 70 10.1039/D3MH00717K 37814922 Filippov SK, et al. Dynamic light scattering and transmission electron microscopy in drug delivery: a roadmap for correct characterization of nanoparticles and interpretation of results. Mater Horiz. 2023;10(12):5354–70. 37814922 10.1039/d3mh00717k

190. Carvalho NK Antibacterial, biological, and physicochemical properties of root canal sealers containing chlorhexidine-hexametaphosphate nanoparticles Dent Mater 2021 37 5 863 74 10.1016/j.dental.2021.02.007 33648745 Carvalho NK, et al. Antibacterial, biological, and physicochemical properties of root canal sealers containing chlorhexidine-hexametaphosphate nanoparticles. Dent Mater. 2021;37(5):863–74. 33648745 10.1016/j.dental.2021.02.007

191. Xu Y Surface modification of lipid-based nanoparticles ACS Nano 2022 16 5 7168 96 10.1021/acsnano.2c02347 35446546 Xu Y, et al. Surface modification of lipid-based nanoparticles. ACS Nano. 2022;16(5):7168–96. 35446546 10.1021/acsnano.2c02347

192. Suk JS PEGylation as a strategy for improving nanoparticle-based drug and gene delivery Adv Drug Deliv Rev 2016 99Pt A 28 51 10.1016/j.addr.2015.09.012 PMC4798869 26456916 Suk JS, et al. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev. 2016;99Pt A:28–51. 10.1016/j.addr.2015.09.012 PMC4798869 26456916

193. Bajracharya R Functional ligands for improving anticancer drug therapy: current status and applications to drug delivery systems Drug Deliv 2022 29 1 1959 70 10.1080/10717544.2022.2089296 35762636 PMC9246174 Bajracharya R, et al. Functional ligands for improving anticancer drug therapy: current status and applications to drug delivery systems. Drug Deliv. 2022;29(1):1959–70. 35762636 10.1080/10717544.2022.2089296 PMC9246174

194. Kozma GT Pseudo-anaphylaxis to polyethylene glycol (PEG)-coated liposomes: roles of anti-PEG IgM and complement activation in a porcine model of human infusion reactions ACS Nano 2019 13 8 9315 24 10.1021/acsnano.9b03942 31348638 Kozma GT, et al. Pseudo-anaphylaxis to polyethylene glycol (PEG)-coated liposomes: roles of anti-PEG IgM and complement activation in a porcine model of human infusion reactions. ACS Nano. 2019;13(8):9315–24. 31348638 10.1021/acsnano.9b03942

195. Chen BM Cheng TL Roffler SR Polyethylene glycol immunogenicity: theoretical, clinical, and practical aspects of anti-Polyethylene glycol antibodies ACS Nano 2021 15 9 14022 48 10.1021/acsnano.1c05922 34469112 Chen BM, Cheng TL, Roffler SR. Polyethylene glycol immunogenicity: theoretical, clinical, and practical aspects of anti-Polyethylene glycol antibodies. ACS Nano. 2021;15(9):14022–48. 34469112 10.1021/acsnano.1c05922

196. Owens DE Peppas NA Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles Int J Pharm 2006 307 1 93 102 10.1016/j.ijpharm.2005.10.010 16303268 Owens DE, Peppas NA. Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm. 2006;307(1):93–102. 16303268 10.1016/j.ijpharm.2005.10.010

197. Almalik A Hyaluronic acid coated chitosan nanoparticles reduced the immunogenicity of the formed protein corona Sci Rep 2017 7 1 10542 10.1038/s41598-017-10836-7 28874846 PMC5585335 Almalik A, et al. Hyaluronic acid coated chitosan nanoparticles reduced the immunogenicity of the formed protein corona. Sci Rep. 2017;7(1):10542. 28874846 10.1038/s41598-017-10836-7 PMC5585335

198. Shin K Polyglycerol and Poly(ethylene glycol) exhibit different effects on pharmacokinetics and antibody generation when grafted to nanoparticle surfaces Biomaterials 2022 287 121676 10.1016/j.biomaterials.2022.121676 35849999 Shin K, et al. Polyglycerol and Poly(ethylene glycol) exhibit different effects on pharmacokinetics and antibody generation when grafted to nanoparticle surfaces. Biomaterials. 2022;287:121676. 35849999 10.1016/j.biomaterials.2022.121676

199. Joseph A Surfactants influence polymer nanoparticle fate within the brain Biomaterials 2021 277 121086 10.1016/j.biomaterials.2021.121086 34481289 PMC8478896 Joseph A, et al. Surfactants influence polymer nanoparticle fate within the brain. Biomaterials. 2021;277:121086. 34481289 10.1016/j.biomaterials.2021.121086 PMC8478896

200. Shreffler JW Overcoming hurdles in nanoparticle clinical translation: the influence of experimental design and surface modification Int J Mol Sci 2019 10.3390/ijms20236056 31801303 PMC6928924 Shreffler JW, et al. Overcoming hurdles in nanoparticle clinical translation: the influence of experimental design and surface modification. Int J Mol Sci. 2019. 10.3390/ijms20236056. 31801303 10.3390/ijms20236056 PMC6928924

201. Ragelle H Nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures Expert Opin Drug Deliv 2017 14 7 851 64 10.1080/17425247.2016.1244187 27730820 Ragelle H, et al. Nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures. Expert Opin Drug Deliv. 2017;14(7):851–64. 27730820 10.1080/17425247.2016.1244187

202. Nel A Toxic potential of materials at the nanolevel Science 2006 311 5761 622 7 10.1126/science.1114397 16456071 Nel A, et al. Toxic potential of materials at the nanolevel. Science. 2006;311(5761):622–7. 16456071 10.1126/science.1114397

