Preliminary Studies On Preparation And Properties Of Folate-Conjugated2-Methoxyestradiol Albumin Nanoparticles

2-Methoxyestradiol(2-ME) is a steroid hormone compound, which has beenconsidered as a new anti-cancer drug. It has relatively high specificity for fast-growing tumor cells and has no marrow and organs toxicity. However, the clinical application of2-ME has been hampered by its low solubility, poor gastrointestinal absorption,shorter half-life and low bioavailability. In this study, the bovine serum albumin(BSA)was selected as drug carriers, small molecule folic acid(FA)was selected as a targeting factor, and2-ME was selected as a model drug. we prepared folate-conjugated2-ME albumin nanoparticles, in order to maintain the high anticancer efficacy, reduce theside effects and prolong the circulation time in bloodstream.1. Preparing2-ME-FA-BSANPs and investigating related properties.In this study,2-methoxyestradiol-loaded albumin nanoparticles(2-ME-BSANPs)were prepared by desolvation method. Single factor design was used to optimize theformula and technology of2-ME-BSANPs, and the influence of concentration ofBSA, concentration of2-ME, volume of glutaraldehyde, the proportion of ethanol andwater, pH were investigated. The preparation condition of2-ME-BSANPs was thatthe concentration of BSA was40mg/ml、the concentration of2-ME was4mg/ml、pH value was10.0、the ratio of ethanol and water was3:1. And then the activatedfolic acid was conjugated to2-ME-BSANPs via surface active amino groups ofBSA(2-ME-FA-BSANPs), to improve its intracellular uptake and ability to targetspecific cells.The size and zeta potential of2-ME-FA-BSANPs were about(217.5±5.1) nmand(-37.59±1.39) mV, respectively; Encapsulation efficiency and loading amount ofthe nanoparticles were(82.50±5.80)%and(7.50±0.50)%; The SEM image indicatedthat2-ME-FA-BSANPs had a uniform size and a ball-like structure; In vitro2-MErelease study,2-ME solution group released quickly, the cumulative release capacitywas85%in12h, while the release of2-ME-FA-BSANPs was relatively slow andlasted for96h, the cumulative release capacity was85%. The results showed that thetargeting drug delivery system had a certain slow-release effect; Moreover,2-ME-FA- BSANPs with3%sucrose was lyophilized for improving its stability, the resultsshowed that the lyophilized powder was stable under4℃and room temperature forsix months.2. Investigating on the vitro antitumor activity and targeted research of2-ME-FA-BSANPsThe MTT assay was performed to measure the impact of2-ME-FA-BSANPs ongrowth inhibition of SMMC-7721tumor cells. When the action time of2-ME-FA-BSANPs was24h、48h and72h, the maximum concentration was40umol/l, thecorresponding tumor inhibition ratio were50.58%、68.53%and75.00%; while thetumor inhibition ratio of2-ME was25.28%、30.31%and35.37%. The results showedthat the tumor inhibition ratio of2-ME-FA-BSANPs were higher than2-MEgroup(P<0.05) at different times and concentrations.The two methods of fluorescence microscopy and flow cytometry were adoptedto investigate the target ability of2-ME-BSANPs and2-ME-FA-BSANPs. The resultsshowed that compared with2-ME-BSANPs group, the fluorecence intensity of2-ME-FA-BSANPs was significantly enhanced and the ratio of2-ME-FA-BSANPs intumor cells was99.8%in4h. The results indicated that2-ME-FA-BSANPs couldtransfer into the tumor cells more faster than2-ME-BSANPs and had high targetability by folate mediated; The cell-cycle arrest of2-ME-FA-BSANPs on theSMMC-7721cells was at G2/M phase and did not change the mechanism of2-ME.Cell apoptosis assay showed that2-ME-FA-BSANPs could induce more numbers ofSMMC-7721cells in apoptotic than2-ME-BSANPs and2-ME group.3. Investigating on the pharmacokinetic study of2-ME-FA-BSANPs.In this suty, sprague-Dawle (SD) rats was selected as animal model, theconcentration of2-ME in rat plasma was measured with HPLC in order to investigatethe pharmacokinetics of2-ME-FA-BSANPs. The results showed that the eliminitionhalf-life of2-ME-FA-BSANPs and2-ME was (124.61±12.00) min and (24.00±6.00)min; the average residence time of two groups was (181.03±5.20) min and(22.66±2.54) min; bioavailability AUC of two groups was (290.53±10.10)(μg/ml)min and (112.99±5.50)(μg/ml) min, respectively. The MRT, t1/2βand AUC of2-ME-FA-BSANPs were significantly higher than2-ME group (P<0.05). The targeted delivery sysytem2-ME-FA-BSNPs could significantly improve the half-life, bioavailability, reduce cytotoxicity and enhance the therapeutic effects of2-ME.