RGD序列肽的合成、脂质体的制备及抗肿瘤作用的研究
摘要
RGD三肽序列是细胞外基质及体内多种粘附蛋白分子所共有的细胞粘附和分子识别位点，是一类含有Arg，Gly和Asp三种氨基酸的小肽。RGD三肽及其衍生物在抗肿瘤、抗血栓、治疗急性肾衰、抗炎、治疗骨质疏松、皮肤再生及生物材料工程等方面的研究已有越来越多的报道。由于RGD具有广泛的应用前景，所以已经成为极具研究价值的生物活性肽之一。本论文主要围绕RGD肽的合成、制剂及生物活性方面进行了研究。
一、Alcalase酶催化下的RGD肽合成
本文以工业用碱性蛋白酶alcalase作为催化剂，在动力学控制下合成了RGD的前体三肽Bz-RGD-(-NH_2)-OH，RGDS的前体二肽Bz-Arg-Gly-NH_2和Z-Asp-Ser-NH_2。通过考察溶剂体系种类、水含量、pH值、温度、反应时间、底物摩尔比等因素对酶促肽合成产率的影响，得出最佳的反应条件。
Bz-RGD-(-NH_2)-OH合成是通过酶法和化学法相结合来完成的。这里所采用的化学法可以低成本、大规模地合成GD-(NH_2)_2。Bz-Arg-OEt和GD-(NH_2)_2之间的键合是通过酶法完成的。以alcalase为催化剂，Bz-Arg-OEt和GD-(NH_2)_2为底物，在水-有机溶剂双相体系中合成目标产物。合成该三肽的最佳反应条件是：Bz-Arg-OEt·HCl(0.05M)，GD-(NH_2)_2(0.25M)，三乙胺(35μl／ml体系)，乙醇／0.1M Tirs-HCl pH 8.0缓冲液体系(85：15，V／V)，35℃，反应时间8小时，产率达73.6％。
Bz-Arg-Gly-NH_2、Z-Asp-Ser-NH_2是合成RGDS的两个前体二肽，本文以alcalase为催化剂，分别以Bz-Arg-OEt·HCl、Z-Asp-OMe为酰基供体，以Gly-NH_2、Ser-NH_2为亲核试剂，在有机溶剂／水双相体系中合成目标产物。Bz-Arg-Gly-NH_2合成的最佳反应条件：Bz-Arg-OEt·HCl(0.05M)，Gly-NH_2(0.35M)，三乙胺(14μl／ml体系)，乙腈／0.1M Na_2CO_3-NaH CO_3 pH10.0缓冲液(90：10，V／V)，45℃，反应时间1小时，产率达82.9％。Z-Asp-Ser-NH_2合成的最佳反应条件：Z-Asp-OMe(0.05M)and Ser-NH_2(0.35M)，三乙胺(28μl／ml体系)，乙腈／0.1 M Na_2CO_3-NaHCO_3 pH 10.0缓冲液(90：10，V／V)，35℃，反应时间6小时，产率达75.5％。
由上述结果我们可以得出如下结论：Alcalase酶在含有少量水的亲水性溶剂中(如乙醇、乙腈等)有很好的稳定性和催化活性，使亲水性小肽酶促合成可以在亲水性的溶剂中进行，解决了在亲水性小肽合成中需要兼顾底物溶解性和酶的活性及稳定性的问题。此外，从经济成本的角度出发，Alcalase是工业化酶，成本低，离心很容易沉淀下来，易于与产物分离并可以考虑重复使用的问题，适合规模化生产。尽管Alcalase酶目前还没有广泛用于肽合成中，但是它有望成为亲水性小肽合成中很有前景的催化剂。
二、木瓜蛋白酶催化下的RGD肽合成
本文研究了在纯水相体系中，碱性pH条件下，木瓜蛋白酶催化的Bz-RGD-OMe的合成，尽管在反应过程中合成的亲水性肽溶于反应体系中，但肽产物的二次水解不显著。最佳反应条件：Bz-RG(-OEt)和D(-OMe)_2·HCl分别为0.05M和0.15M，0.1M KCl／NaOH溶液作为反应介质(含有8mM EDTA和2mM DTT)，pH8.5，40℃，反应时间为60min，最大转化率为62％。
上述结果进一步证实，对于木瓜蛋白酶来说，采用动力学控制，在pH＞8时其水解酶的活性受到抑制，酯酶活性却很明显。在这个条件下能够合成出可溶性肽，并且不会发生二次水解。
三、RGD肽的化学法合成
本文根据RGD结构上有一个Gly这个特点，巧妙地设计了RGD合成的新路线。GD二肽合成采用了新颖的化学法，包括天冬氨酸的氯乙酰化和酰化产物的氨解。三肽的合成采用N-羧基内酸酐法，首先将苄氧羰酰精氨酸用催化剂催化使生成N-羧基内酸酐精氨酸，然后与游离的GD二肽缩合生成RGD三肽。这一路线中采用了较少保护的氨基酸，路线低成本、简单、快速，产率为62％。
四、脂质体制备
本文采用逆相蒸发法制备了RGD脂质体，在制备过程中对各种影响因素进行了考察，采用正交实验进行优化，确定最佳的工艺条件：即卵磷脂／胆固醇比例为5：1，RGD 16mg，PBS 8ml，超声时间5min。制得的脂质体平均粒径为473nm。包封率62.99％。稳定性实验证明RGD脂质体在4℃低温存放时较稳定，温度升高稳定性下降。随时间的延长包封率有下降的趋势。尤其是超过两个月包封率急剧下降。体外释放度实验证明RGD脂质体有一定的缓释效果。
五、RGD抗肿瘤作用
本文对上述化学法合成RGD肽进行了活性检测，细胞增殖实验、细胞粘附实验、细胞迁移实验证明：RGD肽对人纤维肉瘤细胞HT1080、人小细胞肺癌细胞NCI-H446、人肝癌细胞SMMC-7721增殖、在FN上粘附及迁移有一定的抑制作用。
