Redox-responsive palbociclib-loaded self-assembled protein nanoparticles in breast cancer therapy—quality by design approach and efficacy assessment in 2D and 3D <i>in vitro</i> culture systems

Redox-responsive nanocarriers exploit the unique intracellular redox environment to enable targeted and controlled release of therapeutic agents. The present study comprises the fabrication of Palbociclib-bearing Bovine serum albumin-based redox-responsive nanoparticles (RR-PAL-NPs) through self-crosslinking using the desolvation technique. The optimisation of the formulation was achieved using the Box-Behnken design of experiment. The RR-PAL-NPs particle size, PDI, zeta potential, and percentage entrapment efficiency are 195.56 ± 9.99 nm, 0.22 ± 0.01, 3.9 ± 0.12 mV, and 91.23 ± 3.8%, respectively. In the presence of 10 mM glutathione, at pH of 5.5 and 7.4, nearly all the PAL was released from RR-PAL-NPs, making a release of 88.98 ± 3.00 and 82.41 ± 3.9%, respectively. Furthermore, dilution study, turbidimetric analysis, UV and spectrofluorescence analysis, circular dichroism, stability study, pH titration, and hemocompatibility study were also performed, which deemed the formulation satisfactory. RR-PAL-NPs depicted an exceptional <i>in vitro</i> cytotoxic and apoptotic profile. An outstanding mitochondrial depolarisation, ROS generation, inhibition of colony formation, wound healing, and glutathione depletion were seen. In the 3D mammosphere study, it caused a 3.57-fold reduction in the 3D mammosphere diameter as compared to PAL, along with higher apoptosis and uptake efficiency. Immunofluorescence assays depicted an upregulation of BAX and downregulation of Bcl2 protein expression. In the cell cycle study, the RR-PAL-NPs showed remarkable G0/G1 phase arrest due to the decreased phosphorylation of retinoblastoma protein.