Preparation And Evaluation Of Dual Redox And MMP-2 Sensitive PTX-Loaded Nanoparticles

The chemotherapy drug Pclitaxel(PTX)is the fist-line treatment for breat cancer.However,the clinical application of PTX was limited due to its low solubility,poor targeting and severe adverse effect.The first commercial formulation of PTX(Taxol(?))was formulated with polyoxyethylated castor oil(Cremophor(?)EL)and anhydrous ethanol at the ratio of 1:1.But the Cremophor(?)EL is easy to cause strong allergic reactions,renal toxicity,neurotoxicity and low tolerance.In order to enhance the solubility and targeted ability of PTX,improve drug safety,a dual-sensitive nanoparticle was designed to deliver PTX prodrug.According to high concentration of glutathione(GSH)and overexpression of matrix metalloproteinase-2(MMP-2)in the tumor tissues,the nanoparticle is redox and MMP-2 sensitive,and the programmed drug release could be realized.Moreover,the bovine serum albumin(BSA)was used to actively target SPARC receptor on the breat cancer.The project mainly included the following four parts:1.The synthesis and structure identification of PTX prodrug and Gel-SHThe prodrug PTX-SS-COOH was synthesized by the esterification raction between PTX and dithiodipropionicanhydride(DTDPA),and the hydrophilic material Gel-SH was synthesized by the amide raction between Gelatin and 4-meraptobenzoic acid.Finally,the sulfhydrylation amphiphilic polymer Gel-SS-PTX was synthesized by PTX-SS-COOH and Gel-SH.The above products were identified by ultraviolet spectroscopy(UV),nuclear magnetic resonance spectroscopy(1H-NMR)and Fourier transform infrared spectroscopy(FT-IR).The content of sulfydryl was determined by Ellman’s method as 55.88μmol/g.2.The preparation and investigation of physicochemical property of BSA/Gel-SS-PTX/PTX-SS-COOH nanopraticleThe BSA/Gel-SS-PTX/PTX-SS-COOH co-delivery nanoparticle was prepared as the optimal preparation process and the drug loading content(DL%)was determined as 28.71%.The morphology was observed by transmission microscopy(TEM),and the image revealed the nanoparticle was smooth and spherical.The size was determined as 124.4±1.32 nm by dynamic light scattering(DLS),Zeta potential was about-1.47±0.278 mV.Finally,the critical aggregation concentration(CAC)was estimated by fluorescence spectroscopy using pyrene as a probe as 0.0355 mg/mL.3.The in vitro evaluation of BSA/Gel-SS-PTX/PTX-SS-COOH nanoparticleThe BSA/Gel-SS-PTX/PTX-SS-COOH nanoparticle was shrunk after incubation with MMP-2 under different pH value,which indicated the nanoparticle was MMP-2 sensitive.Besides,the in vitro drug release of Gel-SH/PTX-SS-COOH NPs,Gel-SS-PTX NPs and BSA/Gel-SS-PTX/PTX-SS-COOH NPs were determined by dialysis bag method,and the drug release was improved obviously with 10 mM D,L-dithiothreitol(DTT),which demonstrated the redox sensitive of NPs.Moreover,the in vitro anticancer activity,cellular uptake,apoptosis and the ability of depletion of GSH was evaluated on the mouse melanoma cell(B16 cell)and human breast cancer cell(MCF-7 cell),and the active targeting of NPs was also confirmed by flow cytometry(FCM).4.The evaluation of in vivo pharmacodynamics of BSA/Gel-SS-PTX/PTX-SS-COOH nanoparticleThe B16 burdened mice was applicated to evaluate phamacodynamics of BSA/Gel-SS-PTX/PTX-SS-COOH nanoparticle.The normal saline,Taxol(?)and nanoparticle were administrated via tail vein;the tumor volume and the body weight of mice were used to investigate in vivo anticancer activity and safety as indicators.The Taxol(?)and nanoparticle treated mice showed obvious tumor suppression compared to normal saline treated mice,the body weight of nanoparticle treated mice was increased;while the Taxol(?)treated mice got weight loss.Moreover,the hematoxylin-eosin staining(H&E staining)of normal tissues and tumor tissue in mice was used to observe histopathology changes.The results demonstrated no significant toxicity in normal saline and nanoparticle treated mice,while the Taxol(?)treated mice revealed strong hepatotoxicity and nephrotoxicity.The above results indicated the favor in vivo anticancer activity and safety of nanoparticle.