203. Song Y Li X Du X Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma Eur Respir J 2009 34 3 559 67 10.1183/09031936.00178308 19696157 Song Y, Li X, Du X. Exposure to nanoparticles is related to pleural effusion, pulmonary fibrosis and granuloma. Eur Respir J. 2009;34(3):559–67. 19696157 10.1183/09031936.00178308

204. Joyce P A translational framework to DELIVER nanomedicines to the clinic Nat Nanotechnol 2024 19 11 1597 611 10.1038/s41565-024-01754-7 39242807 Joyce P, et al. A translational framework to DELIVER nanomedicines to the clinic. Nat Nanotechnol. 2024;19(11):1597–611. 39242807 10.1038/s41565-024-01754-7

205. Hou X Lipid nanoparticles for mRNA delivery Nat Rev Mater 2021 6 12 1078 94 10.1038/s41578-021-00358-0 34394960 PMC8353930 Hou X, et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078–94. 34394960 10.1038/s41578-021-00358-0 PMC8353930

206. Chopyk DM Grakoui A Contribution of the intestinal microbiome and gut barrier to hepatic disorders Gastroenterology 2020 159 3 849 63 10.1053/j.gastro.2020.04.077 32569766 PMC7502510 Chopyk DM, Grakoui A. Contribution of the intestinal microbiome and gut barrier to hepatic disorders. Gastroenterology. 2020;159(3):849–63. 32569766 10.1053/j.gastro.2020.04.077 PMC7502510

207. Gustafson HH Nanoparticle uptake: the phagocyte problem Nano Today 2015 10 4 487 510 10.1016/j.nantod.2015.06.006 26640510 PMC4666556 Gustafson HH, et al. Nanoparticle uptake: the phagocyte problem. Nano Today. 2015;10(4):487–510. 26640510 10.1016/j.nantod.2015.06.006 PMC4666556

208. Poon W Elimination pathways of nanoparticles ACS Nano 2019 13 5 5785 98 10.1021/acsnano.9b01383 30990673 Poon W, et al. Elimination pathways of nanoparticles. ACS Nano. 2019;13(5):5785–98. 30990673 10.1021/acsnano.9b01383

209. Zhang YN Nanoparticle-liver interactions: cellular uptake and hepatobiliary elimination J Control Release 2016 240 332 48 10.1016/j.jconrel.2016.01.020 26774224 Zhang YN, et al. Nanoparticle-liver interactions: cellular uptake and hepatobiliary elimination. J Control Release. 2016;240:332–48. 26774224 10.1016/j.jconrel.2016.01.020

210. Fornaguera C In vivo retargeting of Poly(beta aminoester) (OM-PBAE) nanoparticles is influenced by protein corona Adv Healthc Mater 2019 8 19 e1900849 10.1002/adhm.201900849 31478348 Fornaguera C, et al. In vivo retargeting of Poly(beta aminoester) (OM-PBAE) nanoparticles is influenced by protein corona. Adv Healthc Mater. 2019;8(19):e1900849. 31478348 10.1002/adhm.201900849

211. Blanco E Shen H Ferrari M Principles of nanoparticle design for overcoming biological barriers to drug delivery Nat Biotechnol 2015 33 9 941 51 10.1038/nbt.3330 26348965 PMC4978509 Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol. 2015;33(9):941–51. 26348965 10.1038/nbt.3330 PMC4978509

212. von Roemeling C Breaking down the barriers to precision cancer nanomedicine Trends Biotechnol 2017 35 2 159 71 10.1016/j.tibtech.2016.07.006 27492049 von Roemeling C, et al. Breaking down the barriers to precision cancer nanomedicine. Trends Biotechnol. 2017;35(2):159–71. 27492049 10.1016/j.tibtech.2016.07.006

213. Juliano R Biological barriers to therapy with antisense and siRNA oligonucleotides Mol Pharm 2009 6 3 686 95 10.1021/mp900093r 19397332 PMC2758224 Juliano R, et al. Biological barriers to therapy with antisense and siRNA oligonucleotides. Mol Pharm. 2009;6(3):686–95. 19397332 10.1021/mp900093r PMC2758224

214. Zhao Z Targeting strategies for tissue-specific drug delivery Cell 2020 181 1 151 67 10.1016/j.cell.2020.02.001 32243788 Zhao Z, et al. Targeting strategies for tissue-specific drug delivery. Cell. 2020;181(1):151–67. 32243788 10.1016/j.cell.2020.02.001

215. Morachis JM Mahmoud EA Almutairi A Physical and chemical strategies for therapeutic delivery by using polymeric nanoparticles Pharmacol Rev 2012 64 3 505 19 10.1124/pr.111.005363 22544864 PMC3400833 Morachis JM, Mahmoud EA, Almutairi A. Physical and chemical strategies for therapeutic delivery by using polymeric nanoparticles. Pharmacol Rev. 2012;64(3):505–19. 22544864 10.1124/pr.111.005363 PMC3400833

216. Krishnan N Fang RH Zhang L Engineering of stimuli-responsive self-assembled biomimetic nanoparticles Adv Drug Deliv Rev 2021 179 114006 10.1016/j.addr.2021.114006 34655662 Krishnan N, Fang RH, Zhang L. Engineering of stimuli-responsive self-assembled biomimetic nanoparticles. Adv Drug Deliv Rev. 2021;179:114006. 34655662 10.1016/j.addr.2021.114006

217. Donahue ND Acar H Wilhelm S Concepts of nanoparticle cellular uptake, intracellular trafficking, and kinetics in nanomedicine Adv Drug Deliv Rev 2019 143 68 96 10.1016/j.addr.2019.04.008 31022434 Donahue ND, Acar H, Wilhelm S. Concepts of nanoparticle cellular uptake, intracellular trafficking, and kinetics in nanomedicine. Adv Drug Deliv Rev. 2019;143:68–96. 31022434 10.1016/j.addr.2019.04.008

