Study On Folate-Modified Bexarotene-Loaded Bovine Serum Albumin Nanoparticles

Bexarotene (BEX) is a novel retinoid drug, which could specifically activate the RXRs. It exhibits efficacious anti-cancer ability through regulating the expression of special genes. In 2000, the Food and Drug Administration (FDA) gave a license to the use of oral and gel bexarotene formulations in clinical treatments of T-cell lymphoma. In recent years, bexarotene has been gradually taken seriously in the study of lung cancer, breast cancer, ovarian. However, the clinical applications of bexarotene are limited because it is a poorly water-soluble agent and always results some side effects and low targeting ability. Therefore, novel and effective drug delivery systems should be developed to increase the drug concentration in tumor and improve the antitumor efficacy of bexarotene.Albumin is a targeting carrier material that possesses non-toxic, no antigenic, biodegradable and good biocompatible properties. Albumin-based drug delivery system is widely used in drug controlled-release and targeting system. Folic acid (FA), one of the targeting factors, could show a high affinity of tumor cells, increase the concentration of the drug in lesion partial through binding to the FA receptors in a wide variety of tumor cells such as ovarian cancer, colorectal cancer, breast cancer.In this study, the model drug was bexarotene and bovine serum albumin (BSA) was the carrier material. Fistly, desolvation technique was performed to prepare the bexarotene-loaded bovine serum albumin nanoparticles (BEX-BSANPs), and then the folate-modified bexarotene-loaded bovine serum albumin nanoparticles (FA-BEX-BSANPs) was obtained via an amidation reaction based on the amino group in the surface of BSA and the carboxyl group in FA. Finally, we investigated the construction method of this drug delivery system, physical and chemical properties in pharmaceutical, the in vitro antitumor activity and in vivo pharmacokinetic characteristics in rats and so on. The main contents of this study included:1. Preformulation study of BEX loaded nanoparticlesAccording to the UV scanning spectrum, the maximum absorption wavelength of BEX was 260 nm. This study established the determination method of BEX by high performance liquid chromatography (HPLC). The results of methodology examination showed that BEX exhibited a good linear relationship in the 0.25-80.00 μg/mL concentration range. Specificity, within-day and between-day precision, and recovery rate conformed to the requirements of the measurement. The entrapment efficiency and drug loading of the BEX loaded nanoparticles were evaluated by refrigerated ultracentrifugation which was simple and feasible. And the recovery rate also conformed to the requirements of the measurement.2. Preparation optimization of folate-modified bexarotene-loaded bovine serum albumin nanoparticles (FA-BEX-BSANPs).The optimized formulation according to single factorial investigation design was as follows:A certain amount of BSA was dissolved in distilled water and the BSA aqueous solution (1.5%, w/v) was obtained. The pH of BSA solution was adjusted to 9.0. A certain amount BEX ethanol solution was added into the BSA solution. After 1h, ethanol was added dropwise at a rate of 1.5 mL/min under 25 ℃ constant water bath and magnetic stirring of 500 r/min. The glutaraldehyde solution was added for 24 h to stabilize nanoparticles. Finally, after rotary evaporation under vacuum at 40 ℃ and three cycles of centrifugation (4 ℃,20,000 rpm,20 min), the pellet of nanoparticles was redispersed to original volum in deionized water and then pure BEX-BSA-NPs were obtained. Results of amidation coupling reaction were detailed as follows:A certain amount of FA was dissolved in DMSO, then an equal molar amount EDC and NHS were added (five-fold of FA). After 20 min, the activated FA solution (NHS-FA) was dropped into BEX-BSANPs solution (pH=10). The reaction was required for 12 h under constant stirring. After three cycles of centrifugation (4℃, 20,000 rpm,20 min), the pellet of nanoparticles was redispersed to original volume in deionized water and then pure FA-BEX-BSANPs were obtained. BEX-BSANPs and FA-BEX-BSANPs were prepared by the simple optimized process with stable entrapment efficiency. The average entrapment efficiency were 78.12±0.45% and 65.45±0.55%, and the drug loading were 7.96±0.46%,5.36±0.79% respectively. The