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-(-NH_2)-OH (precursor tripeptide of RGD)、Bz-Arg-Gly-NH_2和Z-Asp-Ser-NH_2 (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 showed①The optimum conditions for Bz-RGD-(-NH_2)-OH synthesis were Bz-Arg-OEt·HCl (0.05M), GD-(NH_2)_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%.②The optimum conditions for Bz-Arg-Gly-NH_2 synthesis were Bz-Arg-OEt·HCl (0.05M), Gly-NH_2 (0.35M), triethylamine (14μl/ml system), in acetonitrile/0.1M Na_2CO_3-NaHCO_3 pH 10.0 buffer system (90:10, V/V), 45℃, 1h with the dipeptide yield of 82.9%.③The optimum conditions for Z-Asp-Ser-NH_2 synthesis were Z-Asp-OMe (0.05M), Ser-NH_2 (0.35M), triethylamine(28μl/ml system), in acetonitrile/0.1M Na_2CO_3-NaHCO_3 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, N_0-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.
一、Alcalase酶催化下的RGD肽合成
本文以工业用碱性蛋白酶alcalase作为催化剂，在动力学控制下合成了RGD的前体三肽Bz-RGD-(-NH_2)-OH，RGDS的前体二肽Bz-Arg-Gly-NH_2和Z-Asp-Ser-NH_2。通过考察溶剂体系种类、水含量、pH值、温度、反应时间、底物摩尔比等因素对酶促肽合成产率的影响，得出最佳的反应条件。
Bz-RGD-(-NH_2)-OH合成是通过酶法和化学法相结合来完成的。这里所采用的化学法可以低成本、大规模地合成GD-(NH_2)_2。Bz-Arg-OEt和GD-(NH_2)_2之间的键合是通过酶法完成的。以alcalase为催化剂，Bz-Arg-OEt和GD-(NH_2)_2为底物，在水-有机溶剂双相体系中合成目标产物。合成该三肽的最佳反应条件是：Bz-Arg-OEt·HCl(0.05M)，GD-(NH_2)_2(0.25M)，三乙胺(35μl／ml体系)，乙醇／0.1M Tirs-HCl pH 8.0缓冲液体系(85：15，V／V)，35℃，反应时间8小时，产率达73.6％。
Bz-Arg-Gly-NH_2、Z-Asp-Ser-NH_2是合成RGDS的两个前体二肽，本文以alcalase为催化剂，分别以Bz-Arg-OEt·HCl、Z-Asp-OMe为酰基供体，以Gly-NH_2、Ser-NH_2为亲核试剂，在有机溶剂／水双相体系中合成目标产物。Bz-Arg-Gly-NH_2合成的最佳反应条件：Bz-Arg-OEt·HCl(0.05M)，Gly-NH_2(0.35M)，三乙胺(14μl／ml体系)，乙腈／0.1M Na_2CO_3-NaH CO_3 pH10.0缓冲液(90：10，V／V)，45℃，反应时间1小时，产率达82.9％。Z-Asp-Ser-NH_2合成的最佳反应条件：Z-Asp-OMe(0.05M)and Ser-NH_2(0.35M)，三乙胺(28μl／ml体系)，乙腈／0.1 M Na_2CO_3-NaHCO_3 pH 10.0缓冲液(90：10，V／V)，35℃，反应时间6小时，产率达75.5％。
由上述结果我们可以得出如下结论：Alcalase酶在含有少量水的亲水性溶剂中(如乙醇、乙腈等)有很好的稳定性和催化活性，使亲水性小肽酶促合成可以在亲水性的溶剂中进行，解决了在亲水性小肽合成中需要兼顾底物溶解性和酶的活性及稳定性的问题。此外，从经济成本的角度出发，Alcalase是工业化酶，成本低，离心很容易沉淀下来，易于与产物分离并可以考虑重复使用的问题，适合规模化生产。尽管Alcalase酶目前还没有广泛用于肽合成中，但是它有望成为亲水性小肽合成中很有前景的催化剂。
二、木瓜蛋白酶催化下的RGD肽合成
本文研究了在纯水相体系中，碱性pH条件下，木瓜蛋白酶催化的Bz-RGD-OMe的合成，尽管在反应过程中合成的亲水性肽溶于反应体系中，但肽产物的二次水解不显著。最佳反应条件：Bz-RG(-OEt)和D(-OMe)_2·HCl分别为0.05M和0.15M，0.1M KCl／NaOH溶液作为反应介质(含有8mM EDTA和2mM DTT)，pH8.5，40℃，反应时间为60min，最大转化率为62％。
上述结果进一步证实，对于木瓜蛋白酶来说，采用动力学控制，在pH＞8时其水解酶的活性受到抑制，酯酶活性却很明显。在这个条件下能够合成出可溶性肽，并且不会发生二次水解。
三、RGD肽的化学法合成
