Construction Of Ph Sensitive Micelles And Targeting Heterogeneous Cancer Cells

Most tumors are heterogeneous and contain small population of highly tumorigenic and intrinsically drug resistant cancer stem cells(CSCs).CSCs are mainly quiescent and able to repair their DNA when they self renew and differentiate to other tumor cell types,and express high levels of ATP-binding cassette(ABC)drug pumps.Therefore,parts of malignant stem cells can survive during chemotherapy and contribute to relapse.Potential approaches to kill CSCs include delivering therapeutic agents to block essential self-renewal signaling,inhibiting drug efflux transporters by disruption of multiple pathways in an attempt to sensitize CSCs,targeting CSCs surface markers through antibody-based cytotoxic approaches and inducing tumor cell differentiation or apoptosis.The main objectives of the proposal are to design and study a novel polymeric micellar drug delivery systems with triggered release mechanism for targeting heterogeneous cancer cells.Two kinds of polymeric micelles responding to tumor interstitial pH and endosome(or lysosome)pH respectively were constructed using pH responsive copolymer of mPEG-poly(lactic acid)-poly(L-histidine)(mPEG-PLA-PHis).The micelles were used for targeting delivery of resveratrol and doxorubicin in combination.The endosome(or lysosome)pH responsive micelles were designed to release doxorubicin and resveratrol for killing cancer stem cells under the synergistic effect of inducing apoptosis from resveratrol and overcoming multi-drug resistance from Pluronic L61 via EPR effect.The micelles would respond to the acidic pH in the endosome(or lysosome)and local hyperthemia,resulting burst release of the incoporated doxorubicin and resveratrol.The polymeric micellar drug delivery systems for targeting cancer stem cells and cancer cells simultaneously are of great creativity and have not been reported so far.The study of the project will help to improve the design of tumor targeting drug delivery systems.More importantly,the study will reveal the validity of synergistic effect of inducing apoptosis pathways in targeting cancer stem cells.This paper mainly includes five parts:(1)Synthesis and characterization of triblock copolymers mPEG-PLA-PHis;(2)pH induced reassembly of copolymer micelles and mechanism underlying endolysosomal escape for intracellular delivery;(3)Construction and characterization of drug loaded pH sensitive micelles;(4)Study of the drug loaded pH sensitive micelles targeting both tumor and CSCs;(5)In vivo biodistribution and pharmacodynamics of pH sensitive micelles.A series of mPEG-PLA-PHis with different degrees of polymerization of PLA and PHis block were synthesized.The obtained copolymers were characterized by FT-IR,1H-NMR and GPC.The pKa value of the pH-sensitive copolymer measured by titration was 6.8.The copolymer showed good buffer capacity in the pH range of 4.0～7.4 in the titration curve.The CMC values of the copolymers were measured by the change of fluorescence spectrum of pyrene due to its selective partition into hydrophobic micellar core in the copolymer solution.The CMC value was lower enough to keep micelles stable.The self-assembly and reassembly behaviors of the copolymers were characterized using transmission electron microscopy(TEM),1H-NMR,fluorescence probe technique,and dynamic light scattering(DLS).The copolymers self-assembled into micelles with PLA and unprotonated PHis blocks as hydrophobic core and PEG as hydrophilic shell at neutral pH.The changes in TEM images,1H-NMR spectrum of PHis peak,pyrene fluorescene spectrum,and particle size as well as size distribution over the pH range from pH 8.5 to 4.5 suggested that the copolymer micelles reassembled into micelles with PLA as hydrophobic core and protonated PHis and PEG as hydrophilic shell under acidic environment.The pH induced reassembling triggered the release of incoporated doxorubicin(DOX),as indicated by the in vitro accelerated drug release and enhanced cytotoxicity.The integrity of endolysosome membrane during the copolymer facilitated DOX endolysosomal escape was observed by confocal laser scan microscopy(CLSM)and further evaluated by calculation of the critical size of endolysosomal membrane.The results indicate that the endolysosomal membrane remained intact during the copolymer facilitated endolysosomal escape of DOX.It is more reasonable to ascribe the PHis based copolymer facilitation endolysosomal escape to the "proton sponge" hypothesis without rupturing the endolysosomal membrane.Cellular uptake results indicate that mPEG-PLA-PHis copolymer micelles showed higher cellular uptake by MCF-7 cells and endocytosis mediated via clathrin.The pH triggering values of copolymers was determined by in vitro drug release at different pH.The results indicated that the triggering pH decreased with the increasing of the length of the PLA block.Fluorescence spectrophotometry and HPLC method were used to detect the content of DOX and RES,respectively.The DOX and RES loaded copolymer micelles were prepared using film evaporation method.The solvent and solvent volume,ratio of drug to copolymer,pH and volume of hydration medium,temperature of film evaporating were used to optimize the preparation method.The physical characterizations of the DOX and RES loaded mPEG-PLA-PHis were characterized by DLS and TEM.The results indicated that the particle size of both micelles were around 30 nm with good distribution by DLS.The copolymer solution showed spherically shaped and smoothly surfaced micelles by TEM.In vitro studies were performed on the human breast cancer MCF-7 cells.Breast CSCs were isolated by incubation without serum.The mammospheres were sorted from MCF-7 cells and identified with CD44+/CD24-phenotype,which was concident with the previous studies in human breast cancer.To study the inhibitory effects of DOX micelles on breast cancer cells and RES micelles on breast cancer stem cells.The results showed that the inhibitory effect of RES micelles was more potent in cancer stem cells than in cells and of concentration dependence.In vivo evaluation,including distribution and pharmacology were performed on femal BALB/c nude mice bearing MCF-7cancer xenografts.Effect of polymer micelles on the in vivo distribution was studied using non-invasive near-infrared fluorescence(NIRF)imaging.The results indicate that the pH sensitive mPEG-PLA-PHis micelles showed higher passive targeting to the tumor due to the EPR effect.The results of in vivo pharmacodynamic study indicated that DOX+RES+L61/mPEG-PLA-PHis micelles demonstrated significant difference in inhibition of tumor growth from other formulations.In conclusion,the PHis blocks in the copolymers become protonated and keep the proton pump functioning,leading to the influx of Cl-ions and water molecules.This causes an increase of osmotic pressure in endolysosomes and the swelling of endolysosomes,resulting in the release of incorporated drug to the cytosol with the membrane remaining intact.RES loaded micelles were more sensitive in suppressing breast cancer stem cells.The combination therapy using DOX plus RES micelles including Pluronic L61 was capable of producing a stronger inhibitory effect to the tumors,representing a potential strategy for the treatment of breast cancer by eradicating breast cancer cells and CSCs together.