Investigation On Tumor Microenvironment Triggered Nanoscale Drug Delivery System

Chemotherapy is one of the most common methods to treat malignant tumor in clinic.Traditional chemotherapeutic drugs were often associated with ineffective access to tumor local site and severe side effects,which limit their clinical applications.It was well known that the tumor microenvironment has unique physiological characteristics,such as lower pH,oxidative stress,et al.Taking use of these unique features,researchers developed numerous tumor microenvironment stimuli-responsive drug delivery systems with capabilities of tumor-targeting and controlled drug release.And the engineered drug delivery systems could effectively not only reduce side effects of chemotherapeutic drugs,but also improve anti-tumor efficacy.In this study,we prepared redox/pH dual-responsive MnO₂ nanosheets and self-assembly nanoparticles.Then two kinds of endogenous stimuli drug delivery systems were designed based on these stimuli-responsive nanomaterials for targeted drug delivery and controlled drug release.The main contents were as follows:1.Study on the Integration of GSH/pH Double Sensitive Tumor Enhancement TherapyIn this work,A redox/pH dual-triggered tumor theranostic platform based on the degradable manganese dioxide?MnO₂?nanosheets was developed,and folic acid?FA?was employed as the targeting group while doxorubicin?DOX?as a chemotherapeutic agent.Firstly,the MnO2 nanosheets were synthesized and modified with 3-amino-propyl trimethoxy silane?APTMS?to obtain MnO₂-NH₂,and then functionalized with NH₂-PEG2000-COOH.FA was finally conjugated with the PEGylated MnO2 nanosheets.At last,DOX was absorbed onto the modified nanosheets.This multifunctional system was designed as MnO₂-PEG-FA/DOX and it possessed good biocompatibility.The nanoplatform could not only efficiently delivery DOX to tumor cells in vitro and in vivo,but also respond to the slightly acidic and high concentration of reduced glutathione?GSH?tumoral environment,which caused the degradation of MnO₂ into manganese ions with the ability of enhancing magnetic resonance imaging?MRI?.The MnO₂ nanosheets were in shape of diamond with the lattice space of 0.23 nm.The average zeta potential of MnO₂-PEG-FA/DOX was¹1.5 mV.The formulation has good stability and dispersibility.The entrapment efficiency of DOX was 90.2±0.43%.The release behaviours of drug loaded nanosheets showed that 85.0±0.7%DOX was released from MnO₂-PEG-FA/DOX nanosheets within 24 h in pH 5.0 medium containing 2mM GSH while the cumulative release percentage was less than 40%in the pH 7.4medium.These results indicated that the formulation not only could response to the tumor microenvironment stimuli,but also displayed a sustained release profile.The in vitro results indicated that MnO₂-PEG-FA had a good biocompatibility and exhibited no obvious cytotoxicity to HeLa cells at the test concentration range.The MnO₂-PEG-FA/DOX exhibited time-and dose-dependent cytotoxicity against Hela cells.The inhibition effect of MnO₂-PEG-FA/DOX was relatively weak compared to that of DOX at 24 h while the inhibition effect of MnO₂-PEG-FA/DOX was enhanced at 48 h.The apoptosis rate of MnO₂-PEG-FA/DOX group was 50.2±3.24%,which showed significant difference with those from MnO₂-PEG/DOX and DOXgroups.Theinvivoresultsdistributiondemonstratedthat MnO₂-PEG-FA/DOX had good tumor-targeting ability.The in vivo therapeutic efficacy of the nanoplatform was investigated and the results showed that the relative tumor volume of tumor-bearing mice of MnO₂-PEG-FA/DOX group and MnO₂-PEG/DOX group were 1.3±0.3 and 2.1±0.4,respectively,showing a relative decreasing trend.The in vitro and in vivo MRI results showed that the contrast enhancement of T₁-MRI led by the formulation was observed on the condition of redox/pH dual-stimuli.The longitudinal relaxation rate r₁ was 2.26mM^-1.s^-1 at pH 5.0 PBS containing 2 mM GSH.These reduction and pH dual-responsive biodegradable nanosheets combining MRI and chemotherapy provide a strategy for tumor-targeting theranostic application.2.Investigation on the pH Hypersensitivity self-assembled nanoparticles for tumor therapy.Molecular self-assembly drug delivery system had specific tumor recognition and binding ability.However,the non-covalent interactions were weak and easily disassembled during the circulation process in vivo.In this work,DOX loaded self-assembly nanoparticles?BNPs?based on phenylboronic acid?PBA?were constructed,which consisted of pH-responsive cores and detachable poloxamer 188shells.The poloxamer 188 coating could be easily detached when the breakage of the borate ester bonds in the external nanocores were initially triggered in the tumor extracellular weak acid environment.The concealed PBA was subsequently exposed and could react with sialic acids?SA?,which were overexpressed on tumor cells.The combination of PBA and SA enhanced the tumor retention effect of the fresh nanoparticles as well as facilitated the cellular uptake after removing the protective layers.After entering the cells,the nanoparticles with the pH-responsive esters could rapidly release DOX in the acidic tumor environment,which resulted in an enhanced therapeutic efficiency in vitro and in vivo.The characteristics of DOX/BNPs showed that the nanoplatform with round sphere morphology and good stability had an average particle size of 210.0±5 nm,and exhibited a potential of²0 mV.According the in vitro drug release result,within a time period of 24 h,the cumulative DOX release percentages were 92.0±2.9%at pH 5.0 and 61.4±3.7%at pH 6.0,respectively,whereas that was only 22.8±1.8%at pH 7.4.These results suggeted that an acidic stimulus the cleavage of the linkages and catechol borate ester bonds,leading into the disassembly of the nanocomplexes.For both formulations,the inhibition effects were in a time-and dose-dependent manner.At 24 h,the inhibition effect of DOX/BNPs was comparable to that of free DOX.With prolonged incubation time,the drug antitumor effect enhanced to some extent.The result of cell cycle experiments showed that BNPs nanoparticles had no obvious effect on cell cycle.DOX/BNPs significantly arrested cell cycle in S and G₂/M phases,which could be attributed to the elevated celluar uptake of DOX.The result of apoptosis was consistent with that of cytotoxicity.During the in vivo therapeutic period,no obvious body weight losses were observed in any of the groups,indicating that the BNPs nanoparticles had good biocompatibility.The relative tumor volumes of mice in blank control group,BNPs group,DOX group and DOX/BNPs group were 7.2±0.86,6.40±0.68,4.28±0.87 and 2.47±0.51,respectively.H&E staining and TUNEL apoptosis of tumor tissues showed that the DOX/BNPs could inhibit cell growth and induce more cells apoptosis.In summary,this pH dependent behaviour of DOX/BNPs provided new insights for enhanced chemotherapeutic treatment in cancer.