Anti-EGFR liposomal drug delivery system loaded with AGY2 potentiates the anti-cancer effect of AGY2 against EGFR expressed pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies worldwide due to late diagnosis, dense stromal barriers, and resistance to existing chemotherapeutics. To address these limitations, we developed a targeted liposomal delivery system that encapsulates AGY2, a newly synthesized, more potent fluoro pyrimidine nucleoside analog. In this study, epidermal growth factor receptor (EGFR) functionalized liposomal nanoparticles (EGFR-AGY2 LNPs) were optimized using Box-Behnken Design to achieve superior physicochemical stability. The optimized formulation demonstrated nanoscale dimensions (∼121 nm), high entrapment efficiency (∼75%), and strong colloidal stability. Surface antibody conjugation was confirmed by Fourier Transform Infrared Analysis (FTIR) and immunofluorescence analysis, demonstrating successful receptor targeting. The drug release profile best fits the Weibull kinetic model, suggesting sustained release behavior. In-vitro evaluations in 3D pancreatic cancer spheroids and organoids revealed significantly enhanced cytotoxicity. In spheroids, the IC₅₀ values of EGFR-AGY2 LNPs (7.7 ± 2.3 μM in MiaPaCa-2 and 9.63 ± 0.9 μM in PANC-1 cells) compared to free AGY2 (27.58 ± 4.2 μM and 31.51 ± 5.5 μM), non-targeted AGY2 LNPs (16.53 ± 3.7 μM in MiaPaCa-2 and 18.53 ± 5.7 μM in PANC-1), and 5-fluorouracil (36.63 ± 4.8 μM MiaPaCa-2 and 40.78 ± 5.9 μM PANC-1). Similar trend was observed in organoids with highest anti-cancer activity EGFR-AGY2 LNPs (IC₅₀ 10.83 ± 3.3 μM in MiaPaCa-2 and 12.2 ± 3.9 μM in PANC-1) both significantly better than 5-FU (p = 0.0001), Non-targeted AGY2 LNPs (25.09 ± 3.9 μM and 29.09 ± 4.9 μM in MiaPaCa-2 and PANC-1), and free AGY2 achieved IC₅₀ values of 35.18 ± 3.7 μM and 32.56 ± 5.1 μM. Pharmacokinetic analysis showed substantial improvement over 5-FU and non-targeted AGY2 LNPs. The elimination rate constant (k10) decreased from 1.82 h-1 (5-FU) to 0.39 h-1 (EGFR-AGY2 LNPs), while t1/2 increased from 0.4 to 1.8 h. Systemic clearance dropped from 259.6 to 21.3 mL/h, and AUC rose >12-fold (from 2.08 to 25.32 μg*h/mL). In vivo antitumor efficacy studies showed that EGFR-AGY2 LNPs reduced mean tumor volume to ∼900 mm3 compared with 1200 mm3 (AGY2 LNPs), 1500 mm3 (free AGY2), and 2200 mm3 (5-FU), p < 0.001. No significant change in body weight was observed, indicating minimal systemic toxicity. Collectively, these results provide strong evidence that EGFR-targeted delivery of AGY2 offers a promising therapeutic platform capable of overcoming drug delivery barriers and improving outcomes in PDAC therapy.