Preparerition And Anti-tumor Activity Study Of Cabazitaxel-loaded Human Serum Albumin Nanoparticles

Cancer is the second cause of death in the world.At present,chemotherapy is one of the primary therapeutic strategies.Among the chemotherapeutic agents,taxanes have shown good efficacy in the treatment of solid tumors such as breast cancer,lung cancer and prostate cancer.Cabazitaxel is a chemical semi-synthetic small molecular compound of taxanes,which has been widely used in the treatment of prostate cancer in recent years.However,the commercial cabazitaxel has some problems when used in the clinical.On the one hand,the uneven distribution of the drug in the body,such as the accumulation of intestinal cells leads to severe diarrhea and other gastrointestinal toxicity.On the other hand,because of the poor water solubility of catabase,the clinical prescription containing solubilizing polysorbate 80,will produce serious toxic side effects,which limit the application of cabazitaxel.In recent years,more and more attention has been paid to albumin nano drug delivery system because of high solubility,safety,non-toxicity,non-immunogenicity and biodegradability.The preparation of albumin nanoparticles loaded cabazitaxel can effectively improve water solubility of cabazitaxel,which can avoid the use of polysorbitol 80 and reduce the toxicity and side effects.There are some problems in the preparation of albumin nanoparticles.These methods like dissolvation and Nab^TM are complex and may affect the biological activity of albumin.In addition,the use of toxic reagents and crosslinking agents increases the toxicity.In contrast,salting-out method can avoid the use of toxic reagents and cross-linking agents,using albumin unfolding and refolding self-assembly to form nanoparticles,which can maintain the biological activity of albumin and easy to operate.Based on the advantages of the above-mentioned albumin nanoparticles and the salting-out method,the albumin nanoparticles loaded cabazitaxel?Cbz-NPs?were prepared by salting-out method and the formulation were optimized.Cabazitaxel injection solution?Cbz-Tween?was prepared according to the solvent formulation of clinical cabazitaxel injection.Comparing with Cbz-Tween,the in vitro and in vivo evaluation of Cbz-NPs and its anti-tumor effect were studied.The main research contents and results are as follows:1.Study on the properties of cabazitaxel and human serum albumin?HSA?In vitro analytical method of cabazitaxel and human serum albumin were established.Methodological validation tests of cabazitaxel and albumin were carried out.The linear relationship between cabazitaxel and human serum albumin was good in the range of 0.02¹.00mg/mL,and the linear relationship between human serum albumin and sperm albumin was good in the range of 0.10-6.40mg/mL.The density was 1.56%and 0.14%,the mean of recovery was 99.15%and 97.78%,and the RSD of stability was 0.69%and 0.24%,respectively,which met the methodological requirements.2.Preparation and formulation optimization of Cbz-NPsResponse surface methodology?RSM?was used to optimize the composition of nanoparticles.Three independent variables were selected:ratio of drug to HSA?w/w?,pH and temperature of injection,respectively and encapsulation efficacy?EE?were selected as the response variables.The experiment under the recommended optimum conditions with a ratio of drug to HSA?w/w?0.16,pH 7.8 and temperature of injection 66oC was performed.Finally,the experimental encapsulation efficiency of Cbz-NPs was 52.95%,the size is 170.2±1.9nm,and the drug loading capacity is5.07±0.38%.3.The evaluation of Cbz-NPs in vitroCbz-NPs were also evaluated in vitro.In DSC measurements,the typical endothermic peaks remained in the mixture disappeared in the nanoparticles,indicates the formation of new phase.A favorable stability and a sustained release were displayed.Compared with Cbz-Tween,excellent blood biocompatibility of Cbz-NPs were suitable for intravenous administration.The cytotoxicity of Cbz-NPs was evaluated by the cell viability of Human prostate cancer lines PC3 and Human Lung Cancer line A549.Obviously,the single solvent has significant cytotoxicity in these cell lines,with the cell viability of 81.48±5.28%for PC3 and 77.53±5.38%for A549.Nevertheless,drug-free nanoparticles exhibit almost no toxicity as expected.4.Establishment of analysis method of Cbz-NPs in vivoA high performance liquid chromatography?HPLC?method was developed for the determination of cabazitaxel in rat plasma with diazepam as internal standard.The results of methodological validation showed that the precision of the method was good during the day and in the day.The recovery rates at low,medium and high concentrations were 98.41±1.33%,97.23±1.86%and 98.20±3.24%,respectively.The matrix effect was 104.62±5.99%,102.36±2.99%and 101.43±8.01%.In addition,diazepam was also used as an internal standard to establish a method for the determination of cabazitaxel in heart,liver,spleen,lung,kidney and tumor tissues of nude mice bearing tumor.Methodological validation showed that the standard curves of cabazitaxel in various tissues were well linearized in the range of 0.2-10.0?g/mL?R>0.99?,and the precision ranged from 2.19%to 5.43%during the day and in the day.The recovery rates of cabazitaxel in low,medium and high concentrations were more than 95%,and the matrix effect was about 100%.All of them conform to the analysis requirements of biological samples.5.The evaluation of Cbz-NPs in vivoCbz-NPs were evaluated in vivo by pharmacokinetic study in rats,distribution of drug tissues in tumor-bearing nude mice,preliminary pharmacodynamic study of anti-tumor in tumor-bearing nude mice,histological observation and cytokine detection.Compared with Cbz-Tween,the half-life?T1/2?h??of Cbz-NPs injection group was increased by 2.12 times,the area of AUC0-?curve was increased by 0.33times,and the mean retention time?MRT0-??was increased by 0.24 times.Human prostate cancer PC3 tumor model was established in nude mice.The distribution of Cbz-NPs in tumor-bearing nude mice showed that Cbz-NPs could effectively reduce the drug content in heart,spleen and kidney tissues and thus reduce the drug toxicity.However,Cbz-NPs accumulated continuously in the tumor site and effectively increased the drug content in the tumor site.Therefore,Cbz-NPs and Cbz-Tween can significantly inhibit the growth of tumor in nude mice bearing tumor in vivo.The toxicity of Cbz-NPs and Cbz-Tween was evaluated by body weight,pathological sections and cytokine changes.Cbz-NPs therapy can be passively targeted by high permeability and retention effect?EPR?,effectively targeting tumor tissues,and significantly reducing the liver and kidney toxicity and injury caused by Cbz-Tween.In summary,Cbz-NPs were prepared with good particle size distribution and entrapment efficiency by salting-out method,which solved the problem of low water-solubility of cabazitaxel.Compared with Cbz-Tween,Cbz-NPs can reduce the toxic and side effects.It has good application effect and development prospects.