Construction Of Tumor Microenviroment Responsive Drug Delivery System And Antitumor Efficacy For Ovarian Cancer

Background and ObjectiveOvarian cancer,one of the gynecological tumors,has already become a serious threat to life and health of women.Due to the lack of effective screening methods,patients usually have progessed in an advanced stage when they are looking for a doctor.For patients with advanced ovarian cancer,chemotherapy is one of the main treatments.Because of the low bioavailability and dose-dependent side effects of conventional chemotherapy drugs,patients can not get effective treatment,resulting in resistance to chemotherapy,even disease progress.It is critical to find effective first-line drug candidates,in order to improve patient tolerance to the drugs,while reducing the recurrence and avoiding drug resistance.Study strategyAlthough the mechanisms of failure treatment by the first-line ovarian cancer drug cisplatin[Pt(?)],paclitaxel(PTX)and second-line drug adriamycin(ADR)vary,all are associated with the drug resistance,the low drug concentration in the tumor and serious side effects.(1)Design of tumor microenvironment drug delivery system:Based on these points,we designed a tumor microenvironment responsive drug delivery system for the delivery of the drug above to the tumor site,which has the PLGA as the core,the two chains cRGD-PEG1K-PLGA/PEG5Kpeptide-PLGA as the main body,the drugs entrapped in the core of the PEG-PLGA hydrophobic parts.PEG5K makes the system deserve the long circulation characteristics,forbiding the inner part in the carrier exposing to ions or macromolecules in the vascular.It also minimizes the reticuloendothelial system to take up the nanoparticles in the body.Therefore,there are many more nanoparticles leaking to tumor tissues through the irregulated tumor vascular.While nanoparticles reach the tumor tissues,which contain high concentration of matrix metalloproteinases(MMPs)mainly secreted by tumor cells,MMP substrate peptide between PLGA and PEG5K in the PEG5K-peptide-PLGA could be hydrolyzed by MMPs and result in PEG5K separated from the nanopaticles.Also,cRGD in the cRGD-PEG1k-PLGA could be exposed to the environment in tumor tissues,which promoted the uptake of nanoparticles by tumor cells.(2)Au-Pt(?)mediated release of Pt(?)and overcome sulfur thiol-mediated drug resistance:Especially for Pt(?),Pt(?)is transformed into Pt(?),which is connected to the surface of Au nanoparticles.Then Au-Pt(?)nanoparticles are encapsulated into the above polymer matrix.In the blood,the water solubility of Au-Pt(?)@NPs-cRGD is improved and the toxicity is reduced,compared to that of Pt(?).After the Au-Pt(?)@NPs-cRGD reach the tumor tissue,they are ingested by the tumor cell.In the reducing environment,Pt(?)is separated from the surface of Au nanoparticles and transformed into Pt(?).Au nanoparticles are capable of binding to glutathione(GSH),to avoid the generation of Pt(?)-GSH which is related with Pt(?)inactivation of tumor cells,and increase the sensitivity of tumor cells to the drugs.Methods and Results(1)Construction and characteristic of tumor microenvironment drug delivery system:Based on the above design,three kinds of drug delivery system Au-Pt(?)@NPs-cRGD,PTX@NPs-cRGD and ADR@NPs-cRGD are prepared.Optimal preparation conditions of nano-carrier are acquired based on drug loading.Nanoparticles diameter of Au-Pt(?)@NPs-cRGD is about 180 nm,the diameter of PTX@NPs-cRGD and ADR@NPs-cRGD is both about 160 nm,all the three nanoparticle is of electrically neutral charge.Morphology of the nanoparrticles is spherical and uniformly dispersed.Wherein the Au-Pt(?)dispersed in a polymer matrix,the number of each nanoparticle ranges.In 37 C PBS buffer,the nanoparticles could maintain the particle size stable in 18 d,which indicates the nanoparticles have good stability.Au-Pt(?)@NPs-cRGD in Pt release are acid-insensitive,but in a GSH concentration-dependent manner.PTX or ADR release from PTX@NPs-cRGD or ADR@NPs-cRGD is faster under acidic conditions.Compared with the rapid release of free drug,drug-loaded nanoparticles have a sustained release capability for drugs.