Protein-based Nanoparticles Prepared By Radical Oxidation For Poorly Water-soluble Drug Delivery

The safety of the carrier materials of drugs is the most important issue for nano-drug delivery system.Protein-based nanoparticles(NPs)as drug delivery system is increasingly popular in both research pipeline and clinical applications due to their biocompatible,biodegradable,and nontoxic natures.The nab(nanoparticle albumin-bound)technology is the most successful technology for preparing protein-based nanocarriers at present.Abraxane(?),an albumin-bound paclitaxel injection prepared by the nab technology,has become a huge success since it was approved in 2005.However,the nab technology has some disadvantages including complicated process,high energy consumption and use of toxic volatile solvents.Homogenization is the main process of the nab technology.In homogenization process,shearing,cavitation,collision,and other effects occur simultaneously.The resulted mechanical force and chemical effects such as free radical oxidation on protein is complicated.In addition,the duration and strength of these effects are hardly controllable,posing great challenges to the quality control of products by the nab technology.Therefore,it is urgent to develop a more controllable,efficient,and environmentally friendly preparation method for protein-based nanocarriers.In this thesis,the feasibility of preparing protein NPs by hydroxyl radical(·OH)oxidation was testified,with the ·OH produced by the Fenton reaction in a controllable manner.The prepared paclitaxel loaded albumin NPs and piroxicam loaded casein NPs by this method were characterized and evaluated in vitro and in vivo.Furthermore,the mechanism behind the protein NPs formation induced by ·OH was explored.The main research contents and conclusions are listed as follows:1)Preparation and characterization of albumin NPs by radical oxidation.By one-step mixing of the Fenton's reagent and the bovine serum albumin(BSA)solution at room temperature,uniform albumin NPs(BSA NPs)was obtained and the preparation was not affected by temperature in the range of 25 ? 37 °C.Paclitaxel(PTX),a poorly soluble chemotherapeutics,was successfully loaded into the prepared BSA NPs,resulting in the PTX@BSA NPs.Both BSA NPs and PTX@BSA NPs were spheroid in shape,with the mean diameter of which at about 180 nm and 160 nm,the zeta potential of which at-22.1m V and-22.7 m V,respectively.The suspension of the PTX@BSA NPs remained stable at4 ? for 24 h,which meets the requirements of clinical application and subsequent evaluation of the NPs.2)Biological evaluation of the PTX@BSA NPs.The PTX@BSA NPs effectively inhibited the proliferation of 4T1 cells in vitro.The activity of the encapsulated PTX was not impaired by the preparation of PTX@BSA NPs by radical oxidation.Pharmacokinetic studies revealed that the PTX@BSA NPs group showed pharmacokinetic profiles similar to Abraxane by the nab technology.In vivo biodistribution studies revealed that the prepared BSA NPs could increase the accumulation of encapsulated insoluble drugs in tumor tissues.In vivo pharmacodynamics and safety investigations demonstrated that the PTX@BSA NPs exhibited excellent anti-tumor effect and biological safety profiles.3)Preparation and characterization of casein NPs by radical oxidation.By one-step mixing of the sodium caseinate(Na Cas)solution and the Fenton's reagent,casein NPs(Cas NPs)and piroxicam(PIX)loaded casein NPs(PIX@Cas NPs)were obtained.Both Cas NPs and PIX@Cas NPs were uniform and spheroid in shape,with the mean diameter of which at about 250 nm and 180 nm,the zeta potential of which at-32.0 m V and-30.5 m V,respectively.In vitro release studies showed that piroxicam could be slowly released from PIX@Cas NPs in simulated enzyme-containing gastric and intestinal fluids.4)Biological evaluation of the PIX@Cas NPs.Encapsulation of piroxicam in the prepared Cas NPs could promote the uptake of piroxicam by Caco-2 cells.Pharmacokinetic studies revealed that comparing to the orally delivered piroxicam solution,the orally administrated PIX@Cas NPs resulted in a 49% increase of the relative bioavailability and a29% reduction of the peak blood concentration of piroxicam in rats.Results of gastric irritation test demonstrated significant reduction of gastric irritation after oral administration of piroxicam formulated as PIX@Cas NPs,compared to piroxicam solution.5)Exploration of the mechanism of protein NPs formation by radical oxidation.The structures of BSA and sodium caseinate during the formation of the BSA NPs and Cas NPs were investigated.It was found that ·OH caused oxidative modification of the side chains of amino acid residues of protein molecules,resulting in conformational changes and increased surface hydrophobicity of the protein.The intermolecular hydrophobic interaction leads to the non-covalent assembly of proteins,which results in the formation of protein NPs.In summary,a method of preparing protein-based NPs by radical oxidation was reported in the present work.Under the oxidation of hydroxyl radicals,protein NPs can be prepared by non-covalent assembly of protein molecules with intermolecular hydrophobic interaction as a driven force.This work not only helps to clarify the mechanism of the nab technology,but also provides a simple,efficient,controllable,and environmentally friendly method for the preparation of protein-based NPs,which can be applied for the delivery of poorly water-soluble drugs.