218. Kim J Self-assembled mrna vaccines Adv Drug Deliv Rev 2021 170 83 112 10.1016/j.addr.2020.12.014 33400957 PMC7837307 Kim J, et al. Self-assembled mrna vaccines. Adv Drug Deliv Rev. 2021;170:83–112. 33400957 10.1016/j.addr.2020.12.014 PMC7837307

219. Tombácz I Highly efficient CD4 + T cell targeting and genetic recombination using engineered CD4 + cell-homing mRNA-LNPs Mol Ther 2021 29 11 3293 304 10.1016/j.ymthe.2021.06.004 34091054 PMC8571164 Tombácz I, et al. Highly efficient CD4 + T cell targeting and genetic recombination using engineered CD4 + cell-homing mRNA-LNPs. Mol Ther. 2021;29(11):3293–304. 34091054 10.1016/j.ymthe.2021.06.004 PMC8571164

220. Niu H Zhao P Sun W Biomaterials for chimeric antigen receptor T cell engineering Acta Biomater 2023 166 1 13 10.1016/j.actbio.2023.04.043 37137403 Niu H, Zhao P, Sun W. Biomaterials for chimeric antigen receptor T cell engineering. Acta Biomater. 2023;166:1–13. 37137403 10.1016/j.actbio.2023.04.043

221. Ramishetti S A combinatorial library of lipid nanoparticles for RNA delivery to leukocytes Adv Mater 2020 32 12 e1906128 10.1002/adma.201906128 31999380 Ramishetti S, et al. A combinatorial library of lipid nanoparticles for RNA delivery to leukocytes. Adv Mater. 2020;32(12):e1906128. 31999380 10.1002/adma.201906128

222. Patel S Brief update on endocytosis of nanomedicines Adv Drug Deliv Rev 2019 144 90 111 10.1016/j.addr.2019.08.004 31419450 PMC6986687 Patel S, et al. Brief update on endocytosis of nanomedicines. Adv Drug Deliv Rev. 2019;144:90–111. 31419450 10.1016/j.addr.2019.08.004 PMC6986687

223. Liu S Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing Nat Mater 2021 20 5 701 10 10.1038/s41563-020-00886-0 33542471 PMC8188687 Liu S, et al. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing. Nat Mater. 2021;20(5):701–10. 33542471 10.1038/s41563-020-00886-0 PMC8188687

224. Wojnilowicz M Super-resolution imaging of proton sponge-triggered rupture of endosomes and cytosolic release of small interfering RNA ACS Nano 2019 13 1 187 202 10.1021/acsnano.8b05151 30566836 Wojnilowicz M, et al. Super-resolution imaging of proton sponge-triggered rupture of endosomes and cytosolic release of small interfering RNA. ACS Nano. 2019;13(1):187–202. 30566836 10.1021/acsnano.8b05151

225. Ayyadevara V Roh KH Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells Drug Deliv 2020 27 1 805 15 10.1080/10717544.2020.1770371 32489110 PMC8216448 Ayyadevara V, Roh KH. Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells. Drug Deliv. 2020;27(1):805–15. 32489110 10.1080/10717544.2020.1770371 PMC8216448

226. Zheng L Lipid nanoparticle topology regulates endosomal escape and delivery of RNA to the cytoplasm Proc Natl Acad Sci U S A 2023 120 27 e2301067120 10.1073/pnas.2301067120 37364130 PMC10318962 Zheng L, et al. Lipid nanoparticle topology regulates endosomal escape and delivery of RNA to the cytoplasm. Proc Natl Acad Sci U S A. 2023;120(27):e2301067120. 37364130 10.1073/pnas.2301067120 PMC10318962

227. Maus MV Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial apcs expressing ligands for the T-cell receptor, CD28 and 4-1BB Nat Biotechnol 2002 20 2 143 8 10.1038/nbt0202-143 11821859 Maus MV, et al. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial apcs expressing ligands for the T-cell receptor, CD28 and 4-1BB. Nat Biotechnol. 2002;20(2):143–8. 11821859 10.1038/nbt0202-143

228. Harris E Nonviral gene delivery to T cells with lipofectamine LTX Biotechnol Bioeng 2021 118 4 1693 706 10.1002/bit.27686 33480049 Harris E, et al. Nonviral gene delivery to T cells with lipofectamine LTX. Biotechnol Bioeng. 2021;118(4):1693–706. 33480049 10.1002/bit.27686

229. Ghosh B Biswas S Polymeric micelles in cancer therapy: state of the Art J Control Release 2021 332 127 47 10.1016/j.jconrel.2021.02.016 33609621 Ghosh B, Biswas S. Polymeric micelles in cancer therapy: state of the Art. J Control Release. 2021;332:127–47. 33609621 10.1016/j.jconrel.2021.02.016

230. Metkar M Pepin CS Moore MJ Tailor made: the art of therapeutic mRNA design Nat Rev Drug Discov 2024 23 1 67 83 10.1038/s41573-023-00827-x 38030688 Metkar M, Pepin CS, Moore MJ. Tailor made: the art of therapeutic mRNA design. Nat Rev Drug Discov. 2024;23(1):67–83. 38030688 10.1038/s41573-023-00827-x

231. Schmidt C, Schnierle BS. Self-Amplifying RNA vaccine candidates: alternative platforms for mRNA vaccine development. Pathogens. 2023;12(1):138. 10.3390/pathogens12010138 PMC9863218 36678486

232. Yıldız A, Răileanu C, Beissert T. Trans-Amplifying RNA: A journey from alphavirus research to future vaccines. Viruses. 2024;16(4):503. 10.3390/v16040503 PMC11055088 38675846

