PH-Sensitive chitosan-tripolyphosphate nanoparticles increase doxorubicin-induced growth inhibition of cervical HeLa tumor cells by apoptosis and cell cycle modulation

Abstract

Delivery systems responsive to pH variations might allow the exploitation of the various pH gradients within the body, e.g. between healthy and tumor tissue, or between the extracellular space and some cell compartments. In previous studies, we designed doxorubicin-loaded pH-responsive chitosan-tripolyphosphate nanoparticles (DOX-CS-NPs) and also performed an extensive in vitro study evidencing its notable antiproliferative activity against different tumor cells. Here, we focus on the understanding of the mechanisms underlying the improved in vitro antitumor activity of these NPs, using experimental conditions simulating both the physiological environments (pH 7.4) and the extracellular space of tumors (pHe 6.6). CS-NPs were obtained by ionotropic gelation method, using the surfactant 77KS, derived from the amino acid lysine, as a pH-sensitive adjuvant. The apoptotic effects on HeLa tumor cells was analyzed by annexin V-FITC quantification using flow cytometry. Likewise, the modulation of the cell cycle and the NP cell uptake rate were assessed by flow cytometry. pH-Responsive NPs augmented DOX cytotoxicity by increasing the number of apoptosis events, thus causing cell cycle arrest in the G2/M or S phase. The apoptotic effects were notably more evident at pH 6.6. It was also demonstrated that DOX-CS-NPs were internalized by HeLa cells in a greater extent than the non-associated drug, especially at pH 6.6. It was proven that the combined physicochemical and pH-responsive properties of CS-NPs allowed an enhanced DOX cell internalization in a tumor cell model, allowing the entrapped drug to induce greater cell cycle arrest and apoptotic effects.