Design Of Star PLGA-based And Reduction-responsive Nanomedicines For Targeted Tumor Therapy

PLGA,a copolymer of lactide and glycolide,has superior biocompatibility,in vivo biodegradability,and was approved by the US FDA for using in medical devices.PLGA is a commonly used biodegradable polymer that has a wide range of biomedical applications including drug delivery and tissue engineering.Several clinical drug formulations like Lupron Depot,Decapeptyl,and Nutropin Depot used to treat prostate cancer,lung cancer,and growth hormone deficiency are based on PLGA microparticles.Notably,BIND-014,a first active targeting docetaxel nanomedicine tested in human patients,is also fabricated from PLGA.However,nanomedicines based on PLGA have several drawbacks such as inadequate in vivo stability,drug premature release,or slow drug release at tumor sites,which significantly reduce the target ability and therapeutic efficacy.To deal with it,this thesis was set to design PLGA nanomedicines which combine superior stability,long circulation,high tumor cellular uptake and fast intracellular release to achieve efficient antitumor efficacy.Firstly,we fabricated lipoyl terminated star PLGA（sPLGA-LA）as a smart material,and based on sPLGA-LA we designed and constructed several targeted and reduction sensitive reversibly crosslinked PLGA nanomedicines for efficient tumor therapy.In chapter 2,we designed and synthesized lipoic acid functionalized star PLGA（sPLGA-LA）that forms reversibly crosslinked nanoparticles（sPLGA XNPs）.Doxorubicin（DOX）was loaded as a model drug.The stability,reduction sensitivity and in vivo pharmacokinetics of drug-loaded nanoparticles was characterized.Both ¹H NMR spectrum and UV measurement revealed the successful synthesis of sPLGA-LA.The degree of substitution was over 90%.sPLGA XNPs were prepared by co-assembly of PEG-PDLLA and sPLGA-LA with the size of 73.1±1.2 nm,which had excellent stability and good reduction sensitivity.It could also efficiently encapsulate DOX,achieving a high drug loading content of 13.3 wt.%.DOX-loaded sPLGA XNPs（DOX-sPLGA XNPs）were very stable and DOX leakage under physiological conditions was significantly lower than that from both linear and star non-crosslinked PLGA nanoparticles（denoted as lPLGA NPs and sPLGA NPs,respectively）.While DOX release was greatly accelerated under 10 mM glutathione condition.Confocal laser scanning microscopy（CLSM）and flow cytometry（FACS）studies displayed obviously stronger DOX fluorescence in B16F10 melanoma cells treated with DOX-sPLGA XNPs than with those with lPLGA and sPLGA counterparts.MTT assays on B16F10 cells revealed that DOX-sPLGA XNPs had a half inhibition concentration(IC₅₀)of 1.8μg/m L,which was 2.4 and 4.2-fold lower than those of DOX-sPLGA NPs and DOX-lPLGA NPs,respectively.In vivo pharmacokinetics studies showed that DOX-sPLGA XNPs significantly prolonged the circulation time(half elimination time t_1/2,?₌4.94h)and improved the area under curve（AUC）compared to lPLGA NPs and sPLGA NPs.Hence,sPLGA-LA emerges as a simple and smart material for better-controlled anticancer drug delivery.Compared with lPLGA NPs based on traditional linear PLGA,sPLGA XNPs showed several excellent properties including high stability and good reduction sensitivity.Good tumor targeting ability is the key factor for high efficient tumor therapy.In chapter3,in order to expand the active targeting ability of sPLGA XNPs,we prepared cRGD decorated sPLGA XNPs（cRGD-sPLGA XNPs）by introducing cRGD decorated PEG-PDLLA,which could actively target to?_v?₃ over-expressed B16F10 melanoma.DOX-loaded cRGD-sPLGA XNPs（DOX-cRGD-sPLGA XNPs,48%cRGD molar content）with a small size of 91.0±0.6 nm exhibited 2-fold higher cellular uptake in B16F10 cells determined by FACS,and efficiently delivered DOX into the cell nuclei observed by CLSM.MTT assays further confirmed that DOX-cRGD-s PLGA XNPs had an IC₅₀ of 0.92?g/m L,which was 1.95-fold lower than that of DOX-sPLGA XNPs.Biodistribution studies of DOX-cRGD-sPLGA XNPs in B16F10 bearing nude mice displayed an obvious targetability with a high tumor accumulation of 10.96 ID%/g.Furthermore,in vivo anti-tumor experiments showed that DOX-cRGD-sPLGA XNPs could effectively suppress tumor growth and prolong survival time（median survival time 38 