A novel cyclophosphamide microemulsion exhibits enhanced efficacy: Formulation development, evaluation, in-vitro cytotoxicity and in-vivo efficacy studies.

This study presents a novel Cyclophosphamide (CYC) microemulsion designed to overcome limitations of conventional formulations, including poor aqueous solubility, instability, and rapid degradation. The microemulsion was developed using PEG-8 caprylic/capric glycerides (oil phase), Soluplus and Cremophor EL (surfactants), and PEG-400 (co-surfactant), optimized through pseudo-ternary phase diagrams (Smix 5:1). The final formulation achieved a globule size of ∼74 nm, polydispersity index of 0.072, and >99% drug entrapment, with 12-month physical and microbial stability. In- vitro cytotoxicity against MCF-7 breast cancer cells demonstrated enhanced potency (IC₅₀ = 25.0 µg/mL) compared to pure CYC (IC₅₀ = 43.6 µg/mL). Flow cytometry revealed superior cellular uptake (97.6% vs. 70.7%), likely due to increased membrane permeability and endocytosis driven by nanoscale droplet size and surfactant-mediated interaction. Controlled drug release over 6 hours and sterility confirmed formulation integrity for parenteral use. In- vivo, DMBA-induced breast cancer rats treated with CYC microemulsion exhibited excellent tumor regression and restored histological architecture, outperforming the conventional lyophilized CYC injection. These therapeutic outcomes are attributed to enhanced bioavailability, sustained release, and targeted intracellular delivery enabled by the nano-carrier system. This work mechanistically validates a rationally designed microemulsion platform for oncologic drug delivery, providing a clinically viable strategy to improve CYC efficacy while minimizing systemic toxicity. Future efforts will focus on scaling up production, detailed pharmacokinetic studies, and clinical trials to validate its translational potential. This work represents a significant advancement in leveraging nanocarrier systems for effective cancer treatment.