本文根据RGD结构上有一个Gly这个特点，巧妙地设计了RGD合成的新路线。GD二肽合成采用了新颖的化学法，包括天冬氨酸的氯乙酰化和酰化产物的氨解。三肽的合成采用N-羧基内酸酐法，首先将苄氧羰酰精氨酸用催化剂催化使生成N-羧基内酸酐精氨酸，然后与游离的GD二肽缩合生成RGD三肽。这一路线中采用了较少保护的氨基酸，路线低成本、简单、快速，产率为62％。
四、脂质体制备
本文采用逆相蒸发法制备了RGD脂质体，在制备过程中对各种影响因素进行了考察，采用正交实验进行优化，确定最佳的工艺条件：即卵磷脂／胆固醇比例为5：1，RGD 16mg，PBS 8ml，超声时间5min。制得的脂质体平均粒径为473nm。包封率62.99％。稳定性实验证明RGD脂质体在4℃低温存放时较稳定，温度升高稳定性下降。随时间的延长包封率有下降的趋势。尤其是超过两个月包封率急剧下降。体外释放度实验证明RGD脂质体有一定的缓释效果。
五、RGD抗肿瘤作用
本文对上述化学法合成RGD肽进行了活性检测，细胞增殖实验、细胞粘附实验、细胞迁移实验证明：RGD肽对人纤维肉瘤细胞HT1080、人小细胞肺癌细胞NCI-H446、人肝癌细胞SMMC-7721增殖、在FN上粘附及迁移有一定的抑制作用。
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-(-NH_2)-OH (precursor tripeptide of RGD)、Bz-Arg-Gly-NH_2和Z-Asp-Ser-NH_2 (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 showed①The optimum conditions for Bz-RGD-(-NH_2)-OH synthesis were Bz-Arg-OEt·HCl (0.05M), GD-(NH_2)_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%.②The optimum conditions for Bz-Arg-Gly-NH_2 synthesis were Bz-Arg-OEt·HCl (0.05M), Gly-NH_2 (0.35M), triethylamine (14μl/ml system), in acetonitrile/0.1M Na_2CO_3-NaHCO_3 pH 10.0 buffer system (90:10, V/V), 45℃, 1h with the dipeptide yield of 82.9%.③The optimum conditions for Z-Asp-Ser-NH_2 synthesis were Z-Asp-OMe (0.05M), Ser-NH_2 (0.35M), triethylamine(28μl/ml system), in acetonitrile/0.1M Na_2CO_3-NaHCO_3 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, N_0-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.
引文
[1]Pierschbacher, M.D.; Ruolslahti, E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984,309,30-33.
[2] Pierschbacher, M.D.; Hayman, E.G.; Ruoslahti E. Location of the Cell-attachment site in Fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell 1981,26, 259-267.
[3] Yamada, K.M.; Kennedy D.W. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. Cell Biol. 1984, 99,29-36.
[4] Hayman, E.G.; Pierschbacher, M.D.; Ruolslahti, E. Detachment of cells from culture substrate by soluble fibronectin peptides. J.Cell Biol. 1995, 100,1948-1954.
[5] Pierschbacher, M.D.; Ruolslahti, E. Variants of the cell recognition site of fibronectin that retain attachment-promoting activity.Proc. Nalt. Acad. Sci. USA 1984, 81,5985-5988.
[6] Ruolslahti, E.; Pierschbacher, M.D. New perspectives in cell adhesion: RGD and integrins. Science 1987, 238,491-497.
[7] Juliano, R.L.; Haskill, S. Signal transduction from the extracellular matrix. J. Cell Biol. 1993,120, 577-585.
