HPLC METHOD DEVELOPMENT AND VALIDATION FOR METFORMIN AND PHENYL BUTYRIC ACID AND IN VITRO EVALUATION OF THEIR PEGYLATED LIPOSOMAL FORMULATION AGAINST COLORECTAL CANCER

Objective: This study aimed to develop a PEGylated liposomal formulation combining Metformin (MET) and Phenyl Butyric Acid (PBA) for colorectal cancer treatment. Current colorectal cancer therapies face limitations such as poor drug solubility, rapid clearance, and systemic toxicity. Combining MET and PBA in liposomes could potentially overcome these limitations by enhancing drug stability, prolonging circulation time, and improving targeted delivery. Methods: A High-Performance Liquid Chromatography (HPLC) method was optimized and validated for the simultaneous quantification of MET and PBA, achieving calibration curve R² values above 0.99. Liposomal formulations were prepared, and encapsulation efficiencies were evaluated. Sustained drug release was studied over 72 h at 37 °C. Cytotoxicity assays were conducted using colorectal cancer cell lines HCT116 and HT29. Additionally, colony formation assays assessed the formulation's long-term effects on cell proliferation. Results: The liposomal formulation showed encapsulation efficiencies of 48% for Lip-MET and 56.8% and 83.9% for Lip-MET-PBA. Sustained release profiles were observed, with 60% of MET and 55% of PBA released over 72 h. Cytotoxicity assays revealed that Lip-MET-PBA significantly reduced cell viability in HCT116 and HT29 cell lines, with IC50 values of 0.65 mmol and 0.82 mmol, respectively, compared to MET alone (1.1 mmol and 1.3 mmol). Colony formation assays demonstrated a concentration-dependent reduction in colony numbers, highlighting the enhanced anticancer efficacy of the combination. Statistical analysis showed significant differences (p<0.05) in cell viability and colony formation between the liposomal formulation and the free drugs. Conclusion: This is the first PEGylated liposomal formulation combining MET and PBA, offering a promising strategy for colorectal cancer therapy. The formulation combines sustained release, improved cytotoxicity, and a significant impact on colony formation, warranting further in vivo investigations.