Construction And Characterization Of Alginate-based Acid Sensitive Polymeric Prodrug Nanocarriers For Targeted Hepatocellular Carcinoma Cells

Hepatocellular carcinoma has caused serious harm to human health and life safety.Chemotherapy is still an indispensable treatment method in the clinical treatment of hepatocellular carcinoma,but the toxic and side effects of chemotherapy drugs have limited its clinical application.With the interdisciplinary development of medicine,materials chemistry and other disciplines,intelligent nanometer drug delivery systems based on nanotechnology have been widely developed.Using chemical synthesis design and self-assembled nanotechnology,from the perspective of pharmacy,is designed to targeted deliver drugs that are stable in the blood circulation and respond to drug release in the tumor cell microenvironment.It is of great significance to improve the bioavailability of chemotherapeutics and reduce serious side effects.In order to improve the safety and therapeutic effect of chemotherapeutic drugs,we designed and synthesised an active-targeting amphiphilic doxorubicin polymeric prodrug(ASA-DOX with mannose-targeted ability and Bio-ASA-DOX with biotin/mannose dual-targeted ability)based on the natural polysaccharide sodium alginate derivative as drug carrier,and prepared acid-responsive polymeric prodrug nanosystem in process of the solution self-assembly.The main structure and content of this paper include the following sections.ASA-DOX and Bio-ASA-DOX polymeric prodrugs,ASA-DOX and Bio-ASA-DOX nanosystems was prepared.Its chemical structure was characterized by FT-IR and ~1H-NMR.The nanosystem was prepared by the solution self-assembly method,the particle size was measured by dynamic light scattering method(DLS),the nano-morphology was inspected by transmission electron microscope(TEM),the drug loading was determined by ultraviolet and visible spectrophotometry(UV-Vis),and the drug release performance was measured by dynamic diffusion.The results showed the successful synthesis of polymeric prodrugs and the preparation of ASA-DOX and Bio-ASA-DOX nanosystem with an irregular spherical shape and a uniform size.The average particle sizes of ASA-DOX and Bio-ASA-DOX systems was 97.3 nm and 108.7 nm,showing a single-peak normal distribution.The zeta potential values were-21.9 mv and-27.3 mv,respectively,both of which was irregular and spherical.Both of nanosystem maintained stable performance over the past 5 days.Its drug loading was 11.0±1.1%,and the encapsulation rate was 62.2±0.3%of Bio-ASA-DOX nanosystem.Drug loading of ASA-DOX nanosystem was11.3±1.8%,and the encapsulation rate was 56.7±0.4%.The drug release rate and cumulative release amount under simulated cancer cell environment(pH 5.0)was significantly higher than the simulated blood environment(pH 7.4).It showed acid-sensitive drug release properties in both of ASA-DOX and Bio-ASA-DOX.The biocompatibility of the ASA-DOX and Bio-ASA-DOX nanosystems was evaluated.Hemolysis experiment was employed to study its blood compatibility,BSA adsorption experiment was used to research its mutual adsorption with proteins,and MTT assay was performed to detect its cytotoxicity to human umbilical vein endothelial cells HUVEC,human normal liver cells L02 and mouse normal myocardial cells H9c2.The results showed that the hemolysis rates of the ASA-DOX and Bio-ASA-DOX nanosystems were lower than that of free doxorubicin,and no hemolysis occurred at the highest concentration.The blood compatibility of ASA-DOX and Bio-ASA-DOX was favourable.Compared with free doxorubicin,they also had a lower protein adsorption rate,indicating that it can significantly reduce the protein adsorption capacity of DOX.At the same time,compared with doxorubicin,it demonstrated higher survival rate on HUVEC,L02 and H9c2 cells after incubation with ASA-DOX and Bio-ASA-DOX,which means that they can significantly inhibit the cytotoxicity of DOX and reduce liver toxicity and myocardial toxicity.The anti-proliferation and uptake of hepatocellular carcinoma cells were evaluated in vitro.Using hepatocellular carcinoma cells HepG2,SMMC-7721,and Bel-7402 as test cell lines,MTT assay was used to determine the proliferation of hepatocellular carcinoma cells after treated with the ASA-DOX and Bio-ASA-DOX nanosystem.Fluorescence microscope was performed to observe the uptake ability of hepatoma cells to the nanosystem.The results showed that both of the ASA-DOX and Bio-ASA-DOX nanosystem could effectively inhibit the proliferation of hepatocellular carcinoma cells,and showed a higher inhibition rate with the increase of time and concentration,As time and concentration increase,it displayed a higher inhibition rate,and it can be seen that it demonstrated sustained release effect.Compared with ASA-DOX,Bio-ASA-DOX showed higher inhibition rate on liver cancer cells.The results displayed that targeted ASA-DOX with mannose showed good anti-liver cancer effect in vitro,while dual targeted Bio-ASA-DOX with biotin/mannose demonstrated better anti-liver cancer effect.It was further found that over time,the ASA-DOX polymeric prodrug nanosystem can be effectively absorbed by liver cancer cells,but Bio-ASA-DOX showed a higher ability to absorb liver cancer cells,and it was clearly observed that it entered the cellular nuclear.In this study,the amphiphilic alginate-doxorubic acid-sensitive polymeric prodrug ASA-DOX and Bio-ASA-DOX nanosystems were successfully synthesized and prepared.The preparation process of the two is simple and convenient,and has the advantages of high drug loading,controllable particle size,good stability and biocompatibility,acid-sensitive drug release performance,etc.It has good anti-liver cancer effect in vitro.Bio-ASA-DOX acid-sensitive polymeric prodrug nanosystem is expected to be used as an intelligent drug delivery system for precise liver cancer cell targeting.