Synthesis Of RGD-containing Peptides,Preparation Of Liposome And Its Anti-tumor Effect

Many adhesive proteins such as fibronectin, vitronectin, collagens, and osteopontin are present in extracellular matrices and in the blood cells, which contain the Arg-Gly-Asp (RGD) sequence as their cell recognition sites. The RGD motif plays a key role in mediating integrin-matrix interaction and RGD-sequence containing peptides are known to be potent anti-adhesive molecules, as they compete for the integrin-matrix recognition process, their anti-metastatic,anti-thrombotic and anti-inflammatory effects,acute renal failure,osteoporosis and skin regeneration therapy have been largely investigated and showed relevant potential clinical applications. Therefore, the characteristic tripeptide sequence Arg-Gly-Asp has been attracting much attention of investigators.It is difficult to isolate RGD from whole tissue directly, so the adhesiveness of exogenetic RGD has been target in drug design. Many researchers tried to synthesize RGD and RGD-containing peptides by chemical or enzymatic methods. Chemical and enzymatic method has different characteristic and suit different synthesis scale. Chemical method is suitable for synthesis the moderate length peptide and with large-scale. An enzymatic method is suitable for synthesis the short peptide and with small-scale. As compared with the chemical method, the important benefits of enzymatic peptide synthesis are: a) the mild conditions of the reaction; b) the high regiospecifity of enzyme allowing the use of minimally protected substrates; c) the reaction being stereospecificity without racemization. At present, many hydrophobic small peptides were synthesized in high yield using proteases in organic media as largely reported. However, the formation of peptide bonds between hydrophilic amino acids faced many problems. Hydrophilic substrate has low solubility in hydrophobic organic solvents, hindering us to use hydrophobic organic solvents where the enzyme usually shows better activity and selectivity than in hydrophilic organic solvents. Hydrophilic organic solvents are suitable as the reaction media to enhance the solubility of hydrophilic substrates. However, hydrophilic solvents are often harmful to enzyme because it has a greater tendency to strip the tightly bound essential water from the enzyme molecules. The appropriate reaction system should be selected considering the balance of the solubility of substrates and enzymatic activity.In this study, RGD(S) is a good hydrophilic peptide model containing three charged or polar residues (Arg, Asp and Ser) and a neutral one (Gly). Considering the solubility of hydrophilic substrates, We selected hydrophilic organic solvents such as ethanol,acetonitrile as the reaction media. A new and practical enzymatic procedure for peptide synthesis using alcalase as a biocatalyst was studied. Chen et al. successfully synthesized a number of peptides in anhydrous alcohol with yields up to 94% in 1992. Recently, Klein et al also reported the synthesis of peptide bonds catalyzed by subtilisin Carlsberg in different hydrophilic organic solvents with variable water concentration. So we can deduce alcalase has better stability and activity in polar organic solvents such as alcohols, acetonitrile, DMF, etc. Alcalase is a suitable biocatalyst to catalyze peptide bond formation in hydrophilic organic solvents. Alcalase was used to catalyze the synthesis of Bz-RGD-(-NH₂)-OH (precursor tripeptide of RGD),Bz-Arg-Gly-NH₂å’ŒZ-Asp-Ser-NH₂ (precursor dipeptides of RGDS) under kinetic control condition in water-organic cosolvents systems. The synthesis reaction conditions were optimized by examining the effects of several factors including water content, temperature, pH, and reaction time on the yields. The results showedâ‘ The optimum conditions for Bz-RGD-(-NH₂)-OH synthesis were Bz-Arg-OEt·HCl (0.05M), GD-(NH₂)₂ (0.25M), triethylamine (35μl/ml system), in ethanol/0.1M This-HCl pH8.0 buffer system (85:15, V/V), 35℃, 8h with the maximum yield of 73.6%.â‘¡The optimum conditions for Bz-Arg-Gly-NH₂ synthesis were Bz-Arg-OEt·HCl (0.05M), Gly-NH₂ (0.35M), triethylamine (14μl/ml system), in acetonitrile/0.1M Na₂CO₃-NaHCO₃ pH 10.0 buffer system (90:10, V/V), 45℃, 1h with the dipeptide yield of 82.9%.