Self-assembled Nanoparticles Of Poly(D,L-lactide-co-glycolide)/Hyaluronic Acid Block Copolymers For Breast Cancer And Breast Cancer Stem Cells Targeting

Studies have shown breast cancer is initiated and maintained by a small fraction ofbreast cancer stem cells (BCSCs), which are responsible for metastasis, recurrence, drugresistance, and poor patient survival. Current chemotherapeutic agents (such as paclitaxel,doxorubicin or cisplatin) fail to cure breast cancer, because such agents only killdifferentiated breast cells, but the BCSCs produce tumor bulk though continuousself-renewal and differentiation. Therefore, in order to cure breast cancer, it is necessary toeradicate both differentiated breast cancer cells and BCSCs. The objectives of this projectare:(1) To develop a biocompatible and biodegradable copolymer based on poly (D,L-lactide-co-glycolide)/hyaluronic acid (PLGA-b-HA) to encapsulate chemotherapeuticdrug (docetaxel) and BCSCs specific agent (sulforaphane).(2) To eradicate differentiatedbreast cancer cells and stem cells by DTX and SFN loaded PLGA-b-HA nanoparticels.This study included four chapters:(1) Development and identificatio n of poly (D,L-lactide-co-glycolide)/hyaluronic acid self-assembled nanoparticles, including synthesisand characterization of PLGA-b-HA copolymers, preparation and optimization ofPLGA-b-HA nanoparticles, characterization of DTX loaded PLGA-b-HA nanoparticles.(2)In vitro studies of PLGA-b-HA nanoparticles, including CD44receptors specific-targetingability, in vitro anti-breast cancer activity of DTX/SANPs.(3) In vivo studies ofPLGA-b-HA nanoparticles, including in vivo breast carcinoma targeting, pharmacokinetics,in vivo antitumor activity.(4) Combination of DTX/SANPs and SFN/SANPs to eradicatebreast cancer cells and stem cells, including isolation and identification of breast cancerstem cells, in vitro cellular uptake in breast cancer stem cells, preparation andcharacterization of SFN/SANPs, in vitro inhibition of breast cancer and breast cancer stemcells, in vivo antitumor and breast cancer stem cells activity.In the first chapter, the amphiphilic diblock copolymer poly(D,L-lactide–co–glycolide)–block–hyaluronan(PLGA-b-HA) was synthesized by end toend coupling strategy. The chemical structure of PLGA-b-HA was identified by H-NMRand FT-IR. The critical micelle concentrations of PLGA-b-HA copolymers composed ofdifferent molecular weight of PLGA/HA were determined by pyrene as a fluorescenceprobe. The result showed the CMC values increased from2.0mg/L to19.9mg/L withincrease of HA proportion in the whole polymer. We prepared PLGA-b-HA self-assemblednanoparticles by solvent-dialysis based method. The hydrophobic/hydrophilic moieties had great effect on the size and morphology of nanoparticles, one of the polyers,PLGA502H-b-HA5.6kformed the optimized nanoparticles with the smallest size (~111nm),an acceptable CMC (~7.9mg/L), typical core/shell structure, was selected for furtherstudy.The effects of solvent, polymer concentration and pH on the physio-chemicalproperties of the nanoparticles were evaluated. The priority PLGA-b-HA nanoparticles(<200nm, zeta potential-25~-30mV) could be prepared at such condition: DMSO/DMF(v/v=3:1) as solvent, pH=6-8, polymer concentration <3.0mg. Powder X-RD revealed thatDTX existed as the amorphous or molecular state after loaded into PLGA502H-b-HA5.6knanoparticles. Furthermore, DTX/SANPs exhibited a biphasic release pattern at bothpH=5.5and pH=7.4.In the second chapter, the cellular uptake of coumarin-6loaded SANPswas investigatedin MDA-MB-231(overexpressing CD44receptors) and MCF-7cells (lowexpressing CD44recptors) to evaluate the CD44receptor specific targeting ability of SANPs. Comparedwith PLGA502Hnanoparticles, the cellular uptake of SANPs was significantly enhanced inCD44receptors overexpressed MDA-MB-231cells. Furthermore, the internalization couldbe inhibited by free HA, indicating SANPs were internalized into cells by CD44-mediatedendocytosis. In addition, blank SANPs showed negligible cytotoxicity to MDA-MB-231and MCF-7cells at a concentration of50-500μg/ml, however, DTX/SANPs showedenhanced cytotoxicity to MDA-MB-231at48h compared with DTX/PLGA nanoparticles.Cell cycle distribution and cells apoptosis were further evaluated in MDA-MB-231cells,and the results showed DTX/SANPs could more