233. Liu X Circular RNA: an emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines J Control Release 2022 348 84 94 10.1016/j.jconrel.2022.05.043 35649485 PMC9644292 Liu X, et al. Circular RNA: an emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J Control Release. 2022;348:84–94. 35649485 10.1016/j.jconrel.2022.05.043 PMC9644292

234. Pasquier B Autophagy inhibitors Cell Mol Life Sci 2016 73 5 985 1001 10.1007/s00018-015-2104-y 26658914 PMC11108294 Pasquier B. Autophagy inhibitors. Cell Mol Life Sci. 2016;73(5):985–1001. 26658914 10.1007/s00018-015-2104-y PMC11108294

235. Olden BR Identifying key barriers in cationic polymer gene delivery to human T cells Biomater Sci 2019 7 3 789 97 10.1039/C8BM01262H 30633266 PMC6391219 Olden BR, et al. Identifying key barriers in cationic polymer gene delivery to human T cells. Biomater Sci. 2019;7(3):789–97. 30633266 10.1039/c8bm01262h PMC6391219

236. Wang H Polyethylene glycol (PEG)-associated immune responses triggered by clinically relevant lipid nanoparticles in rats NPJ Vaccines 2023 8 1 169 10.1038/s41541-023-00766-z 37919316 PMC10622525 Wang H, et al. Polyethylene glycol (PEG)-associated immune responses triggered by clinically relevant lipid nanoparticles in rats. NPJ Vaccines. 2023;8(1):169. 37919316 10.1038/s41541-023-00766-z PMC10622525

237. Oroojalian F Immune cell Membrane-Coated biomimetic nanoparticles for targeted cancer therapy Small 2021 17 12 e2006484 10.1002/smll.202006484 33577127 Oroojalian F, et al. Immune cell Membrane-Coated biomimetic nanoparticles for targeted cancer therapy. Small. 2021;17(12):e2006484. 33577127 10.1002/smll.202006484

238. Sukumaran S Enhancing the potency and specificity of engineered T cells for cancer treatment Cancer Discov 2018 8 8 972 87 10.1158/2159-8290.CD-17-1298 29880586 PMC6428579 Sukumaran S, et al. Enhancing the potency and specificity of engineered T cells for cancer treatment. Cancer Discov. 2018;8(8):972–87. 29880586 10.1158/2159-8290.CD-17-1298 PMC6428579

239. Könczöl M Cytotoxicity and genotoxicity of size-fractionated iron oxide (magnetite) in A549 human lung epithelial cells: role of ROS, JNK, and NF-κB Chem Res Toxicol 2011 24 9 1460 75 10.1021/tx200051s 21761924 Könczöl M, et al. Cytotoxicity and genotoxicity of size-fractionated iron oxide (magnetite) in A549 human lung epithelial cells: role of ROS, JNK, and NF-κB. Chem Res Toxicol. 2011;24(9):1460–75. 21761924 10.1021/tx200051s

240. Karlsson HL Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes Chem Res Toxicol 2008 21 9 1726 32 10.1021/tx800064j 18710264 Karlsson HL, et al. Copper oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes. Chem Res Toxicol. 2008;21(9):1726–32. 18710264 10.1021/tx800064j

241. Kheirolomoom A In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift Biomaterials 2022 281 121339 10.1016/j.biomaterials.2021.121339 35078042 PMC8892572 Kheirolomoom A, et al. In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift. Biomaterials. 2022;281:121339. 35078042 10.1016/j.biomaterials.2021.121339 PMC8892572

242. Lee Y Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics Exp Mol Med 2023 55 10 2085 96 10.1038/s12276-023-01086-x 37779140 PMC10618257 Lee Y, et al. Immunogenicity of lipid nanoparticles and its impact on the efficacy of mRNA vaccines and therapeutics. Exp Mol Med. 2023;55(10):2085–96. 37779140 10.1038/s12276-023-01086-x PMC10618257

243. Unnikrishnan BS Folic acid-appended galactoxyloglucan-capped iron oxide nanoparticles as a biocompatible nanotheranostic agent for tumor-targeted delivery of doxorubicin Int J Biol Macromol 2021 168 130 42 10.1016/j.ijbiomac.2020.11.205 33278441 Unnikrishnan BS, et al. Folic acid-appended galactoxyloglucan-capped iron oxide nanoparticles as a biocompatible nanotheranostic agent for tumor-targeted delivery of doxorubicin. Int J Biol Macromol. 2021;168:130–42. 33278441 10.1016/j.ijbiomac.2020.11.205

244. Wasti S Ethical and legal challenges in nanomedical innovations: a scoping review Front Genet 2023 14 1163392 10.3389/fgene.2023.1163392 37252668 PMC10213273 Wasti S, et al. Ethical and legal challenges in nanomedical innovations: a scoping review. Front Genet. 2023;14:1163392. 37252668 10.3389/fgene.2023.1163392 PMC10213273

245. Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products . 2024; Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/considerations-development-chimeric-antigen-receptor-car-t-cell-products

246. Regenerative Medicine Advanced Therapy Designation . 2025; Available from: https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/regenerative-medicine-advanced-therapy-designation

247. Breakthrough T. 2018; Available from: https://www.fda.gov/patients/fast-track-breakthrough-therapy-accelerated-approval-priority-review/breakthrough-therapy

248. Chemistry., Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs) . 2020; Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/chemistry-manufacturing-and-control-cmc-information-human-gene-therapy-investigational-new-drug

249. Launch of PRIME – Paving the way for promising medicines for patients . 2016; Available from: https://www.ema.europa.eu/en/documents/press-release/launch-prime-paving-way-promising-medicines-patients_en.pdf

250. Conditional. marketing authorisation . 2016; Available from: https://www.ema.europa.eu/en/human-regulatory-overview/marketing-authorisation/conditional-marketing-authorisation