â‘¢The optimum conditions for Z-Asp-Ser-NH₂ synthesis were Z-Asp-OMe (0.05M), Ser-NH₂ (0.35M), triethylamine(28μl/ml system), in acetonitrile/0.1M Na₂CO₃-NaHCO₃ pH 10.0 buffer system (90:10, V/V), 35℃, 6h with the dipeptide yield of 75.5%.In this paper, synthesis of Bz-RGD-OMe catalyzed by papain at alkaline pH under the kinetic control in full aqueous medium is described. The substrates are N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester. 0.1M KCl/NaOH aqueous solution containing 8mM EDTA and 2mM DTT was selected as the reaction medium. The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process, however, the secondary hydrolysis of the tripeptide product was not considerable. The effects of different factors, including water content, temperature, reaction time and molar ratio of the substrates, on the yield of Bz-Arg-Gly-Asp-OMe were examined. The optimal reaction conditions are 0.05M Bz-Arg-Gly-OEt and 0.15M Asp(-OMe)₂·HCl in 0.1M KCl/NaOH solution(pH 8.5), 40℃and reaction time of 60 min with the maximum conversion yield of 62%. As far as papain is concerned, its peptidase activity at pH＞8 is negligible, while the esterase activity is still significant, it is possible to synthesize soluble peptides in aqueous medium under kinetic control and alkaline pH value without obvious secondary hydrolysis of peptide product.We attempted to design a simple chemical synthesis route. RGD can be synthesized from N-terminal to C-terminal or from C-terminal to N-terminal. In previous literature, mixed anhydrides method,DCC method and activated ester method were generally applied, it is necessary that certain functional groups of amino acid such as the amino groups, the carboxyl groups and side chain groups must be blocked, otherwise this can be the source of serious complications and side reactions will occur. In addition, the protecting groups need to be removed after the peptide bond formation. In our study, we designed a novel and simple synthesis route, Firstly, Gly-Asp was synthesized by a novel chemical method in two steps including chloroacetylation of L-aspartic acid and ammonolysis of chloroacetyl L-aspartic acid. Secondly, N₀-Z-L-Arginine was reacted with Gly-Asp to yield RGD tripeptide by the N-carboxyanhydride method. Less protected amino acids were used in this synthesis. This method possessed low cost,simple and rapidity with a reasonable yield 62% calculated from arginine. In additional, compared with above method, a conventional solid phase method was also used to synthesize RGD in this paper, the yield was 75% calculated from the first amino acid anchored to resin.RGD have small molecular weight and can be easily decomposed by enzyme, their half life in vivo are very short. Some efforts had been made to increase circulating time and enhance their stability by modificating RGD's structure. The synthetic substance such as RGD repetitive sequence, RGD-conjugated with polyethylene glycol or CM-chitin; RGD-containing pseudopeptide and peptidomimetics. However, RGD amounts these linear peptides contained are limited. In this paper, RGD liposome was designed. Liposome is a new drug delivery. It has been widely used in medical area. Liposome has many characters: longest effect, target effect, slow release, reducing dose frequency. RGD liposome was prepared by reverse phase evaporation method. The impact of every factor on the encapsulation efficiency was investigated, and the factors were optimized by orthogonal design The optimum conditions for RGD liposome preparation: the mass ratio of egg phosphatidylcholine to. cholesterol 5:1, RGD 16mg, the volume of phosphate buffer 8ml, sonicating 5min, its mean size was 473nm. the highest encapsulation efficiency was 62.99%. The liposomes should be stored under lower temprerature, because the stability is not good enough under higher temperature. There is no any phenomenon of layering and emulison damage after storing under 4℃, 25℃respectively for 2 months. There is a big decrease of encapsulation efficiency when time is more than 2 months. In vitro, the release test showed RGD liposome had slow release effect.The effects of the synthetic tripeptide RGD on the proliferation,adherence and motility of HT1080,NCI-H446 and SMMC-7721 cells have been studied. The results indicated that RGD peptide had inhibitory effects on the proliferation and adherence of HT1080,NCI-H446 and SMMC-7721 cells to fibronectin. It also could restrain motility of HT1080,NCI-H446 and SMMC-7721 cells on fibronectin.