| Cabazitaxel(CTX),a second-generation semisynthetic taxane derivative,exhibits a broad spectrum and high levels of antitumor activity owing to its ability to overcome the P-glycoprotein-mediated drug resistance.Nonetheless,its insoluble nature and the absence of cell selectivity limit clinical applications of CTX.It's commercial formulation(Jevtana),using Tween 80 and ethanol as cosolvents,shows haematological and gastrointestinal side effects.Polymeric micelles(PMs)are potential to improve the solubility and circumvent the side effects of CTX.In the present study,a series of PEG-PLA derivates were synthesized to prepare CTX-loaded multi-functional PMs.They integrated cytotoxic drugs,angiogenesis inhibitors,tumor-targeted elements,and tumor metabolism-sensitive drug release mechanism.They were novel CTX formulations which were stable in blood circulation,internalized into cancer cells quickly,and then stimulus-responsively release drug to kill cancer more effectively.The study was expected to provide new ideas for the improvement of stability and targeting of PMs and provide a potential candidate for clinical treatment of tumors.The main contents and conclusions are summarized below:1.Novel CTX-loaded PMs with superior stabilitywere designed and developed to improve passive targeting/EPR effect(enhanced permeability and retention effect)of PMs.Firstly,a novel mPEG-PLA derivative,mPEG-PLA-Phe(Boc),was successfully synthesized through simple chemical process by conjugation of N-t-butoxycarbonyl-L-phenylalanine.The grafting rate was high,the molecular weight had a homogeneous distribution,and the critical micelle concentration was far lower than that of PEG-PLA.Then regular spherical mPEG-PLA-Phe(Boc)/CTX micelles with small size(17.34±0.29 nm)and narrow distribution(PDI=0.019±0.008)were prepared by solid dispersion-thin film hydration.The process was simple and beneficial to industrial amplification.The micelles had high entrapment efficiency(EE,97.46±0.66%)and drug loading capacity(DLC,4.83 ± 0.13%).Solubility of CTX was increased by nearly 1000 times due to encapsulation into micelles.Their in vitro storage stability and dilution stability were better than those of unmodified mPEG-PLA/CTX micelles.Release kinetics showed that mPEG-PLA-Phe(Boc)/CTX micelles have delayed and sustained release properties in vitro.Pharmacokinetic study in rats showed that the micelles stably retained CTX in blood circulation against precipitation compared with Jevtana,which will probably improve drug accumulation in tumor sites and bioavailability in vivo.MTT experiments against NCI-H460 cells showed that mPEG-PLA-Phe(Boc)/CTX micelles achieve equivalent cytotoxicity of Jevtana,while the micelle carrier showed better safety.These fndings suggested that mPEG-PLA-Phe(Boc)/CTX micelles were presumably safe and effective nanoscale drug delivery system for CTX with superior stability and long blood circulation time.2.Excessively improving the micelle stability tends to retard the drug release rate at target sites and thus leads to decreased drug efficiency.Thus CTX-loaded PMs with superior stability and controlled drug release at tumor site were designed and developed.Firstly,a novel mPEG-PLA derivative,mPEG-PLA-(LA)4,was successfully synthesized through simple two-step chemical process by conjugation of four lipoic acids.The process was simple and controllable,the grafting rate was high,and the molecular weight had a homogeneous distribution.Then CTX-loaded reversible core cross-linked DCL-CTX micelles were prepared by solvent evaporation-thin film hydration method and addition of dithiothreitol to trigger the formation of intermolecular disulfide linkages.The micelles were regular sphere with small size(21.74±0.45 nm)and narrow distribution(PDI=0.328±0.005).They had high EE(98.65 ± 1.77%)and DLC(9.87 ± 0.02%).The solubility of CTX was increased by nearly 2500 times,circumventing the toxicity of surfactant used in Jevtana.The micelles were quite stable in neutral microenvironment,while glutathione(GSH)-sensitively disaggregated and released CTX in reducing medium.The results of FCM(flow cytometry)and CLSM(confocal laser scanning microscope)showed that they mainly internalized into MCF-7 cells through clathrin-mediated endocytosis and showed more effective endocytosis than Jevtana and non-crosslinked micelles.Owing to GSH-stimulated disulfide bridges cleavage,DCL-CTX micelles showed better cytotoxicity by accumulating more CTX,while blank micelle carriers had favorable biocompatibility.These GSH-responsive core cross-linked micelles were speculated to be stable in blood circulation,while facilitating drug release at tumor site.They improved the delivery and efficacy of CTX through improving stability and drug release controllability.3.To further improve micelle stability,targeting and efficiency,novel CTX-loaded multifunctional PMs were developed.Firstly,a novel mPEG-PLA derivative,HM-PEG-PLA,was successfully synthesized through simple carbodiimide reaction by conjugation of angiogenesis inhibitory peptide containing arginylglycyl-aspartic acid(RGD)sequence,HM-3.The grafting rate was high,and the molecular weight had a homogeneous distribution.Then one-step nanoprecipitation protocol with optimal parameters was used to encapsulate CTX into PMs by mixing HM-PEG-PLA with mPEG-PLA-(LA)4 in proportion of 1:9.Dithiothreitol was added to trigger the formation of intermolecular disulfide linkages.Core cross-linked micelles with polypeptide modified on surface,HM-PMss/CTX micelles,were obtained.The micelle size(78.55±0.11 nm)was very suitable for passive target by EPR effect.The high EE(93.94±4.19%)and DLC(8.39±2.28%)are benificial to reduce the clinical times of administration.HM-PMss/CTX micelles had superior in vitro storage and serum stability,which is good for intravenous administration.In vitro release behavior showed that HM-PMss/CTX micelles were GSH-sensitive.Endocytosis mechnism studies showed that HM-PMss/CTX micelles can actively target MDA-MB-231 cells overexpressing integrin receptors,and then internalized into cells through receptor-mediated endocytosis,which was faster than unmodified micelles.Cytotoxicity experiments showed that HM-PMss/CTX micelles could actively target tumor cells and GSH-responsively release CTX,resulting into stronger anti-proliferative activity than Jevtana and unmodified micelles.Human umbilical vein endothelial cell angiogenesis experiments indicated that micelles retain the angiogenesis inhibitory activity of HM-3 peptide,and were expected to exhibit synergistic effect with cytotoxic CTX.In addition,HM-PMss/CTX micelles effectively inhibited the formation of vasculogenic mimicry of MDA-MB-231 cells through the synergistic effects of HM-3 and CTX.The multifunctional HM-PMss/CTX micelles provided alternative strategy for combination of multiple mechanisms.In conclusion,nanoscaled PMs with improved functions were prepared in the present study for CTX delivery and release.Novel drug delivery pathways were established.These CTX-loaded micelles were potential candidate novel clinical therapeutics for tumor. |
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