Synthesis And Evaluation Of Redox Sensitive Dual Crosslinked Hyaluronic AcidDrug-loaded Nanoparticles

Cancer is one of the most serious diseases endangering human health.Cure of cancer has always been a research topic in the world.Chemotherapy is one of the important means of cancer treatment.However,the use of large dosage of anticancer drugslacking of specific pharmacological activity not only kill cancer cells but also normal cells,which leads to the therapeutic effects as well as serious side effects.The"target nano drug delivery system"concept provides a new way to solve the common problems of chemotherapy drugs.Target nano drug delivery system has a broad application prospect in the aspects of improving the curative effect of chemotherapy drugs and reducing the toxicity and side effect,and is expected to be a new way to cure cancer.Glutathione sensitive crosslinked nano micelle drug delivery system can keep their stable structure in the circulation and avoid or reduce drug leakage.When the drug delivery system is uptaked by cancer cells through the EPR effect andactive target,they can quickly release the loaded drug in the environment of high concentration of glutathione.Thus,this drug delivery system may achieve the purpose of targeted therapy,andhas been widely studied.This studydeveloped a bioreducible shell and core crosslinked nanocarriers which has good circulation stability andcan target to cancer cells.Thehydrophobic core formed of 4-Azido-benzenemethanaminewas crosslinked under UV irradiation,and the hydrophilic shell formed of thiolated hyaluronic acid was crosslinked by bio-reducible disulfide bonds.The crosslinked nanoparticles could enhance the stability of drug loaded nanoparticles in thecirculation,and the loaded drug could bequickly released after the drug loaded nanoparticles entered cancer cells and reach the goal of targeted therapy.The main research contents and conclusions of this study are as follows:?1?4-Azido-benzenemethanamine and 2-?Pyridyldithio?-ethylaminewere grafted onto hyaluronic acid chain.To evaluate the influence of the feed ratio of BnN₃ and PDA to HAontheir degrees of substitution,we synthesized a series of nanoparticles with different feed ratio of BnN₃ and PDA to HA.The prepared amphiphilic copolymer self assembled into non-crosslinked nanoparticles in aqueous solution.Then,the core of the nanoparticles was crosslinked under UV irradiation,and the shell of the nanoparticles was crosslinked by adding a catalytic amount of dithiothreitol to obtainthe core-shell crosslinked nanoparticles.The chemical struc-tures of the copolymers werecharacterized by nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy.Experimental results showed that we successfully synthesized a core-shell crosslinked nanoparticle.The grafting rate of 2-?Pyridyldithio?-ethylamine and4-Azido-benzenemethanamineboth increased along with their feed ratio under low feed ratio.However,when the feed ratio is greater than 0.5:1,their grafting rate no longer increased.?2?The hydrodynamic diameter,size distribution and Zeta potentialof the nano-particles were measured by dynamic laser light scattering.The morphology of the nanoparticles was observed with a transmission electron microscopy.We investigated the influence of the grafting rate of 4-Azido-benzenemethanamine and2-?Pyridyldithio?-ethylamine on the nanoparticles'size.The stability of core-shellcrosslinked nanoparticles in the serum and the buffer solutions containing different concentrations of glutathione were investigated.The methotrexate loaded core-shell crosslinked nanoparticles were prepared,and thedrug release behavior of the drug loaded nanoparticles in the buffer solutions containing different concentrations of glutathione was investigated.The experimental results showed that the structure of the non-crosslinked nanoparticles is loose with large size.The core crosslinkednanoparticles'size decreased with a tighter structure,and the size of the core-shell crosslinked nanoparticles further reduce and their structure was denserand more regular.The core-shellcrosslinked nanoparticles can maintain the structure stability in serum and buffer solutions containing low concentration of glutathione.However,thenanoparticles'size increased significantly in the buffer solutions containing high concentration of glutathione,indicating that the core-shellcrosslinked nanoparticles had good response ability to reduction,and that the core-shell crosslinked nanoparticles has good stability in the blood circulation.The loading and encapsulation efficienciesof methotrexate loaded core-shell crosslinked nanoparticles were 13.98%and 81.24%,respectively.The results of the in vitro re-lease behaviors of the drug-loaded nanoparticles showed that less than 13%of loaded methotrexate was released in buffer solutions containing low concentration of glutathione,while 80%of loaded methotrexatewas released in buffer solutions containing high concentration of glutathione,suggesting that the redox-sensitive shell and core crosslinked nanoparticles were likely to be stable in the plasma without releasing the hydrophobic drug after intravenous administration,whereas rapid release of the drug could be achieved in tumor cells.?3?we evaluated the in vitro cytotoxicity of the blank core-shell crosslinked nanoparticlesand methotrexate loaded core-shell crosslinked nanoparticles in HeLa cells using CCK-8 assays.The uptake behavior of the fluorescent labeled core-shell crosslinked nanoparticles by HeLa cells wasexamined using a laser scanning confocal fluorescence microscopy.The influence of high concentration of hyaluronic acid in the solution on cytotoxicity and cellular uptake behavior were also measured.The experimental results showed that the cytotoxicity of blank core-shell crosslinked nanoparticles is very low,which showed that the core-shell crosslinked nanoparticles has good biocompatibility.Significant cell death was observed when the cells were treated with free methotrexate and methotrexate loaded core-shell crosslinked nano-particles,and the cytotoxicity of the methotrexate loaded core-shell crosslinked na-noparticles was comparable to that of free methotrexate at the same methotrexate concentration.However,when the HeLa cells were pretreated with a high dose of free hyaluronic acid to block CD44 before culture with methotrexate loaded core-shell crosslinked nanoparticles,cell viability was significantly higher.Strong fluorescent signals were detected in the cytoplasm,indicating that the core-shell crosslinked na-noparticles were readily taken up by HeLa cells.In contrast,intracellular fluorescence was very weak when the HeLa cells were pretreated with a high dose of freehyalu-ronic acid to block CD44 before treatment with Nile red-labeled HA-ss-cNPs,suggesting a lack of cellular uptake of the core-shell crosslinked nanoparticles.These in vitro cellular uptake results were consistent with that of cytotoxicity studies and suggested that the core-shell crosslinked nanoparticles prepared in this study could be internalized into HeLa cells possibly via receptor-mediated endocytosis,displaying great potential as a platform for antitumor drug delivery.