Studies On Brain-targeting Delivery System Of Recombinant Anti-neuroexcitation Peptide (ANEP) Loaded Nanoparticles

Recombinant anti-neuroexcitation peptide (ANEP) is a novel recombinant peptide obtained from the venom of the Chinese scorpion Buthus martensii Karsch. Experimental study of pharmacology on ANEP has showed great anti-neuroexcitation activity. In this paper, based on studies of soluble expression, isolation and purification of ANEP, the properties of ANEP was investigated, and a drug delivery system was established using N-trimethyl chitosan (TMC). The aim of this research is to gain an effective drug delivery system for ANEP, which also has some tendency of brain-targeting.In the work of soluble expression, isolation and purification of ANEP, a non-fusion expression plasmid pNJUTRX-1-ANEP-His6 encoding recombinant ANEP with a His6-Tag at its C-terminus was constructed and transformed into E. coli strain BL21 (DE3). The expressed ANEP was almost in soluble form (not less than 90%) and accounted for about 12% of the total cellular proteins. The recombinant ANEP in the cell lysate was purified to homogeneity by His Bind affinity chromatography. His residues were added to ANEP at the C-terminus in order to increase the efficiency of purification. Recombinant ANEP was purified to homogeneity with a purity of over 95%. About 3.0 mg pure recombinant ANEP-His6 was obtained from 1 L of flask culture.In order to develop a specific determination method for ANEP, polyclonal rabbit antiserum was raised against the prepared ANEP after an immunizing period lasting approximately 8 weeks and purified by protein A affinity chromatography. The activity of antibody was confirmed by Western blotting. Using the prepared antibody of ANEP, indirect ELISA and indirect competitive ELISA were established for the determination of ANEP. After optimization and comparison, the indirect ELISA was chosen as the analytical method for ANEP. The assay result was linear over a concentration range of 0.025-1.600μg/mL and R2 was 0.9994. The established Indirect ELISA method was useful to quantify the peptide in vitro and this study is the first report around world.According to gene sequence encoding ANEP, ANEP was constituted of 64 aminos with a molecular weight of 8300 Da confirmed by SDS-PAGE. The isoelectric point of ANEP was 5.2 as determined by isoelectric focusing and ANEP is a subacid peptide. The measured n-octanol/pH 7.4 PBS distribution coefficient of ANEP was 0.17 and ANEP was water-soluble peptide. Studies on stability of ANEP showed that ANEP is thermal stable and sensitive to low pH value, repeatedly freeze-thawing and long time sonification.Because ANEP is water-soluble peptide and it is sensitive to outside environment, nanoparticles based on trimethyl chitosan were designed as ANEP carriers using mild ionic crosslinking technique. Trimethyl chitosan which is the derivative of chitosan was synthesized by reductive methylation of chitosan. The structures of TMC were characterized by IR and 1H-NMR and the degree of quaternization (DQ%) was calculated. The water solubility and electric charge of the product was greatly increased. ANEP loaded nanoparticles were prepared by ionic crosslinking of TMC with tripolyphosphate (TPP). An orthogonal design was carried out at the base of single factor test to obtain higher encapsulation efficiency. The optimized formulation of nanoparticles contained TMC with a DQ of 36.1%, TPP solution with a concentration of 0.6 mg/ml, and the ratio of TMC/TPP (w/w) was 4.5. The mean encapsulation efficiency, and loading capacity ofoptimum formulation was 80.63% and 185.4μg/ml. The mean particle size, Zeta-potential, and pH value were 255 nm,32.0 mV and 6.61, respectively. The investigation of the stability of ANEP-TMC/NPs showed that the particle size of nanoparticles increased greatly after storage at 4℃over 4 weeks. In order to resolve the problem of stability, lyophilization technique could be utilized. Preparation methods and formulation were investigated, and 5% mannitol was applied for protecting nanoparticles during freeze-drying. The freezed-dried nanoparticles had good appearance without conspicuous changes of the physicochemical properties. The security of preparation was evaluated and there was no hemolysis side-effect or blood vessels stimulation observed.In the studies of pharmacokinetics and tissue distribution, ANEP was labeled with fluorescein isothiocyanate (FITC). A fluorospectrophotometry method was established and the characteristics of ANEP and ANEP-TMC/NPs in vivo were investigated. The pharmacokinetics of FITC-ANEP after intravenous administration (5,10 or 20 mg/kg) was evaluated in rats and dose-dependent pharmacokinetics was observed when a high dose (20 mg/kg) was administrated. For low and medium dose(5,10 mg/kg), linear kinetics was observed. The MRT of FITC-ANEP-TMC/NPs was a little extended than FITC-ANEP solution but there’s no significant difference (P>0.05). Studies on tissue distribution of FITC-ANEP solution and FITC-ANEP-TMC/NPs showed that:The Ce value of brain was 2.236. This indicated that the affinity to brain was significantly increased by FITC-ANEP-TMC/NPs compared with FITC-ANEP solution (P<0.01). The brain distribution of ANEP-FITC-TMC/NPs showed that NPs could be transported across BBB. It indirectly proved that the absorptive mediated transcytosis was the main reason of the phenomenon of brain targeting showed in FITC-ANEP-TMC/NPs. Drug in both groups was eliminated mainly through liver and kidney. The experiment of FITC-TMC elimination in blood showed that FITC-TMC is biodegradable and could be eliminated completely.