d）with reduced side-effects in B16F10bearing mice.These cRGD-sPLGA XNPs could serve as a promising nanoplatform for targeted tumor therapy.The results of this chapter have demonstrated that it is a convenient and feasible strategy to afford PLGA nanoparticles with active targeting,reduction sensitivity,reversibly crosslinking as well as high stability by simply introducing targeting ligand modified amphiphilic copolymers,which had a good application prospect.In chapter 4,we constructed multifunctional PLGA systems（HA-sPLGA XNPs）having CD44 targetability,reduction sensitivity and good stability by simply co-assembly of single amphiphilic copolymer HA-PDLLA and sPLGA-LA for active targeting delivery of DTX to A549 lung cancer.HA-sPLGA XNPs could efficiently load DTX with a drug loading content（DLC）of 5.2 wt.%at a theoretical DLC of 10 wt.%,exhibiting a size of 105.5±0.5 nm.Both FACS and CLSM results showed that HA-sPLGA XNPs could quickly enter into A549cells.However,with the pretreatment of HA,the cellular uptake amounts of nanoparticles decreased dramatically.Similarly,the quantitative analysis of FACS showed that after blocking CD44 by HA,the cellular uptake amounts of nanoparticles decreased one fold after4 h incubation.These results demonstrated that HA-sPLGA XNPs entered into A549 cells via a receptor mediated endocytosis mechanism.MTT assays showed that after 48 h incubation,the IC₅₀0 of DTX-HA-sPLGA XNPs was 0.18μg/m L,which was 2.1 and 6.7-fold lower than that of non-crosslinked and linear control groups,respectivly.Furthermore,the in vivo results showed that they had prolonged circulation time(t_1/2?=4.18 h)and high tumor accumulation of 9.49 ID%/g.In vivo anti-tumor experiments in A549 lung cancer xenograft bearing mice demonstrated that DTX-HA-sPLGA XNPs displayed significantly enhanced anti-tumor efficacy with a tumor inhibition rate（TIR）of 86.4%,which was 1.2,1.4 and 1.9-fold higher than that of DTX-HA-sPLGA NPs,DTX-HA-lPLGA NPs and free DTX,respectively.DTX-HA-sPLGA XNPs with facile preparation and multiple functions provided new options for efficient targeted therapy of CD44 over-expressing tumors.The complex microenvironment of the tumor tissues causes inefficient tumor penetration and cellular uptake of PLGA nanomedicines.To solve this problem,in chapter5,we designed and fabricated GE11 and TAT dual ligand modified PLGA nanoparticles（GE11/TAT-sPLGA XNPs）by introducing GE11 and TAT modified PEG-PDLLA（GE11-PEG-PDLLA and TAT-PEG-PDLLA）.GE11/TAT-sPLGA XNPs could not only active targeting to EGFR-overexpressed MDA-MB-231 triple negative breast cells but also have excellent tumor penetration ability.In this chapter,DTX-loaded nanoparticles（DTX-GE11/TAT-sPLGA XNPs）was prepared to evaluate the therapeutic effect to EGFR overexpressed MDA-MB-231 triple negative breast cancer.To our exciting,the FACS studies in MDA-MB-231 cells showed that the cellular uptake of GE11/TAT-sPLGA XNPs was 9.9 and 18.8-fold higher than those of GE11-sPLGA XNPs and non-targeting sPLGA XNPs,respectively.MTT assays revealed that the IC₅₀ of DTX-GE11/TAT-sPLGA XNPs was 0.05μg/mL,which was 4.6 and 9.8-fold lower than that of DTX-GE11-sPLGA XNPs and DTX-sPLGA XNPs,respectively.Fluorescence microscope observations showed that GE11/TAT-sPLGA XNPs had higher accumulation and longer penetration distance in tumor tissue than those of single targeting and non-targeting groups.In vivo studies showed that DTX-GE11/TAT-sPLGA XNPs had a long circulation time(t_1/2,?=4.86 h),and a high accumulation amount of 9.73%ID/g at tumor tissue in MDA-MB-231 tumor bearing nude mice.The in vivo antitumor experiments showed that DTX-GE11/TAT-sPLGA XNPs could completely suppress tumor growth without any systemic side-effects and greatly improve the survival time of the mice,all the mice survived over 60 days（median survival time of DTX-GE11-sPLGA XNPs and DTX-sPLGA XNPs were 42 and 30 days,respectively）.Thus,these dual ligands decorated multifunctional PLGA nanoparticles DTX-GE11/TAT XNPs with good targeting ability,excellent tumor penetration as well as high stability and good reduction sensitivity,may open a new path for highly efficient targeted tumor therapy.Chapter 6 summarizes the results of whole thesis and provides a future perspective in the field.