Enhancing the gastrointestinal stability of phycocyanin through modified PEGylation: Prospects for oral anticancer treatment.

Phycocyanin (PC), a pigment protein from Spirulina platensis, possesses potent anticancer properties but remains susceptible to rapid gastrointestinal proteolysis, severely compromising its oral efficacy. This study proposes a PEGylation-based strategy to synergistically enhance PC's gastrointestinal stability and preserve its therapeutic efficacy through conjugation with polyethylene glycol (PEG) chains ranging from 2 to 20 kDa. Systematic evaluation demonstrated that PEG molecular weight serves as a critical determinant in balancing digestive resistance and cellular uptake of the modified protein. Low-molecular-weight PEGs (2-5 kDa) conferred insufficient protection, whereas steric hindrance from 20 kDa PEG significantly impaired cellular interaction. Notably, 10 kDa PEG-conjugated PC (10kPEG-PC) maintained superior structural and functional integrity under simulated gastric and intestinal conditions. Confocal imaging confirmed the time-dependent internalization of peptides derived from digested 10kPEG-PC by Caco-2 cells, achieving intracellular accumulation parity with intact PC within 24 h. Functional validation through cellular assays confirmed retention of anticancer activity of these peptides, manifested as dose-dependent cell viability suppression, ROS-mediated oxidative stress amplification, and mitochondrial dysfunction induction. These findings highlight PEGylation-particularly with 10 kDa PEG-as a viable strategy to overcome the proteolytic vulnerability of natural bioactive proteins, thereby enabling the development of orally deliverable, protein-based therapeutics targeting intestinal cancers.