251. S SK Regulatory landscape and challenges in CAR-T cell therapy development in the US, EU, japan, and India Eur J Pharm Biopharm 2024 201 114361 10.1016/j.ejpb.2024.114361 38871092 S SK, et al. Regulatory landscape and challenges in CAR-T cell therapy development in the US, EU, japan, and India. Eur J Pharm Biopharm. 2024;201:114361. 38871092 10.1016/j.ejpb.2024.114361

252. Strategy of. SAKIGAKE . 2014; Available from: https://www.mhlw.go.jp/english/policy/health-medical/pharmaceuticals/dl/140729-01-01.pdf

253. Matsushita S A review of the regulatory framework for initiation and acceleration of patient access to innovative medical products in Japan Clin Pharmacol Ther 2019 106 3 508 11 10.1002/cpt.1383 30883696 Matsushita S, et al. A review of the regulatory framework for initiation and acceleration of patient access to innovative medical products in Japan. Clin Pharmacol Ther. 2019;106(3):508–11. 30883696 10.1002/cpt.1383

254. Desai N Nanoparticle therapeutics in clinical perspective: classification, marketed products, and regulatory landscape Small 2025 21 29 e2502315 10.1002/smll.202502315 40454890 PMC12288819 Desai N, et al. Nanoparticle therapeutics in clinical perspective: classification, marketed products, and regulatory landscape. Small. 2025;21(29):e2502315. 40454890 10.1002/smll.202502315 PMC12288819

255. Considering Whether an FDA-Regulated Product Involves the Application of Nanotechnology . 2014; Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/considering-whether-fda-regulated-product-involves-application-nanotechnology

256. Liposome Drug Products: Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labeling Documentation . 2018; Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/liposome-drug-products-chemistry-manufacturing-and-controls-human-pharmacokinetics-and

257. Drug, Products., Including Biological Products, that Contain Nanomaterials - Guidance for Industry . 2022; Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/drug-products-including-biological-products-contain-nanomaterials-guidance-industry 10.1208/s12248-019-0329-7 PMC6470106 30997588

258. Musazzi UM Feeding next-generation nanomedicines to europe: regulatory and quality challenges Adv Healthc Mater 2023 12 30 e2301956 10.1002/adhm.202301956 37718353 PMC11468706 Musazzi UM, et al. Feeding next-generation nanomedicines to europe: regulatory and quality challenges. Adv Healthc Mater. 2023;12(30):e2301956. 37718353 10.1002/adhm.202301956 PMC11468706

259. Reflection paper on the data requirements for intravenous liposomal products developed with reference to an innovator liposomal product 2013; Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-data-requirements-intravenous-liposomal-products-developed-reference-innovator-liposomal-product-revision-2_en.pdf

260. Reflection paper on surface coatings: general issues for consideration regarding parenteral administration of coated nanomedicine products 2013; Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/reflection-paper-surface-coatings-general-issues-consideration-regarding-parenteral-administration-coated-nanomedicine-products_en.pdf

261. Joint MHLW. EMA reflection paper on the development of block copolymer micelle medicinal products 2013; Available from: https://www.ema.europa.eu/en/documents/scientific-guideline/joint-mhlwema-reflection-paper-development-block-copolymer-micelle-medicinal-products_en.pdf

262. Bleeker EAJ Towards harmonisation of testing of nanomaterials for EU regulatory requirements on chemical safety - a proposal for further actions Regul Toxicol Pharmacol 2023 139 105360 10.1016/j.yrtph.2023.105360 36804527 PMC10060946 Bleeker EAJ, et al. Towards harmonisation of testing of nanomaterials for EU regulatory requirements on chemical safety - a proposal for further actions. Regul Toxicol Pharmacol. 2023;139:105360. 36804527 10.1016/j.yrtph.2023.105360 PMC10060946

263. Publication of Joint MHLW/EMA reflection paper on the development of block copolymer micelle medicinal products 2014; Available from: https://www.pmda.go.jp/files/000272265.pdf

264. Reflection paper on nucleic acids (siRNA)-loaded nanotechnology-based drug products 2016; Available from: https://www.pmda.go.jp/files/000272264.pdf

265. Foulkes R The regulation of nanomaterials and nanomedicines for clinical application: current and future perspectives Biomater Sci 2020 8 17 4653 64 10.1039/D0BM00558D 32672255 Foulkes R, et al. The regulation of nanomaterials and nanomedicines for clinical application: current and future perspectives. Biomater Sci. 2020;8(17):4653–64. 32672255 10.1039/d0bm00558d

266. Ku KS Current advancements in anti-cancer chimeric antigen receptor T cell immunotherapy and how nanotechnology May change the game Int J Mol Sci 2024 10.3390/ijms25105361 38791398 PMC11120994 Ku KS, et al. Current advancements in anti-cancer chimeric antigen receptor T cell immunotherapy and how nanotechnology May change the game. Int J Mol Sci. 2024. 10.3390/ijms25105361. 38791398 10.3390/ijms25105361 PMC11120994

267. Kanp T Exploring the potential of nanocarriers for cancer immunotherapy: insights into mechanism, nanocarriers, and regulatory perspectives ACS Appl Bio Mater 2025 8 1 108 38 10.1021/acsabm.4c01797 39791993 Kanp T, et al. Exploring the potential of nanocarriers for cancer immunotherapy: insights into mechanism, nanocarriers, and regulatory perspectives. ACS Appl Bio Mater. 2025;8(1):108–38. 39791993 10.1021/acsabm.4c01797

268. Xin T -Vivo induced CAR-T cell for the potential breakthrough to overcome the barriers of current CAR-T cell therapy Front Oncol 2022 12 809754 10.3389/fonc.2022.809754 35223491 PMC8866962 Xin T, et al. -Vivo induced CAR-T cell for the potential breakthrough to overcome the barriers of current CAR-T cell therapy. Front Oncol. 2022;12:809754. 35223491 10.3389/fonc.2022.809754 PMC8866962

