Abstract
The therapeutic potential of biotherapeutics and natural compounds in cancer treatment is increasingly attracting interest due to their specificity, safety profiles, and ability to overcome resistance linked to traditional chemotherapeutics. Trastuzumab (Tmab), a monoclonal antibody (mAb), has shown remarkable success in treating HER2+ breast cancer (BC), while natural compounds such as betulinic acid (BA) and taraxerol (TA) have demonstrated significant anticancer effects across various malignancies. However, translating these agents into clinical use often faces challenges due to their physicochemical limitations, including low water solubility, poor stability, and limited bioavailability, which reduce their effectiveness and hinder clinical application. To overcome these issues, this study investigated the encapsulation of Tmab, BA, and TA within biodegradable and biocompatible polyester-based carriers, specifically poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL), because of their advantageous degradation profiles, ability to protect sensitive therapeutic agents, and suitability for drug delivery. Encapsulation was carried out using single and double emulsion techniques, resulting in nanocarriers with improved physicochemical properties, sustained release capabilities, and better therapeutic efficacy compared to their free forms.
The PLGA and PCL nanoparticles (NPs) encapsulating BA and TA, as well as PLGA NPs encapsulating Tmab, displayed hydrodynamic particle sizes within the nanoscale range, measured by dynamic light scattering (DLS). Their low polydispersity index (PDI) values confirmed narrow and uniform particle size distributions, indicating well-dispersed NP systems. Zeta potential measurements showed a favorable surface charge, supporting good colloidal stability. Fourier-transform infrared spectroscopy (FTIR) analysis verified the presence of polymeric functional groups and encapsulated agents (BA, TA, and Tmab), while also indicating no interactions between the polymers (PLGA/PCL) and payloads. X-ray diffraction (XRD) patterns revealed predominantly amorphous characteristics, suggesting enhanced solubility and improved potential bioavailability of the incorporated compounds. Nanoparticle morphology examined via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) showed spherical, uniformly sized particles. Encapsulation efficiencies were optimized to ensure sufficient loading of BA, TA, and Tmab. Notably, the double-emulsion formulation preserved the secondary
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structure and biological function of Tmab. All NP formulations demonstrated sustained release profiles, enabling controlled delivery of BA, TA, and Tmab over extended periods.
The MTT assay showed increased cytotoxicity of the encapsulated BA and TA formulations compared to their free forms across various cancer cell lines, indicating enhanced cellular uptake and activity. Meanwhile, the encapsulated Tmab maintained its specificity for HER2-overexpressing cells and exhibited a significant reduction in cellular viability, comparable to or better than the free antibody. These results highlight the strong potential of nanocarrier-based delivery systems for the effective and targeted treatment of cancers, especially those resistant to traditional therapies. Additionally, it provides a foundation for future preclinical and clinical development of such formulations, offering a promising approach for creating highly effective, stable, and targeted nanotherapeutics for cancer treatment.