Study On Redox-Sensitive Drug Delivery Systems Based On MPEG-PBLG-SS-DTX

Cancer has received more and more concern through the world,since that it has become one of the main factors that menace human health.At present,many kinds of treatments including radiation,chemotherapy and surgery,have been extensively utilized to treat various kinds of cancers in the clinic.Docetaxel(DTX),as a semi-synthetic derivative of paclitaxel,is a kind of chemotherapeutics and has been wildly used in the clinic.DTX has wide anti-cancer spectra,and it can treat a great diversity of cancers,including prostate,breast,ovarian,lung,and head/neck cancer.However,a certain amount of Tween 80 is added as a solubilizer in some parenteral solution of DTX used in the clinic because of its poor solubility,which can lead to serious side effects,such as hypersensitivity reactions.In addition,it can also do harm to normal cells considering non-specificity in distribution of DTX.In order to improve the water solubility of DTX and its ability to target to tumour tissues,various kinds of drug delivery systems,including liposomes,nanoparticles,polymeric micelles,polymer-drug conjugates micelles and so on,have been reported.Polymeric micelle is a kind of self-assembly drug delivery systems with core-shell structure,which is based on amphiphilic blocks.The outer shell is consisted of the hydrophilic block,while the core is composed of hydrophobic block.And the core is the part to load hydrophobic drugs.Polymer-drug conjugates are formed by conjugating drugs with hydrophilic or amphiphilic blocks via chemical bonds.They display outstanding anti-cancer efficiency and many merits,including high drug loading contents,high stability,and slow drug release rate.Besides,the polymer-drug conjugates reveal redox-sensitivity,pH-sensitivity and sensitivity to some kinds of enzyme through conjugating these two parts via chemical bonds which are sensitive to corresponding factors.For the sake of increasing the solubility and decreasing the side effects of DTX,we conjugated DTX to amphiphilic blocks mPEG-PBLG via a linkage containing disulfide bond to form redox-sensitive mPEG-PBLG-SS-DTX micelles.Furthermore,we modified it and prepared the multi-functional mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles,which revealed redox-sensitivity,active targeting property as well as excellent anti-cancer efficiency.This study is consisted of three parts which are listed as follows:(1)The synthesis and characterization of two kinds of mPEG-PBLG-SS-DTX with different molecular weight.We synthesized mPEG-PBLG with different molecular weight(mPEG2000-PBLG1750 and mPEG5000-PBLG1750)via the ring open polymerization of y-Bzl-L-Glu-NCA,which was initiated by mPEG-NH2 with different molecular weight.Then,we conjugated the DTX to mPEG-PBLG via a linkage containing disulfide bond to form redox sensitive polymer mPEG-PBLG-SS-DTX.1H-NMR and FT-IR were used to verify the successful synthesis of the intermediate products and mPEG-PBLG-SS-DTX.(2)The preparation,characterization and evaluation of redox-sensitive mPEG-PBLG-SS-DTX micelles.The mPEG-PBLG-SS-DTX conjugates self-assembled into micelles with core-shell structure in an aqueous environment because of its amphipathy.We prepared the micelles through dialysis method.The micelles revealed spherical structures with uniform size under the TEM.And the mean particle sizes of the mPEG-PBLG-SS-DTX micelles measured by DLS were 101.3 ± 1.4 nm and 148.9 ± 1.4 nm,respectively.The zeta potential of the mPEG-PBLG-SS-DTX micelles were-20.1 ± 0.1 mV and-14.5 ± 0.1 mV,respectively,demonstrating their outstanding stability.The drug loading contents measured by ultraviolet spectrometer were(13.9 ± 0.8)%and(9.2 ± 0.4)%,respectively.We employed the pyrene fluorescent probe method to investigate the CMC of the micelles and they were 3.98 and 6.94 ?g/mL,respectively.The low critical micelle concentrations of the micelles demonstrated they had the ability to remain stable against the dilution in the blood circulation.The micelles possessed redox-sensitivity.The results of drug release study demonstrated that only approximately 10%of the DTX was released from the micelles after 120 h without DTT,while the release rate was accelerated obviously with DTT and the accumulative release amount of DTX reached about 40%after 120 h.The results of hemolysis study illustrated that the mPEG-PBLG-SS-DTXs micelles showed inappreciable hemolysis toxicity(<5%)to red blood cells and excellent blood compatibility.The A549 and MCF-7/ADR cells were served as model cells in the in vitro evaluation of micelles.The mPEG-PBLG-SS-DTX micelles showed more excellent anti-cancer efficiency to A549 and MCF-7/ADR cells compared with that of DTX.And the IC50 of the former against MCF-7/ADR cells for 24 h was roughly a 15th of that of the latter.Moreover,the cellular uptake study illustrated that the mPEG-PBLG-SS-DTX/C-6 micelles could be taken up by A549 and MCF-7/ADR cells more efficiently when compared with C-6,which was coincident with the results of cytotoxicity study.(3)The preparation,characterization and evaluation of multi-functional mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles.We mixed mPEG2000-PBLG1750-SS-DTX,which had higher drug loading content,with actively targeting mPEG-FA,and prepared multi-functional mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles via dialysis method.The mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles showed redox-sensitivity and active targeting effect at the same time.The mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles possessed a spherical structure with uniform size.Its mean particle size and drug loading content were 129.7 ± 2.1 nm and(9.0 ± 1.7)%,respectively.The CMC of the mixed micelles measured by pyrene fluorescent probe method was 5.08 ?g/mL,which was similar to that of mPEG-PBLG-SS-DTX micelles.This could guarantee stability of the mixed micelles in the circulation.In the hemolysis study,the hemolytic potential of the mixed micelles at various concentrations was less than 5%,which demonstrated that the mixed micelles was not possible to lead to hemolysis and it was safe for intravenous administration.The mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles showed redox-sensitivity in the in vitro drug release study.Furthermore,we employed FR-negative A549 cells and FR-positive MCF-7 cells to evaluate the anti-cancer efficiency of the mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles.In the cytotoxicity study,mPEG-PBLG-SS-DTX micelles as well as mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles revealed similar anticancer efficiency against A549 cells,while the cell inhibition capacity of the mixed micelles against MCF-7 cells was visibly higher than that of mPEG-PBLG-SS-DTX micelles.The results demonstrated the active targeting mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles possessed higher anti-tumour efficiency against FR-positive cancer cells.There was no obvious distinction between the cell uptake efficiency of mPEG-PBLG-SS-DTX/C-6 micelles,mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles and mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles plus 2 mM FA in A549 cells,while the mixed micelles showed a significantly higher level of uptake efficiency than that of others in MCF-7 cells.These listed above demonstrated that the mixed micelles could be taken up into the cancer cells through FR-mediated endocytosis,which improved its uptake efficiency dramatically.Analogous results were seen in the apoptosis analysis.The level of cellular apoptosis of A549 induced by mPEG-PBLG-SS-DTX micelles and mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles was identical after 12 h,whereas the latter displayed to be more effective inducer of apoptosis than the former in MCF-7 cells.All of these results demonstrated that active targeting mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles possessed more superior anti-tumour efficiency against FR-positive cells.