Studies On PH-responsive Glycolipid-like Nanocarrier For Improving The Focal Intracellular Drug Release

With the development of nanotechnology, drug delivery systems have achieved accumulation at the tumor site by the passive targeting of the EPR effect and active targeting. Although this has brought about new alternative treatments for cancer therapy, the therapeutic effects are far from perfect. The unsatisfactory results are mainly due to the limited drug release of the nanocarriers. Only when the nanocarriers release the drug in tumor cells can the drug interact with its target sites and exert pharmacological activities. Therefore, we proposed a chitosan-based glycolipid-like polymer that included a Schiff-base bond, which has a fast degradation rate in acidic environments. So the therapeutic efficacy was improved with the rapid released doxorubicin and raised intracellular concentration of drug.4-formylbenzoic acid-linked stearic acid grafted chitosan (CSO-FBA-SA) was fabricated by conjugating stearyl alcohol (SA) to chitosan (CSO) with the linkage of 4-formylbenzoic acid (FBA). The structures of the intermediate and the final product were confirmed by the 1H-NMR. With stearyl alcohol acting as the hydrophobic core of CSO-FBA-SA, and whose degrees of amino-substitution were 7.6%, the chitosan based glycolipid-like polymer could self-assemble into micelles, which had a lower critical micelle concentration (CMC) compared with the low molecular weight surfactants. The CMC value of the CSO-FBA-SA was measured as 194.9μg/mL according to the inflection point for a sharp decrease. The dynamic light scattering (DLS) measurement was taken to determine the hydrodynamic size and zeta potential of the CSO-FBA-SA. Furthermore, the CSO-FBA-SA was characterized by transmission electron microscopy (TEM), and the spherical morphologies were confirmed. The Encapsulation Efficiency of the CSO-FBA-SA/DOX attained 73.2%. And the CSO-FBA-SA/DOX have a better releasing ability in the acidic environment compared with the CSO-SA/DOX.Intracellular drug transport was indicated by the drug internalization of the MCF-7 and SKOV-3 cells and imaged by a confocal laser-scanning microscope (CLSM). And CSO-FBA-SA/DOX had a time-dependent increase in internalization into the two tumor cell lines. Nile Red was applied as the model drug for monitoring the behavior of the intracellular drug release. When the MCF-7 and SKOV-3 cells were incubated with the CSO-FBA-SA/NR, the red fluorescence increased with time, and the fluorescence intensity was higher than that in the cells treated with the CSO-SA/NR within 24 h in our observation. In comparison with the CSO-SA/DOX and DOX, the cytotoxicity of the CSO-FBA-SA/DOX increased by 3.75-fold and 4.77-fold, respectively, due to the fast release of DOX. The curves described the free DOX accumulation in the tumor cells relative to time, indicated a time-dependent change of the drugs. Free DOX accumulation increased as time progressed over 24 h in the CSO-FBA-SA/DOX, CSO-SA/DOX and DOX groups, and the CSO-FBA-SA/DOX had a higher accumulation than the CSO-SA/DOX and DOX in 24 h, namely having larger Areas under the Curve. The free DOX accumulation was attributed to the internalization of the nanocarrier and the intracellular release of the drug.In addition, the CSO-FBA-SA showed rapid intra-tumor drug release, which confers significant advantages over CSO-SA. The CSO-FBA-SA was stable in the normal pH environment, so little drug was observed in the liver and heart. The results revealed that the drug-loaded CSO-FBA-SA had relatively minimal cytotoxicity towards normal tissues. Moreover, as the major side effect of Adriamycin, cardiotoxicity, was further explored in the H&E-stained cross-sections. Mice treated with the CSO-FBA-SA/DOX showed the absence of cardiotoxicity, which was similar to the saline-treated mice.In vivo efficacy in the MCF-7 mice models showed that the group treated with the CSO-SA/DOX had a tumor inhibition rate of 60.9%. The CSO-FBA-SA/DOX and Adriamycin could decrease the growth of the tumor in the nude mice, and the difference was not statistically significant between the two groups. The tumor inhibition values of the mice injected with the CSO-FBA-SA/DOX and Adriamycin were 70.1% and 74.2%, respectively. The CSO-FBA-SA/DOX could selectively respond to the acidic environment and release DOX in tumor only, which had relatively minimal cytotoxicity towards normal tissues. So the newly developed carrier might serve as an effective and potential platform with low systemic toxicity for cancer therapy.