Construction And In Vitro Anticancer Evaluation Of Redox-Sensitive Polymeric Micelles Based On PEO-PPO-SS-DTX Conjugate

Objective:Cancer is one of the most difficult diseases to cure and poses a great threat to human health all over the world,which drives many scientists keep studying new drugs and dosage forms to fight against it.There are many types of cancer,and there are many treatments for different pathological tissues,organs,and pathological properties,including surgical therapy,targeting therapy,radiotherapy and chemotherapy,etc.,the chemotherapy method refers to the use of chemical drugs to treat cancers.Docetaxel(DTX)is a kind of chemotherapy drug,deriving from European berries yew needles.It is a kind of semi-synthetic taxane derivative,which has a stonger pharmacological effects than that of paclitaxel and a good curative effect on many kinds of cancers,such as non-small cell cancer,breast cancer,head and neck cancer,ovarian cancer,pancreatic cancer,gastric cancer,and so on.However,the water solubility of DTX is poor.Currently,polyoxyethylene dehydrated sorbitol monooleate(Tween-80)and ethanol are often added in clinically applied DTX injections to increase its solubility,which results in severe side effects such as allergic reactions,neurotoxicity,etc.In order to give full play to the advantages of DTX in the treatment of various cancers and increase its solubility and tumor targeting effect,scientists have prepared DTX into various new dosage forms,such as polymer micelles,liposomes and vesicles.The polymer-drug graft is formed by the covalent conjugate between the functional groups of amphiphilic block copolymers and drugs.They can spontaneously assemble into micelles in water due to their amphiphilic nature.The hydrophobic block is located inside the micelle and the hydrophilic block is outside the micelle.The hydrophobic blocks of the polymers are inside the micelles while the hydrophilic blocks are outside the micelles.At present,many biodegradable polymer-drug grafts have been widely used in the biomedical field.Polymer-drug graft micelles can greatly improve the solubility of hydrophobic drugs,improve the safety and targeting effect of drugs,thus becoming an effective new dosage form for treating cancers.In addition,scientists have used various kinds of chemical bonds that are sensitive to the tumor microenvironment,such as redox-sensitive bond,acid-sensitive bond,and enzyme-sensitive bond,to link the polymer functional groups to drugs to form amphiphilic polymer-drug grafts.In addition to their ability to self-assemble in water to form micelles,they also have the characteristic of tumor microenvironment sensitive drug release behavior,which can significantly reduce the damage of the drugs to normal tissues.This strategy has become an attractive platform for the emerging drug delivery systems.Based on the above investigation,this study selected biocompatible polyoxyethylene-polyoxypropylene(PEO-PPO)amphiphilic block copolymers as carrier materials,which are linked to anti-cancer drug DTX through redox-sensitive disulfide bonds,thus resulting in PEO-PPO-SS-DTX grafts.The structures of the intermediate products and PEO-PPO-SS-DTX graft were characterized.The physicochemical properties of the PEO-PPO-SS-DTX micelles were characterized and their in vitro antitumor capacity were evaluated.Methods:This topic were divided into two major parts.The first part was the synthesis and characterization of the redox sensitive PEO-PPO-SS-DTX polymer.The PEO-PPO and the anticancer drug DTX are connected by a disulfide bond to form a redox-sensitive PEO-PPO-SS-DTX polymer-drug graft.Then the chemical structures of DTDPA,DTX,PEO-PPO,DTDP,PEO-PPO-SS-COOH and PEO-PPO-SS-DTX were characterized by the 1H-NMR.The chemical structures of DTX,PEO-PPO,PEO-PPO-SS-COOH and PEO-PPO-SS-DTX were characterized by the FT-IR to confirm the successful synthesis of PEO-PPO-SS-DTX graft.The second part was the preparation and characterization of redox sensitive PEO-PPO-SS-DTX micelles.We studied the assembly behavior of PEO-PPO-SS-DTX in aqueous solution and observed the appearance of the micelles.The micromorphology of PEO-PPO-SS-DTX micelles were observed by transmission electron microscope(TEM).The particle size of the micelles was measured by transmission electron micrograph.The critical micelle concentration(CMC)of the micelles was measured by pyrene probe method.The ultraviolet spectrophotometry(UV)was used to measure the drug loading content of the micelles.The in vitro evaluation of the redox sensitive polymer-drug conjugate PEO-PPO-SS-DTX micelles.The hemolysis rate was measured by hemolysis test to evaluate the biocompatibility of the micelles.The in vitro cytotoxicity assay was used to evaluate the killing ability of the micelles to tumor cells,and the cellular uptake experiment was used to evaluate the endocytosis ability of the micelles by the tumor cells.Results:PEO-PPO and DTX were connected by disulfide bond to form the redox sensitive PEO-PPO-SS-DTX polymer-drug conjugate through a series of reactions.The chemical structures of DTDPA,DTX,PEO-PPO,DTDP,PEO-PPO-SS-COOH and PEO-PPO-SS-DTX were characterized by the 1H-NMR.The chemical structures of DTX,PEO-PPO,PEO-PPO-SS-COOH and PEO-PPO-SS-DTX were characterized by the FT-IR to confirm the successful synthesis of PEO-PPO-SS-DTX graft.The redox sensitive polymer-drug graft PEO-PPO-SS-DTX micelles were successfully prepared and characterized.We studied the assembly behavior of PEO-PPO-SS-DTX in aqueous solution.The polymers spontaneously assembled into spherical micelles in aqueous solution.We observed the appearance of the micelles,it can be observed that the micelle solution is milky white and has a Tyndall effect.The PEO-PPO-SS-DTX micelles were observed by transmission electron microscope(TEM).The micelles are spherical with a uniform size.The particle size of the micelles was measured by transmission electron micrograph,and the mean diameter was 145.3 ± 13.1 nm.The mean diameter measured by dynamic light scattering instrument(DLS)was 159.0 ± 4.4 nm,which is slightly larger than that of the TEM micrograph observed.The polydispersity index(PDI)was 0.176 ± 0.014.The critical micelle concentration(CMC)was about 5.0 ?g/mL.The small CMC value ensured the anti-dilution ability and the blood circulation stability of the micelles.The drug loading content was about 8.29%.We evaluated the in vitro properties of redox sensitive polymer-drug conjugate PEO-PPO-SS-DTX micelles.The hemolysis rates of all the concentrations of PEO-PPO-SS-DTX micelle sulutions were less than 5%,indicating the good biocompatibility of the synthesized polymer-drug conjugate.The in vitro cytotoxicity test results showed that compared with DTX free drugs,when the concentration of DTX is 0.002 mol/L and 0.02 mol/L,PEO-PPO-SS-DTX micellar solution showed higher cell inhibition rate,and PEO-PPO-SS-DTX micelle had faster cell penetration rate.The cellular uptake results showed that the PEO-PPO-SS-DTX/IR-780 micelles had faster uptake rate than that of the free IR-780 on Hep 1-6 cells.Conclusion:PEO-PPO-SS-DTX conjugate micelles have good performance and good application prospects.