Preparation And Evaluation Of Tumor Extracellular PH-sensitive Charge-reversal Nano-preparations

Traditional pH-sensitive nanodrugs usually release drugs based on the hydrophilic-hydrophobic transition or weak chemical bond breakage in the tumor microenvironment.Their defect is that it can not promote their tumor cellular internalization,which significantly influences their antineoplastic efficacy both in vitro and in vivo.Based on the characteristics of weakly acidic microenvironment in tumor area and cells,we focus on how to develop charge-reversal nanodrugs,where the initial negative surface charge endows nanodrugs with a longer circulation time and the generated positive surface charge ensures improved cellular.Based on this,we develop tumor extracellular pH-sensitive nanoparticle（es NP）loaded anticancer agents having acid-cleavable amide linkages of 2,3-dimethyl maleamidic acid（DMMA）pendants in micellar shells,thus attaining stimuli responses at tumor site.Charge-reversal nanocarriers loaded with free anticancer agents and triggered by various biological signals（pH and redox）associated with tumor microenvironment.Hoping to solve the defects of traditional anticancer agents in solubility,stability,systemic toxicity and bioavailability and increase targeting ability between normal cells and tumor cells.This paper mainly consists of the following parts:The first part:the pH-sensitive amphiphilic triblock copolymer PEG₄₅-PCL₄₀-PAEA₃₃-DMMA was synthesized,in which the PAEA₃₃-DMMA segment is a pH responsive segment.Through self-assembly,pH responsive nanoparticles with size of 178.3 nm and Zeta potential of-10m V were obtained.Under TEM observation,pH responsive nanoparticles with spherical structure,uniform dispersion and smooth surface were observed.When adjusting the pH from 7.4 to 6.8,the charge reversal behavior of the nanoparticles was observed,and the nanoparticles had good stability.Furthermore,fluorescein was used to label nanoparticles,cellular uptake assays showed that es NP exhibited similar cellular uptake to pH-insensitive nanoparticles（is NP）with succinamic acid（SA）pendants at pH 7.4,while displayed 6-folds cellular uptake as is NP at pH 6.8。The second part:using pH-sensitive amphiphilic polymer as carrier,the anti-tumor drug gambogenic acid was encapsulated by carrier.The anti-tumor nanomedicine was obtained by self-assembly.The drug loading conteng of nanomedicine was 8.1%.The results of drug release in vitro showed that the cumulative drug release rates were less than 30%when the pH was adjusted from 7.4 to 6.5,indicating the stability of nanoparticles.The uptake of nanoparticles by hepatoma Hep G2 cells was studied and the results showed that the uptake of nanoparticles in es NP-GNA group was about 3times that of is NP-GNA group and the cytoxibity study showed that when pH 7.4 and the concentration of es NP-GNA was less 16μg·m L^-1,the nanodrug had no obvious effect on the cells,but when pH was 6.5,the inhibitory effect was obvious,IC50 was4.137±0.04,indicating had stronger cytotoxicity than free drug.The third part:we develop tumor extracellular pH-sensitive nanoparticle（es NP）loaded Pt（IV）prodrugs（es NP-Pt）having acid-cleavable amide linkages and GSH-cleavable Pt（IV）pendants in micellar shells,thus attaining dual-stimuli responses at two locations.The loaded Pt（Ⅳ）pendants could be reduced and release cisplatin.Under the stimuli of 10 mmo L GSH,the release amount of cisplatin increased to 6 folds.MTT assays demonstrated that es NP-Pt possessed preferential inhibition,indicating preferential toxicity against tumor and weak systemic toxicity.Tumor extracellular pH-sensitive charge-reversal nanomedicine possesses many favorable traits for preferential toxicity to cancer cells,for example,minimizing nonspecific uptake in blood circulation and enhancing cellular uptake at tumor site in response to tumor extracellular level of pH,improved intracellular drug delivery to tumor cell cytoplasm（TCC）,increase water solubility and decrease the systemic toxicity of Small molecular anticancer drugs,in situ release of cisplatin in response to TCC reductive potential,which endow it as a promising candidate for cancer therapy.