Preparation Of Human Serum Albumin Bound Nanoparticles Of Docetaxel Fatty Acid Derivative And Evaluation Of Anticancer Activities

Objective:Docetaxel?DTX?is a semi synthetic taxoid derivative,which plays an important role in the cancer therapy.Despite its highly antitumor activity,poor water solubility of docetaxel limits the clinical development and application.Commercially available docetaxel injection uses Tween 80 and ethanol as stabilizers,which can cause severe adverse reactions such as allergies and fluid retention.Prior to the administration of docetaxel injection,corticosteroids should be used in advance to prevent allergies.In addition,the use of PVC material intravenous infusion device is forbidden,which is inconvenience in the clinical application.Therefore,it is of great significance to develop a new formulation with low toxicity and high efficiency by derived docetaxel as a prodrug.Human serum albumin?HSA?bound nanoparticles was prepared with a Nab^TM technology.After intravenous administrated,the nanoparticles will disintegrate into albumin-drug complexes,whose size is similar to that of endogenous albumin in blood.These complexes could be transported via the natural albumin pathways,including gp60 channel and caveolae-mediated transcytosis and accumulated at intratumoral sites and tumor cells.Through association with tumor over-express SPARC?Secret protein,acidic and rich in cysteine?,enhanced drug targeting and penetration in tumors are achieved.The aim of this study is to synthesize docetaxel fatty acid derivatives and prepare albumin bound nanoparticles.While applying the prodrug of docetaxel,it is not only avoid the usage of surfactant has been avoided and benefits of reducing toxicity and tumor targeting have been achieved.Furthermore,new ideas has been provided and experimental basis for the following research have been established.Methods:Docetaxel derivatives are synthetized by esterification reaction to link different carbon chain with lengths of 2,4,6,8,10 to the 2'-hydroxyl site of docetaxel.Albumin bound nanoparticles of docetaxel fatty acid derivatives were prepared by Nab^TM technology using HSA as excipients and docetaxel derivatives as active substance.HSA concentration,drug concentration in oil phase and evaporation temperature were investigated using polydispersity index?PDI?,encapsulation efficiency?EE?%and recovery after filtration as response variables.A Central composite design-Response surface methodology?RSM-CCD?with three factors at five levels were used in formulation screening and process optimization.The quality studies including the particle size distribution,Zeta potential,EE%,drug loading?DL?%and assay et al were conducted on albumin bound nanoparticles prepared by the optimal formation and process.The stability of albumin bound nanoparticles for 24 hours and the stability of lyophilized product for 6 months were investigated.Animal toxicity study was performed on the series of albumin bound nanoparticles of docetaxel fatty acid derivatives prepared.The maximum tolerated doses?MTDs?have been specified and the gastrointestinal and blood toxicity of derivatives with different fatty acid groups were evaluated.MTT method was used to compare the antitumor efficacy of butyryl docetaxel?C4-DTX?and docetaxel?DTX?in vitro.Furthermore,intracellular convertion from C4-DTX to DTX in 3 cell lines including BEL-7402,HT-29and SK-OV-3 was investigated and the mRNA expression of carboxylesterase and SPARC protein in tumor cells was detected.In addition,SGC-7901?human gastric cancer?cells were selected as xenograft tumor model according to indications of docetaxel to investigate the in vivo efficacy and tissue distribution of HSA bound nanoparticles of docetaxel fatty acid derivatives.Results:A series of docetaxel derivatives were sussessfully synthesized and confirmed by MS,FT-IR and ¹H-NMR.Using C4-DTX as model drug and CCD-RSM design,the formulation and process after optimization were as follows:HSA concentration was 0.05g/mL,drug concentration in oil phase was 151.19 g/mL,and evaporation temperature was 46?.As to the nanoparticles prepared through optimized conditions,the particle size was?76.43±2.09?nm the PDI was?0.143±0.944?;drug binding ratio was?96.50±0.87?%;recovery after filtration was?97.42±2.09?%;the drug loading was?22.43±2.35?%;zeta potential was-?30.5±2.05?mV.The were no significate difference in particle size and distribution,assay and drug bingding ratio after place the reconstituted C4-DTX-NP placed for 8 hours at ambient temperature and for 24 hours by refrigerating or freezing.There were no significant changes in test items for lyophilized products stored at 20 to 30?for 6 months.The results of toxitity demonstrated that the MTDs for C4-DTX-NP,C6-DTX-NP,C8-DTX-NP,C10-DTX-NP and DXT-inj were 80,65,30,30 and30?mol/kg.At same toxicity dose level,nanoparticles showed no effect on food and water uptaken.Compared to DXT-inj group,C6-DTX-NP exhibited the lowest hematotoxicity?P<0.05?,C4-DTX-NP took the second place?P<0.05?,C8-DTX-NP and C10-DTX-NP showed no significate differenc?P>0.05?.The results of in vitro test showed that C4-DTX exhibited inhibitory activity in HT-29,SGC-7901,MCF-7,CFPAC,SPC-A-1,BEL-7402 cells.In vivo efficacy study,the antitumor effects of C4-DTX-NP was significately better than that of the DXT-inj?P<0.05?;the C6-DTX-NP showed better efficacy towards DXT-inj and C10-DTX-NP?P<0.01?;C8-DTX-NP,C10-DTX-NP and DXT-inj showed no significate differenc?P>0.05?.In the tissue distribution test,the DTX concentration in tumor tissues after C4-DTX-NP administration for 24 hours was significantly higher than that in other sites.Conclusions:In this study,a series of docetaxel derivatives were sussessfully synthesized.Using C4-DTX as model drug,albumin bound nanoparticles were successfully prepared by Nab^TMM technology,and then formulation screening and process optimization were conducted.The advantages of the C4-DTX-NP obtained including uniform particle size,high binding rate,high drug loading,stable physical properties.Among the nanoparticles prepared,C4-DTX-NP and C6-DTX-NP demonstrated significantly reduced toxicity compared to DTX-inj.In addition,the in vivo study showed that better efficacy than DTX-inj.Move over,its tissue distribution showed obviously tumor targeting effect after C4-DTX-NP administration.