This research study aimed to enhance the therapeutic potential of the drug Doxorubicin (DX) by incorporating it into PL NPs and HyAc/PEG/PL NPs, (nanoparticle composed of hyaluronic acid, polyethylene glycol, and polycaprolactone) while optimizing their composition. The developed NPs were utilized to encapsulate DX and subsequently subjected different evaluation parameters including evaluation of surface morphology, DSC, XRD, entrapment efficiency), in vitro drug release, hemolytic toxicity, tissue distribution study, cell viability, and stability. The nanoparticles exhibited a spherical shape as observed through TEM examinations. The zeta potential measurements for HyAc/PEG/PL NPs and PL NPs were determined to be 16.4±0.84 mV and -4.9±0.3 mV, respectively, with corresponding PDI values of 0.648 and 0.553 for both formulations. The particle sizes of HyAc/PEG/PL NPs and PL NPs were found to be 268±3nm and 142±1.5nm, respectively. The NPs exhibited sustained release of DX, with DX-loaded HyAc/PEG/PL NPs releasing a significant amount of DX over a period of 96 hours, while DX-loaded PL NPs achieved nearly complete drug release within the same time frame. Evaluation of stability based on residual drug content indicated that the NPs formulations remained more stable at temperatures of 4±2°C and subsequently at 28±2°C. The hemolytic toxicity assay demonstrated the high hemocompatibility of DX-loaded HyAc/PEG/PL NPs. Moreover, biodistribution analysis revealed higher concentrations of DX in the colon and tumor when delivered using DX-loaded HyAc/PEG/PL NPs, suggesting enhanced targeting ability. Notably, due to the affinity of HyAc for over expressed CD44 receptors on HT-29 cells, the DX-loaded HyAc/PEG/PL NPs demonstrated superior cytotoxicity, indicating enhanced internalization of the formulated drug.