FOLATE-FUNCTIONALIZED LIPOSOMES CO-ENCAPSULATING DOXORUBICIN AND CURCUMIN FOR POTENTIAL APPLICATION IN CANINE MAMMARY CANCER

Canine mammary cancer is one of the most prevalent neoplasms in veterinary oncology, accounting for 50–70% of tumors diagnosed in intact female dogs and representing a major therapeutic challenge due to its metastatic potential. Although doxorubicin (DOX) is widely used in treatment, its clinical application is limited by severe adverse effects, particularly cumulative cardiotoxicity. The natural polyphenol curcumin (CUR) has demonstrated promising antitumor activity; however, its poor aqueous solubility and low bioavailability restrict its therapeutic efficacy. Liposomes have emerged as attractive nanocarriers capable of improving drug stability, reducing systemic toxicity, and enhancing therapeutic outcomes. Furthermore, active targeting strategies may increase treatment specificity. In this context, folic acid is of particular interest due to the overexpression of folate receptor alpha (FR-α) in mammary tumor cells. This study aimed to develop and characterize DSPE-PEG2000-Folate- functionalized liposomes co-loaded with DOX and CUR for targeted canine mammary cancer therapy. Liposomal formulations were prepared and characterized by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), Proton Nuclear Magnetic Resonance (1H-NMR), and Cryogenic Electron Microscopy (Cryo-EM). Particle size, polydispersity index (PDI), zeta potential (ZP), particle concentration, morphology, encapsulation efficiency, and DSPE-PEG2000-Folate conjugation were evaluated. Drug release was investigated using Franz diffusion cells, and stability was assessed during refrigerated storage (2–8 °C). 1H-NMR confirmed the successful conjugation of folic acid to DSPE-PEG2000, then used to functionalize the liposomes. Liposomes exhibited mean diameters ranging from 350 to 400 nm, low polydispersity (PDI ~ 0.3), particle concentrations on the order of 10¹² particles/mL, and negative ZP (−17 to −19 mV). Cryo-EM analysis revealed spherical vesicles in which DOX encapsulation efficiency reached 78%. The formulations remained stable for at least 150 days under refrigerated conditions. In vitro release studies demonstrated sustained drug release, (approximately 90%, after 48 h), whereas free DOX was completely released within 4 h. These findings demonstrate that folate-targeted liposomes possess suitable physicochemical properties, prolonged stability, and controlled drug-release behavior, highlighting their potential as a targeted nanotherapeutic platform for canine mammary cancer treatment.