269. Oualikene-Gonin W Regulatory assessment of nano-enabled health products in public health interest. Position of the scientific advisory board of the French National agency for the safety of medicines and health products Front Public Health 2023 11 1125577 10.3389/fpubh.2023.1125577 36935690 PMC10018019 Oualikene-Gonin W, et al. Regulatory assessment of nano-enabled health products in public health interest. Position of the scientific advisory board of the French National agency for the safety of medicines and health products. Front Public Health. 2023;11:1125577. 36935690 10.3389/fpubh.2023.1125577 PMC10018019

270. Tan P Artificial intelligence aids in development of nanomedicines for cancer management Semin Cancer Biol 2023 89 61 75 10.1016/j.semcancer.2023.01.005 36682438 Tan P, et al. Artificial intelligence aids in development of nanomedicines for cancer management. Semin Cancer Biol. 2023;89:61–75. 36682438 10.1016/j.semcancer.2023.01.005

271. Raj GM Dananjayan S Gudivada KK Applications of artificial intelligence and machine learning in clinical medicine: what lies ahead? Medicine Advances 2024 2 2 202 4 10.1002/med4.62 Raj GM, Dananjayan S, Gudivada KK. Applications of artificial intelligence and machine learning in clinical medicine: what lies ahead? Medicine Advances. 2024;2(2):202–4.

272. Ali H Generative pre-trained transformer 4 in healthcare: challenges, opportunities, and recommendations Medicine Advances 2023 1 2 163 6 10.1002/med4.21 Ali H. Generative pre-trained transformer 4 in healthcare: challenges, opportunities, and recommendations. Medicine Advances. 2023;1(2):163–6.

273. Cai W Deepseek AI: transforming medical AI with cost-efficiency, transparency, and privacy preservation Intell Oncol 2025 1 2 172 5 10.1016/j.intonc.2025.03.006 Cai W. Deepseek AI: transforming medical AI with cost-efficiency, transparency, and privacy preservation. Intell Oncol. 2025;1(2):172–5.

274. Chen Z, Saw PE. Integration in biomedical science 2024: emerging trends in the Post-Pandemic era. BIOI. 2024;5(1):1–2.

275. Odeh-Couvertier VY Predicting T-cell quality during manufacturing through an artificial intelligence-based integrative multiomics analytical platform Bioeng Transl Med 2022 7 2 e10282 10.1002/btm2.10282 35600660 PMC9115702 Odeh-Couvertier VY, et al. Predicting T-cell quality during manufacturing through an artificial intelligence-based integrative multiomics analytical platform. Bioeng Transl Med. 2022;7(2):e10282. 35600660 10.1002/btm2.10282 PMC9115702

276. Hort S Toward rapid, widely available autologous CAR-T cell therapy - Artificial intelligence and automation enabling the smart manufacturing hospital Front Med Lausanne 2022 9 913287 10.3389/fmed.2022.913287 35733863 PMC9207622 Hort S, et al. Toward rapid, widely available autologous CAR-T cell therapy - Artificial intelligence and automation enabling the smart manufacturing hospital. Front Med Lausanne. 2022;9:913287. 35733863 10.3389/fmed.2022.913287 PMC9207622

277. Yamankurt G Exploration of the nanomedicine-design space with high-throughput screening and machine learning Nat Biomed Eng 2019 3 4 318 27 10.1038/s41551-019-0351-1 30952978 PMC6452897 Yamankurt G, et al. Exploration of the nanomedicine-design space with high-throughput screening and machine learning. Nat Biomed Eng. 2019;3(4):318–27. 30952978 10.1038/s41551-019-0351-1 PMC6452897

278. Gottschlich A Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia Nat Biotechnol 2023 41 11 1618 32 10.1038/s41587-023-01684-0 36914885 PMC7615296 Gottschlich A, et al. Single-cell transcriptomic atlas-guided development of CAR-T cells for the treatment of acute myeloid leukemia. Nat Biotechnol. 2023;41(11):1618–32. 36914885 10.1038/s41587-023-01684-0 PMC7615296

279. Martarelli N Artificial intelligence-powered molecular docking and steered molecular dynamics for accurate ScFv selection of anti-CD30 chimeric antigen receptors Int J Mol Sci 2024 10.3390/ijms25137231 39000338 PMC11242627 Martarelli N, et al. Artificial intelligence-powered molecular docking and steered molecular dynamics for accurate ScFv selection of anti-CD30 chimeric antigen receptors. Int J Mol Sci. 2024. 10.3390/ijms25137231. 39000338 10.3390/ijms25137231 PMC11242627

280. Daniels KG Decoding CAR T cell phenotype using combinatorial signaling motif libraries and machine learning Science 2022 378 6625 1194 200 10.1126/science.abq0225 36480602 PMC10026561 Daniels KG, et al. Decoding CAR T cell phenotype using combinatorial signaling motif libraries and machine learning. Science. 2022;378(6625):1194–200. 36480602 10.1126/science.abq0225 PMC10026561

281. Adir O Integrating artificial intelligence and nanotechnology for precision cancer medicine Adv Mater 2020 32 13 e1901989 10.1002/adma.201901989 31286573 PMC7124889 Adir O, et al. Integrating artificial intelligence and nanotechnology for precision cancer medicine. Adv Mater. 2020;32(13):e1901989. 31286573 10.1002/adma.201901989 PMC7124889

282. Zhu M Machine-learning-assisted single-vessel analysis of nanoparticle permeability in tumour vasculatures Nat Nanotechnol 2023 18 6 657 666 10.1038/s41565-023-01323-4 36781994 Zhu M, et al. Machine-learning-assisted single-vessel analysis of nanoparticle permeability in tumour vasculatures. Nat Nanotechnol. 2023;18(6):657–66. 36781994 10.1038/s41565-023-01323-4

