Study On Oridonin-Loaded Galactosylated Bovine Serum Albumin Nanoparticles

Oridonin (ORI), a diterpenoid compound, is isolated from Chinese herb Rabdosia rubescens. In recent years, a lot of studies have indicated that oridonin exhibits remarkable anti-tumor effects on a wide variety of cancer cells especially hepatic carcinoma and leukemia. The only marketed oridonin (Aminobterin) had been used to treat many kinds of cancer in clinic in last few years. Due to the poor water-solubility of ORI, surfactants and organic solvents were added in the injection formulation in order to improve its solubility. However, long-term intravenous injection of this product could cause many adverse reactions. Owing to its low therapeutic index, aminobterin was forced to be withdrawn from the market. No anti-cancer preparation of ORI has been on market so far. Therefore, there is a need to develop new drug delivery technologies to overcome the limits of poor water-solubility and short biological half-life of ORI and so that it can be reused in clinic.The particular advantages of serum albumin nanoparticles have attracted more and more attentions of pharmaceutical experts. Nanoparticle albumin-bound (nab) paclitaxel (Abraxane(?)) was approved and marketed in2005. It is mainly used for patients of metastatic breast cancer, and has achieved excellent effects in the clinic. This study aims to prepare ORI-loaded bovine serum albumin nanoparticles (ORI-BSA-NP). Furthermore, galactose is introduced on the surface of bovine serum albumin nanoparticles with expectation to promote the liver-targeting behavior by asialoglycoprotein receptor mediated active target effects.In this study, firstly, galactosylated bovine serum albumin (GB) was prepared by the amidation reaction of the amino group in bovine serum albumin and the carboxyl group in lactonic acid. Then, ORI loaded galactosylated bovine serum albumin nanoparticles were prepared with GB as the carrier and ORI as the model drug. The formulation was optimized by the single factorial design with the entrapment efficiency and drug loading efficiency as the main criteria. Then the development of the nanoparticles were performed by optimizing critical formulation parameters such as particle size, Zeta potential, entrapment efficiency, drug loading, morphologies and stability were examined. In order to extend the shelf life and to increase storage stability of the nanoparticles, they were freeze-dried and the type and amount of its cryoprotectants were optimized. DSC and XRD studies were used to analyze the crystalline state of ORI in the ORI-GB-NP lyophilized powder. The in vitro release behaviour and storage stability of ORI-GB-NP lyophilized powder were evaluated. Finally, pharmacokinetics and biodistribution of the ORI-GB-NP were carried out after intravenous administration in Wistar rats and Kunming mice, respectively, with the ORI-BSA-NP and ORI solution as the control groups.The optimized formulation screened by single factorial design experiments was as follows:Firstly,75mg GB was dissolved in distilled water (pH=9). Next, ORI dissolved in ethanol was added into the GB solution. Ethanol was added into the above GB solution drop by drop at a rate of0.7ml·min-1under magnetic stirring of500～600r·min-1. The nanoparticles were stabilized by the addition of0.5%glutaraldehyde solution. The crosslinking process was performed under continuous stirring of24h. Then, ethanol was removed by rotary evaporation under vacuum at40℃. Finally, free drug and glutaraldehyde were eliminated by washing the nanoparticle solution using an AmiconUltra-4centrifugal filter. ORI-BSA-NP was prepared under the same protocol as that used for ORI-GB-NP. The only difference between two types of nanoparticles was that GB was replaced by BSA. The optimized preparation was simple and convenient. The entrapment efficiency and drug loading were consistent among different batches. The average entrapment efficiencies of ORI-BSA-NP and ORI-GB-NP were66.7±0.4%.63.5±0.7%, respectively. The average drug loading of ORI-BSA-NP and ORI-GB-NP were4.26±0.03%、4.06±0.04%, respectively. The mean particle sizes of ORI-BSA-NP and ORI-GB-NP were164.5±6.5nm172.0±8.3nm, respectively. The zeta potentials were-25.39±1.62mV、-31.48±2.15mV, respectively. The morphologies of ORI-BSA-NP and ORI-GB-NP visualized by TEM were nearly spherical with the distribution of unimodal and relatively narrow particle sizes.ORI-GB-NP was freeze-dried as the following process. Firstly,5%mannitol, the cryoprotectant, was added into ORI-GB-NP and then mixed until completely dissolved. The nanoparticle dispersion was filled into each cillin-glass bottle. Then, the cillin-glass bottles were pre-frozen at-80℃C in a ultra-low temperature freezer for24h. The frozen nanoparticles were freeze dried for48h (-50℃、0.25mbar) to obtain the ORI-GB-NP lyophilized powder. Differential scanning calorimetry (DSC) and X-ray diffraction confirmed the amorphous state of ORI in the ORI-GB-NP freeze-dried powder. After stored under the condition of4℃and20-25℃for three months, the ORI-GB-NP lyophilized powder was still dispersed uniformly with no change in the entrapment efficiency and drug loading. The lyophilized powder exhibited good storage stability. Dialysis method was used to evaluate the in vitro release behavior of ORI-GB-NP. Results indicate that the release of ORI-GB-NP behaved in a biphasic pattern with the initial burst and subsequently sustained release.The study of the pharmacokinetic property in rats showed that the blood concentration-time curves of ORI-GB-NP and ORI-BSA-NP were obviously different from that of the ORI solution. The blood concentration-time curve of the ORI solution was steeper than that of ORI-GB-NP and ORI-BSA-NP. The blood concentration of ORI was rapidly declined with the MRT of2.56h, CLz of0.678L·h-1·Kg-1and AUC of29.51h·μg·mL-1. Compared with ORI solution, ORI-BSA-NP had a prolonged MRT of5.65h, lower CLz of0.286L·h-1·Kg-1and higher AUC of48.96·μg·ml-1. Compared with the ORI solution and ORI-BSA-NP, ORI-GB-NP had a slightly longer MRT of6.57h, a little lower CLz of0.224L·h-1·Kg-1and higher AUC of62.42h·μg·mL-1. It could be concluded that ORI-GB-NP and ORI-BSA-NP could improve the availability of oridonin by the slow release of ORI and prolong the drug circulation time in vivo.The results of biodistribution in different tissues of mice with the tail intravenous injection of the ORI solution, ORI-BSA-NP and ORI-GB-NP were as follows: compared with the ORI solution, ORI-BSA-NP had marked different relative uptake efficiencies of3.65,2.61,0.92,0.91and0.78in the mouce liver, spleen, heart, kidney and lung, respectively. The relative uptake efficiencies of ORI-GB-NP were5.47,1.74,0.82,0.71and0.63in the mouce liver, spleen, heart, lung and kidney, respectively. The tissue distribution data indicates that both of ORI-BSA-NP and ORI-GB-NP had liver-targeting characteristics. Moreover, the liver-targeting effect of ORI-GB-NP was more pronounced than that of ORI-BSA-NP. The results demonstrated that ORI-BSA-NP and ORI-GB-NP, especially ORI-GB-NP could enhance the accumulation of ORI in the mouse liver and reduce the toxicity to other tissues.The novel liver targeting effect of oridonin loaded galactosylated bovine serum albumin nanoparticles were first discovered in this work. This study will certainly provide the experimental methodologies for the development of drug delivery systems with bovine serum albumin nanoparticles and galactose mediated liver targeting. In addition, the further nonclinical and clinical evaluations of the novel oridonin nanoparticles described in this work may bring a new oridonin clinical medicine into the market for treatment of hepatocellular carcinoma.