Preparation And Antitumor Actions Of Folate-Decorated Human Serum Albumin Loaded With Nano-Hydroxycamptothecin Nanoparticles

The project regards human serum albumin(HSA)and folate(FA)as drug carrier and targeted group,we have prepared folate-decorated human serum albumin loaded with nano-hydroxycamptothecin nanoparticles(FA-HSA-Nano-HCPT-NPs)which shows strong antitumor activity.And the hydroxycamptothecin-human serum albumin nanoparticle has a small mean particle size and little distribution of particle size.The hydroxycamptothecin nanoparticle also displays well slow release effect and strong tumor targeting.In the process of preparing folate-decorated human serum albumin loaded with nano-hydroxycamptothecin,first,we prepared the micro powder of hy-droxylcamptothecin by an anti-solvent method.The mean particle size of the hydro-xycamptothecin micro powder is inferior to 1000 nm.Then we prepared hydroxycamptothecin nanoparticles using the micro powder of hydroxycamptothecin by high pressure homogenization technique.The optimum condition of high pressure homogenization technique is that concentration of hydroxycamptothecin:0.5 mg·mL-1;pressure:80 MPa;15 cycles.The mean particle size of nHCPT is 67.9 ± 4.3 nm(range:61.2 nm to 78.3 nm).The X-ray diffraction results of raw HCPT and Nano-HCPT shows raw HCPT has a crystalline structure and nano-HCPT reflect low crystallinity.The result of FT-IR shows the functional group of HCPT has no difference in the prepared process.Then,we prepared folate-decorated human serum albumin loaded with nano-hydroxycamptothecin nanoparticles with antitumor activity using anti-solvent method and surface coating method.The optimum condition of FA-HSA-Nano-HCPT-NPs prepared process is that the concentration of hydroxycamptothecin,hydroxycamptothecin:human serum albumin(mass ratio),reaction time,volume of 80%ethanol and PH are 0.33 mg·mL-1?1:10?18h?37 mL and PH = 6.2.Mean particle size of FA-HSA-Nano-HCPT-NPs was 197.2±2.6 nm(range:174.6 nm to 238.5 nm)(Figure 1B),The zeta potential,an important parameter of colloidal nanoparticle stability in solution,was-27.43 ± 2.14 mV for FA-HSA-Nano-HCPT-NPs,suggesting that these nanoparticles are highly stable.Scanning electron microscopy revealed that almost all of the FA-HSA-Nano-HCPT-NPs were spherical.FA-HSA-Nano-HCPT-NPs had no detectable peaks,suggesting that that almost all of these nanoparticles exist in an amorphous structure and that the nHCPT had been loaded by FA-HSA.In vitro release of HCPT from the FA-HSA-Nano-HCPT-NPs was studied by membrane dialysis against PBS(pH = 7.4).The temporal characteristics of HCPT release from FA-HSA-Nano-HCPT-NPs were in accordance with the Higuichi equation of y =-0.26 e(-x/7.46)-8.50 e(-x/1752.34)+8.80(R2 = 0.999)(Figure 4A).An initial burst over the first 24 h(41.4%release)was followed by a sustained-release stage,such that the cumulative release of HCPT was>75%by 96 h.These results suggest the FA-HSA-Nano-HCPT-NPs have a high controlled-release efficacy.Cell proliferation assay was performed using the MTT assay.The research of cell proliferation assay studied the effects of HSA-NPs,raw HCPT,and FA-HSA-Nano-HCPT-NPs on MCF-7 cell.Cytotoxicity of FA-HSA-Nano-HCPT-NPs toward MCF-7 cells was concentration and time-dependent.The cytotoxic effects FA-HSA-Nano-HCPT-NPs were greater than those of raw HCPT.Cell cycle analysis of MCF-7 cells treated with FA-HSA-Nano-HCPT-NPs demonstrated a significant increase in the percent of cells in the G2/M phase compared to control(from 13.46%to 39.18%).These results suggest that the mechanism of cell toxicity by the FA-HSA-Nano-HCPT-NPs is similar to that of raw hydroxycamptothecin.That indicated FA-HSA-Nano-HCPT-NPs took HCPT into cells and released drug in cells.The antitumor assay chose nude mice inoculated MCF-7 cells and the dosage was 8 mg HCPT-kg-1 weight.The result of antitumor test in vivo shows that the antitumor activity of FA-HSA-Nano-HCPT-NPs was greater than HCPT injection and TPT.That explains FA-HSA-Nano-HCPT-NPs shows antitumor activity obviously.The result of drug tissue distribution displays in FA-HSA-Nano-HCPT-NPs group,the concentration of hydroxycamptothecin in tumors is higher than the HCPT injection group;and the drug concentrations of heart,liver,spleen and kidney in the HCPT injection group are higher than those in the FA-HSA-Nano-HCPT-NPs group.Thus it can be says the drug delivery system shows well tumor targeting.The result of bioavailability test shows the plasma concentration of HCPT derived from HCPT injection and FA-HSA-Nano-HCPT-NPs.After 30 min,the plasma concentrations of HCPT derived from HCPT injection and FA-HSA-Nano-HCPT-NPs were 0.37 and 0.54 ?g·mL-1 respectively.By 4 h,no HCPT could be detected from the HCPT injection;conversely,HCPT release from the nanoparticles persisted for up to 6 h.And the t1/2,AUC and bioavailability are all higher than HCPT injection.In conclusion,the research has prepared folate-decorated human serum albumin loaded with nano-hydroxycamptothecin nanoparticles.The folate-decorated human serum albumin loaded with nano-hydroxycamptothecin nanoparticles have a homogeneous particle size and particle size distribution.The FA-HSA-Nano-HCPT-NPs also displays controlled release in vitro and in vivo.The in vivo test indicates the folate-decorated human serum albumin loaded with nano-hydroxycamptothecin nanoparticles demonstrate high bioavailability and tumor targeting.