Stimuli Responsive Polymeric Carriers For Protein Delivery

Protein therapy has recently attracted much attention as promising anti-tumor drug candidates due to high bioactivity,high specifity and low toxicity,for example,antibodies for cancer treatment bevacizumab,rituximab;small molecule proteins granzyme B,cytochrome C,RNase A;protein drugs for autoimmune diseases IL-6,IL-17 and CD20.However,the clinical translation of protein drugs was greatly limited due to unstability and poor membrane permeability.Based on the physiological and pathological characteristics of tumor microenvironment,this dissertation developed various protein delivery systems nanogels and implantable scaffold for maintaining bioactivities of protein drugs and achieving controlled release at the targeted site.The main contents and conclusions are as follows:(1)Glucose and pH dual-responsive nanogels for intracellular protein delivery.Direct delivery of protein suffers from their in vitro and in vivo instability,immunogenicity and a relatively short half-life within the body.To overcome these challenges,pH and glucose dual-responsive biodegradable nanogels comprised of dextran and poly(L-glutamic acid)-g-methoxy poly-(ethylene glycol)/phenyl boronic acid(PLG-g-mPEG/PBA)were designed.The cross-linked network imparted drug-loading efficacy of a-amylase up to 55.6%and hyaluronidase up to 29.1%.In vitro protein release profiles revealed that the release of protein was highly dependent on the pH or glucose concentrations,i.e.less amount of protein was released at pH 7.4 or healthy blood glucose level(1 mg mL-1 glucose),while quicker release of protein occurred at pH 5.5 or diabetic blood glucose level(above 3 mg mL-1 glucose).Circular dichroism spectra showed that the secondary structure of released protein was maintained compared to naive protein.(2)Hypoxia-responsive nanogels for intracellular protein delivery.Ribonuclease A(RNase),an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA,has attracted intense interest due to its high efficacy and specificity.However,RNase suffers from instability,a short half-life in the circulation and poor membrane penetration.To overcome these challenges,we designed a supramolecular nanogel for the cytosolic delivery of RNase.The nanogels were fabricated using host-guest interactions between azobenzene(Azo)and ?-cyclodextrin(?CD)conjugated to poly(L-glutamic acid)-graft-poly(ethylene glycol)methyl ether(PLG-g-mPEG).RNase could be loaded inside the nanogels in mild aqueous conditions.Following optimization,the RNase-loading content and efficiency of the nanogel were 23.5 wt%and 50.4%,respectively.In the presence of nitroreductase(NTR),the cross-linking point between Azo and ?CD was destroyed due to the conformation transition of Azo,ensuring the hypoxia-sensitive release of cargo from the nanogels in tumors in which NTR is overexpressed.In vitro release profiles revealed that 75.0%of the RNase was released under hypoxic conditions in 72 h,whilst only 19.7%was released under normoxic conditions.Cytotoxicity assays showed that the RNase-loaded nanogels(nano-RNase)were more efficient in inhibiting the proliferation of 4T1 cells than free RNase.In vivo studies showed 68.7%tumor suppression rates(TSR%)in the nano-RNase treated group,whilst free RNase treatment led to a lack of tumor inhibition.To further enhance the hypoxia status of tumors,we combined nano-RNase with a nanoformulation of vascular disrupting agents PLG-g-mPEG/combretastatinA4(nano-CA4)and obtained a TSR of 91.7%.(3)pH-responsive implantable scaffolds for local and sustained release of antibodies.Antibody therapies have become an important component in the management of cancer therapy.However,a notable challenge for the application of antibodies in vivo is severe side effects induced by strong immunogenicity.Hence,there is an urgent need for developing protein carriers for increasing the accumulation in tumor sites and reducing exposure to normal tissues.Hydrogels are cross-linked polymers with hydrophilic groups which enable them to absorb large amounts of water.Their porosity and compatibility with aqueous environments make them highly attractive biocompatible drug delivery vehicles.Specially,there is no restrictions for small molecule drugs loading and protein macromolecules drug loading for hydrogels.Herein,we developed a bio-implant for delivery of antibodied anti-PD-1(aPD-1).The bio-implant is fabricated with oxidized dextran(ODEX)and 4-arm poly(ethylene glycol)amine(4-arm PEG-NH2)by Schiff s base reaction at mild conditions,with DOX and aPD-1 loaded inside during and after the fabrication process,respectively.In vitro studies confirmed the slow and sustained release of DOX and aPD-1 from the bio-implants.In vivo studies showed that the bio-implants could be gradually degraded and maintain relatively high concentrations of therapeutic agents in the mouse abdomen.In a murine CT26 PMC model,the BI@DOX+aPD-1 resulted in a 89.7%tumor suppression rate after peritoneal implantation.Importantly,the combination therapy of DOX and aPD-1 in the bio-implant showed an excellent synergistic effect with a Q value of 2.35.Through the investigation of this dissertation,we hope to develop an effective protein delivery carrier for anti-tumor therapy,the design of this protein drug carrier provides much guidance for protein application and anti-tumor treatment.