Exploring the Anticancer Potential of Chitosome-Encapsulated Theobromine Analogue: Integrated Computational, Physiochemical and <i>In vitro</i> Study

Background Theobromine derivatives have emerged as promising anticancer agents, but their therapeutic potential is limited by poor bioavailability. This study investigates a chitosome-based delivery system for a novel theobromine derivative, N -(4-Chlorophenyl)-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1 H -purin-1-yl)acetamide ( T-1-PCPA ), to evaluate its anticancer potential. Methods T-1-PCPA -loaded chitosomess ( T-1-PCPA loaded chitosomes) were formulated using the thin film hydration technique. Molecular docking was employed to computationally assess the stability of the T-1-PCPA loaded chitosomes. Experimentally, various physicochemical properties, including particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency, were analyzed. Transmission electron microscopy (TEM) was used to investigate morphological features. In vitro , the cytotoxicity of the T-1-PCPA -PC-CS was evaluated on HCT-116 and A549 cancer cell lines. Further mechanistic studies included cell cycle analysis, apoptosis detection, and cell migration assays were performed. Results The T-1-PCPA loaded chitosomes exhibited good colloidal stability, with well-defined spherical particles averaging 142.6 ± 4.3 nm in size, a zeta potential of +32.4 ± 1.6 mV, and an entrapment efficiency of 89.2 ± 2.1%, indicating efficient drug loading and formulation stability. In vitro cytotoxicity assays revealed significantly enhanced anticancer activity, with IC 50 values of 8.05 ± 0.06 μg/mL for HCT-116 and 9.55 ± 0.15 μg/mL for A549 cells—substantially lower than those of free T-1-PCPA and blank formulations. Flow cytometric cell cycle analysis demonstrated arrest at G0-G1 and S phases, while apoptosis analysis revealed a significant increase in total apoptosis (39.55%) in treated HCT-116 cells. Cell migration assays showed a 63.4% reduction in migration compared to control, supporting the formulation's anti-migratory potential. Conclusion T-1-PCPA -loaded chitosomes system shows strong potential as a targeted anticancer nanotherapy, demonstrating enhanced cytotoxicity, apoptosis induction, cell cycle arrest, and anti-migratory activity. With high drug loading, stability, and selectivity toward cancer cells, it addresses key limitations of conventional chemotherapy. These findings warrant further preclinical evaluation for colorectal and lung cancer treatment.