Preparation Of Amphiphilic Micelles Of Hyaluronic Acid-Poly (Lactic-Co-Glycolic Acid) And Its Application In Anti-Tumor Drug Carriers

In recent years,with the improvement of chemical industry,the incidence of cancer in China has been increasing year by year.So far,the development of cancer has seriously threatened the well-being index of the nation,and cancer,as a major problem in the world,has become a major research hotspot for the whole society.Drug delivery system could solve the problem of damage to normal tissues and organs of human body by traditional drug formulation,protect the drug from early degradation and improve the drug utilization and therapeutic effect,so it is widely concerned by experts.Compared with other carrier materials,micelles could significantly improve the drug solubility,prolong blood circulation time,increase EPR accumulation,significantly improve the effect of anti-cancer drug action,and have great potential in cancer treatment.Based on this,studies have been carried out successively for the microenvironment of tumor sites.The intracellular p H of tumors is about 5.4,and the concentration of reduced glutathione is much higher in tumor cells than in normal tissues.Therefore,we constructed an amphiphilic hyaluronic acid-poly（lactic co-glycolic acid）micelle with hyaluronic acid as the targeting ligand（which could actively target to CD44 receptor that highly expressed in tumor cells）,and the drug can be released by stimulation in tumor microenvironment（p H 5.4,high GSH content）.The amphiphilic hyaluronic acid-poly（lactic acid-hydroxyacetic acid）micelles were constructed to achieve active tumor targeting and selective drug release from the carrier.In this work,HA-SS-PLGA copolymer（reduction-sensitive type）was prepared by using hyaluronic acid（HA）with good biocompatibility as the hydrophilic end,poly（lactic co-glycolic acid）（PLGA）as the hydrophobic end,and cystine（SS）as the bridge,which could self-assemble in aqueous solution to form amphiphilic micelles with the hydrophilic end outside and the hydrophobic end inside.The carriers were characterized using Fourier transform infrared spectroscopy（FTIR）and Nuclear magnetic resonance hydrogen spectroscopy（¹H NMR）,and the results indicated the successful synthesis of HA-SS-PLGA.The morphology and size structure were characterized by laser particle size measurement,Transmission electron microscopy（TEM）and Scanning electron microscopy（SEM）,and the results showed that the prepared micelles were spherical particles with a particle size of about 151.1 nm and homogeneous particle size.The critical micelle concentration was determined by surface tension method,and the critical micelle concentration of HA-SS-PLGA was 0.0559 mg·m L^-1,which had good micelle formation ability.As a control,the non-reduction-sensitive micelles HA-EN-PLGA were also prepared as a control group with ethylenediamine as a bridge,and their reduction sensitivity was tested.The results showed that both HA-SS-PLGA and HA-EN-PLGA were p H-sensitive,and the particle size of micelles changed greatly under acidic conditions,indicating that the morphology was partially disrupted;in p H 5.4 PBS solution with 20 m M GSH,only the disulfide bonds in HA-SS-PLGA micelles were disrupted and the particle size changed greatly,confirmed that HA-SS-PLGA with great reduction sensitivity.Curcumin（Cur）is an active substance extracted from Curcuma longa,a plant of the ginger family,Cur has various pharmacological effects such as anticancer,anti-HIV,and antibacterial.In this study,curcumin was used as a model drug,Cur-HA-SS-PLGA（a reduction-sensitive micelle）and non-reduction-sensitive micelle（Cur-HA-EN-PLGA）were prepared by dialysis,respectively.The drug loading and encapsulation rates of Cur-HA-SS-PLGA were 28.1%and70.8%for Cur-HA-EN-PLGA,and 25.9%,68.25%for Cur-HA-EN-PLGA.After,the drug release behavior of the drug-loaded micelles was investigated by simulating the tumor microenvironment.Firstly,the p H sensitivity of the carriers was investigated,and the drug release of Cur-HA-EN-PLGA as well as Cur-HA-SS-PLGA in PBS solution at p H 5.4 was about45%,both of which were greater than that in solution at p H 7.4,so both of which had p H-responsive.The reduction responsiveness of the carriers was then investigated,the release behavior of Cur-HA-SS-PLGA as well as Cur-HA-EN-PLGA in p H 5.4 containing 20 m M GSH PBS solution were studied,showing that the drug release capacity of Cur-HA-SS-PLGA（approximately 85%）was greater than that of Cur-HA-EN-PLGA（～50%）,which confirmed that the reduction-responsive drug release performance of Cur-HA-SS-PLGA.Therefore,the drug release of Cur-HA-SS-PLGA was faster Cur-HA-EN-PLGA in the tumor microenvironment and had better tumor inhibition ability.The biocompatibility of drug carriers is crucial to its clinical application,therefore,the biocompatibility of the carrier was investigated by MTT assay,live-dead cell staining assay and blood compatibility assay.The results of MTT and live-dead cell staining assay showed that HA-SS-PLGA was less toxic to MC3T3 osteoblasts,and at high concentrations,Cur-HA-SS-PLGA was more effective in inhibiting lung cancer cells A549 as well as MCF-7 breast cancer cells,which had a significant ability to inhibit the growth of tumor cells.The results of hemocompatibility test showed that the hemolysis rate of HA-SS-PLGA was less than 5%at a concentration below 1 mg·m L^-1,with great hemocompatibility.Cellular uptake experiments were conducted using laser confocal to investigate the distribution of Cur-HA-SS-PLGA in cancer cells.The results showed that the drug was uniformly dispersed in the cytoplasm,and the drug-loaded micelles Cur-HA-SS-PLGA could actively target to the tumor cell surface overexpression receptor（CD44 receptor）and enter MCF-7 cells through endocytosis,thus cancer cell growth was inhibited.