283. Dahlman JE Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics Proc Natl Acad Sci U S A 2017 114 8 2060 5 10.1073/pnas.1620874114 28167778 PMC5338412 Dahlman JE, et al. Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics. Proc Natl Acad Sci U S A. 2017;114(8):2060–5. 28167778 10.1073/pnas.1620874114 PMC5338412

284. Hamilton S Kingston BR Applying artificial intelligence and computational modeling to nanomedicine Curr Opin Biotechnol 2024 85 103043 10.1016/j.copbio.2023.103043 38091874 Hamilton S, Kingston BR. Applying artificial intelligence and computational modeling to nanomedicine. Curr Opin Biotechnol. 2024;85:103043. 38091874 10.1016/j.copbio.2023.103043

285. Teachey DT Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia Cancer Discov 2016 6 6 664 79 10.1158/2159-8290.CD-16-0040 27076371 PMC5448406 Teachey DT, et al. Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Cancer Discov. 2016;6(6):664–79. 27076371 10.1158/2159-8290.CD-16-0040 PMC5448406

286. Rubin DB Clinical predictors of neurotoxicity after chimeric antigen receptor T-cell therapy JAMA Neurol 2020 77 12 1536 42 10.1001/jamaneurol.2020.2703 32777012 PMC7418044 Rubin DB, et al. Clinical predictors of neurotoxicity after chimeric antigen receptor T-cell therapy. JAMA Neurol. 2020;77(12):1536–42. 32777012 10.1001/jamaneurol.2020.2703 PMC7418044

287. Haradhvala NJ Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma Nat Med 2022 28 9 1848 59 10.1038/s41591-022-01959-0 36097221 PMC9509487 Haradhvala NJ, et al. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nat Med. 2022;28(9):1848–59. 36097221 10.1038/s41591-022-01959-0 PMC9509487

288. Kim M Detection of ovarian cancer via the spectral fingerprinting of quantum-defect-modified carbon nanotubes in serum by machine learning Nat Biomed Eng 2022 6 3 267 75 10.1038/s41551-022-00860-y 35301449 PMC9108893 Kim M, et al. Detection of ovarian cancer via the spectral fingerprinting of quantum-defect-modified carbon nanotubes in serum by machine learning. Nat Biomed Eng. 2022;6(3):267–75. 35301449 10.1038/s41551-022-00860-y PMC9108893

289. Tahara K Pharmaceutical formulation and manufacturing using particle/powder technology for personalized medicines Adv Powder Technol 2020 31 1 387 92 10.1016/j.apt.2019.10.031 Tahara K. Pharmaceutical formulation and manufacturing using particle/powder technology for personalized medicines. Adv Powder Technol. 2020;31(1):387–92.

290. Zhou Z Deciphering the tumor immune microenvironment from a multidimensional omics perspective: insight into next-generation CAR-T cell immunotherapy and beyond Mol Cancer 2024 23 1 131 10.1186/s12943-024-02047-2 38918817 PMC11201788 Zhou Z, et al. Deciphering the tumor immune microenvironment from a multidimensional omics perspective: insight into next-generation CAR-T cell immunotherapy and beyond. Mol Cancer. 2024;23(1):131. 38918817 10.1186/s12943-024-02047-2 PMC11201788

291. Huo T Deep learning-based multimodal data fusion in bone tumor management: advances in clinical decision support Intell Oncol 2025 1 3 204 15 10.1016/j.intonc.2025.06.005 Huo T, et al. Deep learning-based multimodal data fusion in bone tumor management: advances in clinical decision support. Intell Oncol. 2025;1(3):204–15.

292. Zhang Y Cancer research revolutionized: unveiling the power of organoids and their therapeutic potential in oncology hLife 2025 3 5 216 36 10.1016/j.hlife.2024.05.002 Zhang Y, et al. Cancer research revolutionized: unveiling the power of organoids and their therapeutic potential in oncology. hLife. 2025;3(5):216–36.

293. Yadav D Malviya R Novel nanomaterials as photo-activated cancer diagnostics and therapy Med Adv 2023 1 3 190 209 10.1002/med4.36 Yadav D, Malviya R. Novel nanomaterials as photo-activated cancer diagnostics and therapy. Med Adv. 2023;1(3):190–209.

294. Wang Y Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity Cell Prolif 2024 57 11 e13683 10.1111/cpr.13683 38830795 PMC11533075 Wang Y, et al. Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity. Cell Prolif. 2024;57(11):e13683. 38830795 10.1111/cpr.13683 PMC11533075

295. Liu Y Mesothelin CAR-engineered NK cells derived from human embryonic stem cells suppress the progression of human ovarian cancer in animals Cell Prolif 2024 57 12 e13727 10.1111/cpr.13727 39136096 PMC11628735 Liu Y, et al. Mesothelin CAR-engineered NK cells derived from human embryonic stem cells suppress the progression of human ovarian cancer in animals. Cell Prolif. 2024;57(12):e13727. 39136096 10.1111/cpr.13727 PMC11628735

296. Beach MA Polymeric nanoparticles for drug delivery Chem Rev 2024 124 9 5505 616 10.1021/acs.chemrev.3c00705 38626459 PMC11086401 Beach MA, et al. Polymeric nanoparticles for drug delivery. Chem Rev. 2024;124(9):5505–616. 38626459 10.1021/acs.chemrev.3c00705 PMC11086401

297. Shah S Liposomes: advancements and innovation in the manufacturing process Adv Drug Deliv Rev 2020 154–155 102 22 10.1016/j.addr.2020.07.002 32650041 Shah S, et al. Liposomes: advancements and innovation in the manufacturing process. Adv Drug Deliv Rev. 2020;154–155:102–22. 32650041 10.1016/j.addr.2020.07.002