effectively inhibited cells in G2/M phaseand induced more cells apoptosis than DTX/PLGA nanoparticles.In the third chapter, we focused on the in vivo breast cancer targeting and antitumoractivity. First, using MDA-MB-231tumor-bearing female nude mice as CD44receptorsoverexpressed breast cancer xeno-graft mode, we evaluated the biodistribution and tumortargeting of DiR loaded SANPs and PLGA nanoparticles using small animal in vivoimaging system. Results showed such two kinds of nanoparticles were mainly distributedin live, lung, spleen and tumor. In detail, PLGA nanoparticle accumulated in live whileSANPs mainly accumulated in lung. As for tumors, compared with PLGA nanoparticles,SANPs could be more efficiently accumulated in tumors, indicating SANPs had goodtumor targeting ability for CD44receptors overexppressed breast cancer cells. Then, weevaluated the pharmacokinetics of DTX, DTX/PLGA and DTX/SANPs in SD mice. Theseresults showed both DTX/PLGA and DTX/SANPs could increase the circulation time of DTX in blood stream. Compared with DTX/PLGA, DTX/SANPs showed a relative longert1/2and larger AUC as well as lower clearance rate, indicating DTX/SANPs couldeffectively improved the circulation time of DTX. Finally, we evaluate the in vivoantitumor activity of DTX, DTX/PLGA and DTX/SANPs in MDA-MB-231tumor-bearingfemale nude mice. Results showed tumor inhibitory rate of DTX, DTX/PLGA andDTX/SANPs were77.10±6.87%，85.96±3.24%，92.22±2.06%, respectively. On the otherhand, both DTX/PLGA and DTX/SANPs showed little toxicity to nude mice at the periodof experiment, indicating DTX/SANPs showed priority anti tumor activity without toxicityin vivo.In the last chapter, breast cancer cell specific drug (SFN) was loaded into SANPs, andcombined with DTX/SANPs to eradicate both differentiated breast cancer cells and BCSCs.The main contents and results of this chapter are described as follows:(1) BCSCs wereisolated and identified as ESA+CD44+CD24-phenotype from MCF-7cells usingserum-free medium culture method. Furthermore, the proportion of ALDH+cells in thesecells are significantly higher than that of MCF-7cells, indicating the isolated BCSCs (alsocalled mammosphere cells, MCF-7MS) exhibited properties of cancer stem cells.(2)Coumarin-6, a fluorescence probe, was loaded into PLGA502Hnanoparticles and SANPs toevaluate the cellular uptake of PLGA nanoparticles and SANPs in BCSCs. Compared withPLGA502Hnanoparticles, the cellular uptake of SANPs was significantly enhanced, andcould be inhibited by free HA, indicating SANPs were internalized into BCSCs byCD44-mediated endocytosis.(3) We prepared SFN/SANPs and DTX/SANPs usingsolvent-dialysis based method. Compared with blank SANPs, the size of SFN/SANPs wasincreased and the morphology of SFN/SANPs was subsphaeroidal. DTX/SANPs showed abiphase release profile and97%of total SFN was released at72h.(4) MCF-7MS wasmore sensitive to SFN, while exhibited significantly resistant to DTX. Drug (DTX or SFN)loaded nanoparticles exhibited enhanced cytotoxicity than free drugs towards bothdifferentiated breast cancer cells and BCSCs.(5) In vitro anti cancer stem cells activity wasevaluated by mammosphere formation and β-catenin as well as cyclin D1expression.Results showed SFN loaded nanoparticles were more effective to reduce the number andthe size of mammospheres than free SFN in vitro. Compared with MCF-7cells, β-cateninand cyclin D1were over expressed in MCF-7MS, and was significantly reduced afterMCF-7MS treated with SFN formulation, while DTX formulations failed.(6) Thecombination of DTX and SFN was more effective to inhibit MCF-7cells than that of single drug, and such enhanced effect was not observed in MCF-7MS.(7) In vivo antitumor activity showed drug loaded nanoparticles were more effectively to inhibit thegrowth of breast cancer than free drug. In particular, combination of DTX and SFN loadednanoparticles achieved the best therapeutic effect.(8) In vivo anti cancer stem cells activitywas also evaluated by mammosphere formation and β-catenin as well as cyclin D1expression in excised tumors. DTX and DTX/SANPs could reduce the size but increasedthe number of mammospheres, and could not down-regulate the expression of β-catenin.SFN and SFN/SANPs could reduced both size and number of mammospheres, furthermoredown-regulate the expressionof β-catenin.