morphologies showed that the nanoparticles were spherical with a narrow size distribution. The mean particle sizes were less than 200 nm. The zeta potentials were-25.51±1.42 mV,-33.64±1.97 mV. The average folic acid coupling efficiency was 71.28±1.93 μg FA/mg BSA.3. The formulation optimization of the FA-BEX-BSANPs lyophilized powderFreeze-dry formula of FA-BEX-BSANPs was as follows:5%mannitol was chosen as the protective agent and the pre-freeze time was 24 h, the freeze-dried time was 48 h and the freeze-dried temperature was -80 ℃. DSC and XRD of the freeze-dried nanoparticles indicated that BEX was in amorphous form rather than in crystal structure. It was proved that BEX had been completely entraped or attached into the carrier with a novel phase rather than simple physical mixture. The FA-BEX-BSANPs freeze-dried powder was stored at 4℃ and room temperature (20～25℃) for 3 months, and there was no obvious change in the appearance, particle size and distribution, entrapment efficiency and drug loading. No impact was observed on the stability of FA-BEX-BSANPs. The in vitro drug release of the freeze-dried FA-BEX-BSANPs was studied by dialysis method. The results showed in pH 7.4 PBS, FA-BEX-BSANPs exerted an apparent sustained-release profile, with an accumulated drug release rate less than 40% in 12 h,76.21±1.65% in 72 h and the durg was almost completely released in 96 h.4. In vitro antitumor activity study of BEX-loaded albumin nanoparticles on cancer cellsResults of the MTT assay performed against A549 cells and MCF-7 cells indicated that BSANPs and FA-BSANPs had no obvious toxicity on the two kinds of tumor cells. BEX-SOL, BEX-BSANPs and FA-BEX-BSANPs could inhibit proliferation of tumor cells. The inhibition capacity was significantly enhanced with FA-BEX-BSANPs in comparison to BEX-BSANPs and BEX-SOL. And compared with A549 cells [FR-negative, FR(-)], FA-BEX-BSANPs exhibited a higher inhibitory effect on MCF-7cells [FR-positive, FR(+)]. Rresults of the cellular uptake process evidenced an increasing fluorescence intensity of the cells treated with coumarin-6-BSANPs and FA-coumarin-6-BSANPs compared with free coumarin-6 for the two cells. For MCF-7cells [FR(+)], the intake of FA-coumarin-6-BSANPs was much stronger than the non-modified nanoparticles. Besides, the cellular uptake of FA-coumarin-6-BSANPs was inhibited for MCF-7 cells by free FA. These characteristics were unobservable on A549 cells [FR(-)].It could be inferred that the FA-modified nanoparticles could be taken into cells depending on the endocytosis process mediated by FA receptors on the surface of the tumor cells.5. In vivo pharmacokinetic study of BEX-loaded albumin nanoparticles in ratsOur study utilized the HPLC method and explored the in vivo pharmacokinetic behavior in rats after the tail intravenous injection of BEX solution, BEX-BSA-NPs and FA-BEX-BSANPs. The results showed that BEX-loaded nanoparticles significantly altered the drug pharmacokinetics in rats, reduced the initial concentration of the drugs and made the blood drug concentration-time curve more abiding. After the injection of BEX-BSANPs and FA-BEX-BSANPs, the MRT (o-∞) was prolonged from 2.365 h to 6.085 h and 6.435 h, the AUC(o-∞)was increased from 28.445 mg/L·h to 49.305 mg/L·h and 52.527 mg/L·h, the CL was decreaed from 0.476 L/h/Kg to 0.283 L/h/kg and 0.267 L/h/kg. The results indicated that the folic acid modified albumin nanoparticles could significantly reduce drug eliminating rate, prolong the half life and cycle time in the body of the drug, which was helpful for sustained drug release and higher bioavailability.In conclusion, this topic successfully builded a dual targeting drug delivery system of FA-BEX-BSANPs for the first time. Passive targeting was realized depending on certain particle size of the nanoparticles and active targeting was achieved by the overexpressed folate receptor in many kinds of cancer cells, which could transport the drugs to the the lesion sites. The study of this paper has not yet been reported at home and abroad, which provided new ideas to improve the therapeutic efficacy of poorly water-soluble drugs and their targeting properties, provided a safe and effective way for clinical treatment of lung cancer, breast cancer and other tumor related diseases, and had an important practical significance for the development of novel receptor-mediated tumor targeting drug delivery systems.