(2)Antitumor efficacy of tumor microenvironment drug delivery system in vitro:Taking SKOV3 cells as the cell model in vitro cytological study,PEG5K chain scission of the nanoparticles in the tumor microenvironment,the cellular uptake pathways,intracellular distribution and the growth inhibition of SKOV-3 cells by the drug-loaded nanoparticle are investigated.PEG5K scission results show that SKOV-3 cells with high expression of MMP-2 are easier uptake of nanoparticles.For MDA-MB-231 cells with low expression of MMP-2,nanoparticles gathered at the cell surface.This result suggests that PEG5K could affect the nanoparticles into tumor cells.In turn,it confirmed that the MMP-2 secreted by SKOV-3 cells is capable of inducing PEG5K chain scission.The entry of nanoparticles into cells predominantly depends on clathrin endocytic pathway and caveolin mediated pathway.The colocalization studies of endocytic organelles and the nanoparticles have shown that nanoparticles could avoid or escape from lysosomes,releasing the drug into the cytoplasma.The three kinds of nanoparticles could inhibit the growth of SKOV-3 cells obviously and empowed the antitumor effects of free drugs.(3)Relationship between cell cycle or apoptosis and the antitumor efficacy of tumor microenvironment drug delivery system:The mechanisms of Au-Pt(?)@NPs-cRGD,PTX@NPs-cRGD and ADR@NPs-cRGD inhibitory effects of SKOV-3 cell growth are analyzed.In the asynchronous model,the cell cycle is tested after the cells incubating with the drugs.The results show that Au-Pt(?)@NPs-cRGD can make cell cycle arrest in S phase,while Pt(?)and the nanoparticles as the control were no significant cycle arrest changes.PTX@NPs-cRGD illustrate that the more obvious cell cycle arrest in M phase,compared with that of PTX.ADR@NPs-cRGD suggest that more obvious in the S phase cell cycle arrest,compared to that of ADR.In synchronous model,the results show all phase of synchronized cells were arrested in S phase after incubating with Au-Pt(?)@NPs-cRGD,while G0/G1 cell appeared cycle arrest in G0/G1 phase and the others arrest in S phase after incubating with Pt(?).Compared with the free drugs,PTX@NPs-cRGD and ADR@NPs-cRGD show that the similar results with those in the non-synchronized cells.The results of study confirmed that the drug-loaded nanoparticles can be more evident in certain cell cycle arrest of tumor cell,compared to that of the free drugs.Studies have shown that drug-loaded nanoparticles are easier to induce apoptosis,compared to that of the free drugs.(4)Antitumor efficacy of tumor microenvironment drug delivery system in vivo:In SKOV-3 tumor-bearing mice,the nanoparticles entrapped fluorescent dye are followed up over time by imaging in vivo.The results show that the nanoparticles are increasingly gathered at the tumor site,which indicates the drug-loaded nanoparticles have outstanding long circulation effects and tumor targeting.Taking Au-Pt(?)@NPs-cRGD as example,the therapeutic effect of the drug-loaded nanoparticles to SKOV-3 tumor-bearing mice is investigated.The results illustrate Au-Pt(?)@NPs-cRGD can significantly inhibit tumor growth.Histopathology and apoptosis analysis indicate that the drug-loaded nanoparticles can more effectively induce apoptosis of tumor cells.In safety assessment,the results suggest that the drug-loaded nanoparticles don t cause organ toxicity.ConclusionAu-Pt(?)@NPs-cRGD,PTX@NPs-cRGD and ADR@NPs-cRGD were successfully prepared,which are multifunctional long-circulating nanoparticles and could response to matrix metalloproteinase in the tumor microenvironment.All of the three kinds of nanoparticles could inhibit the growth of SKOV-3 cells ovarian cancer.The action of the antitumor efficacy is related to the stronger cycle arrest and induction of apoptosis.Tumor targeting is excellent in vivo.Au-Pt(?)@NPs-cRGD show promising therapeutic effect in SKOV-3 tumor-bearing mice.