298. Eygeris Y Chemistry of lipid nanoparticles for RNA delivery Acc Chem Res 2022 55 1 2 12 10.1021/acs.accounts.1c00544 34850635 Eygeris Y, et al. Chemistry of lipid nanoparticles for RNA delivery. Acc Chem Res. 2022;55(1):2–12. 34850635 10.1021/acs.accounts.1c00544

299. Song N Ferritin: a multifunctional nanoplatform for biological detection, imaging diagnosis, and drug delivery Acc Chem Res 2021 54 17 3313 25 10.1021/acs.accounts.1c00267 34415728 Song N, et al. Ferritin: a multifunctional nanoplatform for biological detection, imaging diagnosis, and drug delivery. Acc Chem Res. 2021;54(17):3313–25. 34415728 10.1021/acs.accounts.1c00267

300. Nooraei S Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers J Nanobiotechnol 2021 19 1 59 10.1186/s12951-021-00806-7 PMC7905985 33632278 Nooraei S, et al. Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers. J Nanobiotechnol. 2021;19(1):59. 10.1186/s12951-021-00806-7 PMC7905985 33632278

301. Kesharwani P Gold nanoparticles and gold nanorods in the landscape of cancer therapy Mol Cancer 2023 22 1 98 10.1186/s12943-023-01798-8 37344887 PMC10283337 Kesharwani P, et al. Gold nanoparticles and gold nanorods in the landscape of cancer therapy. Mol Cancer. 2023;22(1):98. 37344887 10.1186/s12943-023-01798-8 PMC10283337

302. Dadfar SM Iron oxide nanoparticles: diagnostic, therapeutic and theranostic applications Adv Drug Deliv Rev 2019 138 302 25 10.1016/j.addr.2019.01.005 30639256 PMC7115878 Dadfar SM, et al. Iron oxide nanoparticles: diagnostic, therapeutic and theranostic applications. Adv Drug Deliv Rev. 2019;138:302–25. 30639256 10.1016/j.addr.2019.01.005 PMC7115878

303. Wickline SA Hou KK Pan H Peptide-based nanoparticles for systemic extrahepatic delivery of therapeutic nucleotides Int J Mol Sci 2023 10.3390/ijms24119455 37298407 PMC10253970 Wickline SA, Hou KK, Pan H. Peptide-based nanoparticles for systemic extrahepatic delivery of therapeutic nucleotides. Int J Mol Sci. 2023. 10.3390/ijms24119455. 37298407 10.3390/ijms24119455 PMC10253970

304. Rajana N Multifunctional hybrid nanoparticles in diagnosis and therapy of breast cancer J Control Release 2022 352 1024 47 10.1016/j.jconrel.2022.11.009 36379278 Rajana N, et al. Multifunctional hybrid nanoparticles in diagnosis and therapy of breast cancer. J Control Release. 2022;352:1024–47. 36379278 10.1016/j.jconrel.2022.11.009

305. Fang RH Gao W Zhang L Targeting drugs to tumours using cell membrane-coated nanoparticles Nat Rev Clin Oncol 2023 20 1 33 48 10.1038/s41571-022-00699-x 36307534 Fang RH, Gao W, Zhang L. Targeting drugs to tumours using cell membrane-coated nanoparticles. Nat Rev Clin Oncol. 2023;20(1):33–48. 36307534 10.1038/s41571-022-00699-x

306. Liu X Xiao C Xiao K Engineered extracellular vesicles-like biomimetic nanoparticles as an emerging platform for targeted cancer therapy J Nanobiotechnol 2023 21 1 287 10.1186/s12951-023-02064-1 PMC10463632 37608298 Liu X, Xiao C, Xiao K. Engineered extracellular vesicles-like biomimetic nanoparticles as an emerging platform for targeted cancer therapy. J Nanobiotechnol. 2023;21(1):287. 10.1186/s12951-023-02064-1 PMC10463632 37608298

307. Parayath NN In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo Nat Commun 2020 11 1 6080 10.1038/s41467-020-19486-2 33247092 PMC7695830 Parayath NN, et al. In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo. Nat Commun. 2020;11(1):6080. 33247092 10.1038/s41467-020-19486-2 PMC7695830

308. Zhao X Imidazole-based synthetic lipidoids for in vivo mRNA delivery into primary T lymphocytes Angew Chem Int Ed Engl 2020 59 45 20083 9 10.1002/anie.202008082 32662132 Zhao X, et al. Imidazole-based synthetic lipidoids for in vivo mRNA delivery into primary T lymphocytes. Angew Chem Int Ed Engl. 2020;59(45):20083–9. 32662132 10.1002/anie.202008082

309. Kumar ARK Materials Improving Immune Cell Transfection Adv Mater 2021 33 21 e2007421 33860598 10.1002/adma.202007421 Kumar ARK, et al. Materials Improving Immune Cell Transfection Adv Mater. 2021;33(21):e2007421. 33860598 10.1002/adma.202007421

310. Irving M Choosing the right tool for genetic engineering: clinical lessons from chimeric antigen Receptor-T cells Hum Gene Ther 2021 32 19–20 1044 58 10.1089/hum.2021.173 34662233 PMC8697565 Irving M, et al. Choosing the right tool for genetic engineering: clinical lessons from chimeric antigen Receptor-T cells. Hum Gene Ther. 2021;32(19–20):1044–58. 34662233 10.1089/hum.2021.173 PMC8697565

311. Lukjanov V Koutná I Šimara P CAR T-Cell production using nonviral approaches J Immunol Res 2021 2021 p6644685 10.1155/2021/6644685 PMC8019376 33855089 Lukjanov V, Koutná I, Šimara P. CAR T-Cell production using nonviral approaches. J Immunol Res. 2021;2021:p6644685. 10.1155/2021/6644685 PMC8019376 33855089