Preparation Of 10 - Hydroxycamptothecin Albumin Nanoparticles By Liquid Drug Loading And Its Preliminary In Vitro And In Vivo Evaluation

Background:Albumin nanoparticles drug delivery system is array of nano-preparations by fabricating nanoparticles based on albumin materials. Due to the superordinary properties like nontoxic, non-immunogenic, biocompatible and biodegradable, as well as high binding capacity of various drugs, human serum albumin has been shown to be natural drug carrier. Meanwhile, accompany with the upsurge research on nano drug delivery(NDDS), human serum albumin nanoparticles(HSA-NPs) is also a focal point for loading drugs to make desired NDDS. Ever since the postmarketing of Abraxane®, the human serum albumin paclitaxel nano formulation patent by American Bioscience, human serum albumin nano preparation had become a major trend in new drug delivery reseach, which had a great financial potency.Camptothecin(CPT) is a natural alkaloid found in camptotheca acuminate who is known to be a potent anti-tumor active pharmaceutical ingrediet. It has the broad antitumor spectrum that inhibits the activity of DNA topoisomerase I which leads to hindrance of DNA synthesis. One of the camptothecin analogs, hydroxycamptothecin(HCPT), compared to CPT, has demonstrated strong antitumor activity against gastric, lung, ovarian, breast, and pancreatic carcinomas. However, the poor water solubility, structure shift from lactone form to the carboxylate form when stays in plasma environment, all affect the far-reaching clinical application of HCPT. Thus, there already some formulation researches like liposome, nanosuspension and nano microemulsion had been done, in order to address this problem. At the same time, HSA formulation was also widely investigated. Howecer, ill ideal solubility in chloroform and soybean oil makes loading procedure less effective when applied emulsification and nab-technology method, which are usually aiming to perform packing lipophilic drugs. Since low preparation effiency, toxic regants involvement(organic solvents and toxic crosslinkers) is still issue for current loading methods, which mean there is yet a technical barrier for fabricating HCPT loaded HSA-NPs.Objective: To overcome the drawbacks like low loading efficiency and toxic regants involvement, this study aimed to search for a new loading method. First, this study applied reduced-glutathione(r-GSH) as a crosslinker to fabricate blank HSA-NPs and low weight polyethylene glycol(l-PEG) HCPT liquid compound method to accomplish loading process. This novel method was taking extensive hydrogen bonds between l-PEG and HCPT to increase drug concentration as liquid HCPT compound. The research was conducted with understanding the novel property of the HCPT liquid compound.To successfully creating human serum albumin based HCPT nanoparticles, this research firstly optimized the disulfide bond stabilized HSA-NPs by craft, as it included protein concentration, water/ethanol ratio and prepare scheme. For results, the blank HSA-NPs were near 200 nm and stable. Next, this study had investigated the parameters of preparation of l-PEG-HCPT, and obtained stable compound. The liquid compound had high concentration(80mg/ml) and characteristic of extremely vulnerable to water. Correspondingly this study also developed a loading strategy for guaranteeing incorporation between HCPT and disulfide bond stabilized HSA-NPs. Consequently, the procedures of preparation were emerged as below:(1) Step1, the blank HSA-NPs were obtained using coacervation method and r-GSH as crosslinker then followed with lyophilization.(2) Step 2, applied ethanol as the menstruum to prepare l-PEG-HCPT liquid compound under reduced pressure distillation process.(3) Step 3, sufficiently mix blank HSA-NPs powder with l-PEG-HCPT under dry condition.(4) Step 4, re-disperse the mixture back to water to acquire desired HCPT-HSA-NPs.After fabricated HCPT-HSA-NPs, several in vitro and in vivo tests were performed to in-depth interpret of loading mechanism and feasibility of this loading method. Such as particle size, distribution, zeta potential, appearance characterization, stability evaluation, solution stability test, release pattern, X-ray powder diffraction(XRD) and differential scanning calorimetry(DSC) tests. Subsequently, the coming in vivo experiments such as cytotoxicity assay, tumor growth inhibition and biodistribution were also performed between HCPT-HSA-NPs and HCPT injection.Results: As all the results suggest, the obtained HCPT-HSA-NPs were suited to the nano characteristic. And l-PEG-HCPT liquid compound method preparation was practicable that the entrapment efficiency about 99%, mean size was under 200 nm and content about 7%. Meanwhile it had overcome drawbacks from conventional method with less toxicant involvement. The polydispersity index indicated it had narrow distribution and zeta potential was fitable. The obtained HCPT-HSA-NPs had long release pattern that sustained near 100 hours. The solution stability test had approved the role of HSA-NPs; meanwhile, XRD and DSC results were also reinforced the successful immersion of l-PEG-HCPT to HSA-NPs matrix. The in vivo experiments such as cytotoxicity assay, tumor growth inhibition and biodistribution also valided HCPT-HSA-NPs that was functional as nano preparation in vivo. The cytotoxicity assay and tumor growth inhibition had shown the HCPT-HSA-NPs were outstripping the HCPT injection.Conclusion: As all the results suggest, the l-PEG-HCPT liquid compound method preparation had overcome drawbacks from conventional method. Meanwhile, simple procedure and non-toxic regents applied in this new perspective of drug loading method send a clue for solving such problems similar to model drug whereby refine and extend realm of packing drugs to HSA-NPs.