Docetaxel-loaded Albumin Nanoparticle Drug Delivery System

Docetaxel(DTX),a semi-synthetic taxane,is a promoter of microtubule polymerization leading to cell cycle arrest at G2/M,apoptosis and cytotoxicity.It has significant activity in tumor chemotherapy.Because of the bulky,extended fused ring with several hydrophobic substitutes in chemical structure,DTX provides high lipophilicity with very poor aqueous solubility,which limits its clinical use.Up to now,all formulations of DTX approved are formulated in the surfactant polysorbate 80,which has been reported in the contribution of acute hypersensitivity reactions and peripheral neuropathy.Therefore,investigation of alternative formulations of DTX without polysorbate 80 is an ongoing research.In this case,protein-based nanoparticles which employ endogenous plasma proteins,especially albumin,come to the best option of drug vectors.Albumin is the most abundant protein in blood plasma,and is playing an increasing role as a drug carrier in the clinical setting.Besides the traditional passive tumor targeting via the enhanced permeability and retention(EPR)effect arisen from the abnormalities in vasculatures in tumor,active targeting is also integrated by taking albumin as drug vectors.Albumin-based nanoparticles have been shown to selectively accumulate in tumor tissues because of its high affinity with albumin receptors overexpressed on tumor cell surface,such as gp60 and secreted protein,acidic and rich in cysteine(SPARC).Therefore,albumin could be an ideal vector for delivering DTX.At present,albumin nanoparticles loading DTX have been successfully fabricated by the nanoparticle albumin-bound(nabTM)technology.However,many problems still exist.Firstly,the fabrication method is too complex to be carried out in industrial scale production.Besides,toxic organic solvent dichloromethane is used in nanoparticle preparation,which cannot be removed completely.A certain amount of residual chloroform may lead to high toxicity in liver,kidney,lung and neural system.Therefore,it is necessary to develop a new method to prepare DTX-loaded albumin nanoparticles.To overcome above-mentioned barriers,in this study,we had pioneered a novel simple "protein unfolding/refolding" strategy to fabricate DTX-loaded albumin nanoparticles.This strategy is based on the hydrophobic interactions between DTX and internal regions of albumin.In the structure of albumin,large hydrophobic cavities hide in the molecule inner,and the protein framework is maintained by 17 disulfide bonds.By reducing disulfide bonds,more hydrophobic regions are exposed and will interact with hydrophobic drugs resulting in nanoparticles.This method eliminated the complicated preparation procedures and toxic residual solvents.It maintained a mild process condition for protein and drug,by which the prepared nanoparticles were proven to be highly effective in antitumor activity.By using the unfolding/refolding method,we successfully fabricated DTX-loaded albumin nanoparticles.All parameter details during the fabrication process were readjusted through a series of screening experiments.It was proved by practice that the method was reasonable,effective and suitable for industrialization produce.The resulting nanoparticles showed a mean diameter size of 150 nm.In the TEM and SEM images,the nanoparticles had a spherical shape and smooth surfaces.DTX was amorphous in nanoparticles,which indicated a readily bioavailable.No significant changes were observed in albumin secondary structures.The desirable drug loading content and high encapsulation efficiency were also achieved.The nanoparticles displayed high stability in suspension solutions and slow,continuous release profile,which could reduce the peak concentration and prolong the therapeutic drug level.The formation mechanisms were alsd characterized by using mass spectrum,fluorescence spectrum and ultraviolet spectrum.The analyze methods of related substances,docetaxel,albumin and residual solvent were established.The methods had been validated and were suitable for the quality control of DTX-loaded albumin nanoparticle.The tability was confirmed by stability experiments.After being encapsulated into nanoparticles,DTX displayed similar cytotoxicity to traditional injection,and showed an uptake behavior of active transport.Since polysorbate 80 was not involved in nanoparticles,the hemolysis was completely eliminated.The maximal tolerance dose of nanoparticles was also increased,which allowed a higher dose to be safely intravenously injected and produced ideal antitumor effects.Tumor targeting effect and long circulating effect were observed in in vivo pharmacokinetics and biodistribution experiments.These effects further caused the higher antitumor effects of nanoparticles in three tumor-bearing animal models than that of traditional injection.Therefore,DTX-loaded albumin nanoparticles fabricated by our strategy showed higher promise in their safety and effectiveness than the traditional DTX injection.