Design,Synthesis And In Vitro Properities Of Novel Reduction-sensitive Anti-cancer Drug Systems

In recent years,as the comprehesion and research for cancer chemotherapy have been deepened,the design of drug delivery systems and their performance requirements that match them have also been greatly improved.Various stimulus-responsive new polymers which have been used as drug carriers have attracted the scientific interest of researchers.Such drug given forms of polymer complexes by organic combination of different forces have many advantages,such as greatly increasing the solubility of hydrophobic drugs in blood and maximizing the effect of drugs utilization on specific tumor tissue and reducing its side effects.Under the guidance of this core tenet,all kinds of blossoming drug given forms give birth.Therefore,under the influence of its ever-deepening design concept,the key points and difficulties of drug delivery system design are summarized as follows:First,the carrier must have a high degree of safety and reliability,that is,it must have the biodegradability and the degradation product must be safe and non-toxic along with the blood circulation and will be excreted finally;Second,it must have a intelligent responsive behavior for controlled release,which is demanding both long-term stability in the process of circulation and effective drug release in a specific tumor tissue site;Third,the synthesis process of carriers needs to be precise and controllable so that it can maintain repeatability maximizingly when maintaining the homogeneity of its chemical structure.In order to sovle above-mentioned scientific issues,this master thesis firstly provided two kinds of grafting brush-like copolymers with similar composition but different hydrophilic segments through the ring-opening polymerization and controlled radical polymerization based on low molecular weight chitooligosaccharides.On one hand,in the first work of chapter two,by usingε-caprolactone（ε-CL）as the hydrophobic segment monomer and oligoethylene glycol methacrylate（OEGMA）as the hydrophilic segment monomer,the amphiphilic polymer micelles bridged with disulfide bond have been constructed,and they have suitable ratio of hydrophilic and hydrophobic segments.On the other hand,in the third chapter of the next work,the hydrophilic segment monomer is basically replaced by glycidyl methacrylate（GMA）.After it was synthesized,oligoamine tetraethylene pentamine（TEPA）was used to modify the corresponding products to produce the amphiphilic polymer micelles further.The behavior of self-assembly of these two types of block copolymer and its release in simulated intracellular reducing environment has been studied systematically and the cytotoxicity of drug-loaded micelles to A549 cells has been tested.The details include the following research contents:1.Based on small molecular weight chitooligosaccharides（CSO）as the substrate,one-step ring-opening polymerization（ROP）method was used to conduct the ring-opening polymerization ofε-caprolactone under the protection of the amino group.CSO-g-PCL was synthesized after chitooligosaccharides have been grafted with polycaprolactone（PCL）segments.Subsequently,a reducing sensitive double-head reagent HOOC-SS-iBuBr was synthesized which has two functional reactive sites including a carboxyl group at the end and an atom-transfer radical polymerization initiation site at the other end,both of which are linked through the intermediate disulfide bond.Then the carboxyl group of the double-head reagent was connected by a condensation reaction to the amino group of chitooligosaccharides,so themacromolecularinitiatorchitooligosaccharidespolycaprolactone CSO-g-PCL-SS-iBuBr whose amino group was modified by double-head reagent was obtained.Finally,atom transfer radical polymerization（ATRP）of the oligomerized ethylene glycol monoester（OEGMA）monomer was initiated by the macromolecular initiator.At the end of the double-head reagent linked to the oligochitosan ring,a hydrophilic fragment polyethylene glycol dimethacrylate（POEGMA）was introduced into the final product.Dynamic light scattering（DLS）was used to determine the particle size of the amphiphilic grafting copolymer in PBS（pH 7.4,150 mM）.The mean hydrodynamic diameter at a concentration of 0.2 mg/mL was less than 200 nm.The experimental results of in vitro anticancer drug doxorubicin（DOX）loaded showed that two copolymers prepared have similar ability to encapsulate DOX,whereas in drug release experiments in simulated tumor environment,the polymer micelle CSO-g-PCL₄₁-SS-POEGMA₆₀0 is more sensitive to reducing environment than the polymer CSO-g-PCL₁₈-SS-POEGMA₆₅.The results of MTS experiments showed that the cytotoxicity of the former is also higher than the latter.2.The macromolecular initiator chitooligosaccharides polycaprolactone CSO-g-PCL-SS-iBuBr whose amino group was modified by double-head reagent was prepared by a similar method,and the macromolecular initiator was used to initiate atom transfer radical polymerization（ATRP）of glycidyl methacrylate（GMA）monomer and oligoethylene glycol methacrylate（OEGMA）monomer to achieve random copolymerization of two different monomers.At last active epoxy groups on GMA units were reacted with tetraethylenepentamine（TEPA）.Cationic polymers including two different ratios of hydrophilic and hydrophobic segments have been successfully prepared.Dynamic light scattering（DLS）results showed that both polymers formed micelles less than 200 nm in diameter in PBS（pH 7.4,150 mM）at a polymer concentration of 0.2 mg/mL.The results of transmission electron microscopy（TEM）showed that the polymer CSO-g-PCL₄₁-SS-P{（GMA-TEPA）₅₆-st-OEGMA₁₄}could be assembled into a more regular globular structure in water while the polymer CSO-g-PCL₁₈-SS-P{（GMA-TEPA）₅₆-st-OEGMA₁₄}could only be assembled into a strip-like structure in the water probably because of its larger propotion of positive hydrophilic segments.In vitro drug loading results showed that the former polymer encapsulation efficiency for DOX was higher than the latter,and in the simulation of intracellular reducing environment（including 10 mM GSH）the release speed of DOX increased slowly over time,and the release contents of DOX was still maintained at a low level,indicating that this type of cationic carrier had a rather high stability to the structure after encapsulation of the drug,which made the disulfide bond difficult to break.In the studying of the changes of particle size along with time,it was found that the particle size of these polymers in PBS（pH 7.4,10 mM DTT）environment remained basically unchanged and continued up to 96 h.The reason was presumably due to the strong hydrogen bond impacts between the amino groups and the thiol groups so as to weaken the ability of thiol groups to reduce disulfide bonds.In acid titration experiments and measurements of Zeta potential of polymer solutions,these two types of polymer solutions have strong buffering capacities in the pH range from7.4 to 5.0,and their Zeta potentials have both positive values,so these previous results can support that they have great potentials as gene carriers.