[8] Plow, E.F.; Haas, T.A.; Zhang, L.; Loftusi, J.; Smith, J.W. Ligand Binding to Integrins. J. Biol. Chem. 2000, 275(29), 21785-21788.
[9] Ruoslahti, E. Integrins. J Clin Invest 1991, 87, 1-5.
[10] Larson, R.S.; Springer, T.A. Structure and function of leukocyte integrins. Immunol. Rev. 1990,114, 181-217.
[11] Luscinskas, F.W.; Lawler, J. Integrins as dynamic regulators of vascular function. FASEB J 1994, 8, 929-938.
[12] Hemler, M.E. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 1990, 8, 365-400.
[13]Spinger, T.A. Adhesion receptors of the immune syntem. Nature 1990, 346, 425-434.
[14]Elices, M.J.; Osborn, L.; Takada, Y.; et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 1990, 60, 577-584.
[15]Mould, A.P.; Wheldon, L.A.; Komoriya, A.; et al. Affinity chromatographic islation of the melanoma adhesion receptor for the ⅢCS region of fibronectin and its identification as the integrin α_4β_1. J. Biol. Chem. 1990, 265, 4020-4024.
[16]Ginsberg, M.H.; Loflus, J.C.; Plow, E.F. Cytoadhesions, integrins, and platelets. Thromb. Haemostasis 1988, 59, 1-6.
[17]Litta, L.A.; Steter Stevenson, W.G. Tumor invation and metastasis: an inbalance of positive and negative regulation. Cancer Res. 1991, 51 (supp 1), 5054-5061.
[18]汤钊猷主编，现代肿瘤学。上海医科大学出版社，1993年，1-5．
[19]细胞生物学教程，绍兴文理学院生命科学学院，田润刚博士主讲。http://www.cella.cn/jxck/index.htm.
[20]Liotta, L.A. Tumor Invasion and Metastases-Role of the Extracellullar Martrix: R.hoacls Memorial Award Lecture. Cancer Res. 1986, 46, 1-7.
[21]Aznavoorian, S.; Syetler-Stevenson W.G.; Liotta, L.A. Molecular aspects of tumor cell invasion and metastasis. Cancer, 1993, 71, 1368-1383.
[22]Honn, K.V.; Tang, D.G.; Crissman, J.D. Platelets and cancer metastasis: a causal relationship. Cancer Metastasis Rev. 1992, 11(3/4), 325-351.
[23]Mohri, H.; Ohkubo, T. The role of RGD peptides and the Y chain peptides of fibrinogen on fibrinogen binding to activated platelets. Peptides 1993, 14(2), 353-357.
[24]王树人。细胞粘附分子的生理和病理生理。四川生理科学杂志 1996，18(3)，24—28．
[25]Hillis, G.S.; MacLeod, A.M. Integrins and disease. Clin Sci (Lond). 1996(Dec), 91(6), 639-650.
[26]Price, J.T.; Bonovich, M: T.; Kohn, E. C. The biochemistry of cancer dissemination. Crit. Rev. Biochem. Mol. Biol. 1997, 32(3), 175-253.
[27]Naruo, K. Organ-specific adhesion of neuroblastoma cells in vitro: correlation with their hepatic metastasis potential. J. Ped. Surg. 1997, 32(4), 546-551.
[28]Humphries, M.J.; Olden, K.; Yamada, K.M. A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science 1986, 233(4762), 467-470.
[29]Hirasawa, M.; Shijubo, N.; Uede;.T.; et al. Integrin expression and ability to adhere to extracellular matrix protein and endothelials cells in human lung cancer lines. Br. J. Cancer 1994, 70(3), 466-472.
[30]Kang, I.C.; Kim, D.S.; Jang, Y.; et al. Suppressive mechanism of salmosin, a novel disintegrin in B16 melanoma cell metastasis. Biochem Biophys Res Commun. 2000, 275(1), 169-173.
[31]吴泽志，黄岂平，肖国辉等。肝癌细胞—胞外基质粘附性与粘附识别的相关性。生物物理学报，2000，16(4)，831-838．
[32]Cui, G.P.H.; Puddefoot, J.R.; Vision, G.P.; et al. Altered cell-matrix contact: a prerequisite for breast cancer metastasis. Br. J. Cancer 1997, 75(5), 623-630.
[33]Nierodzik, M.L.; Klepfish, A.; Karpatkin, S.; et al. Role of platelets, thrombin, integrin Ⅱb/Ⅲa, fibronectin, and von Willebrand factors on tumor adhesion in vitro and metastasis in vivo. Thromb Haemost 1995, 74(3), 282-489.
[34]赵明，彭师奇，迪丽努尔等。含RGD四肽的合成与功能。药学学报，1997，32(4)，271．
[35]Scherbarth, S.; Orr, F.W. Intravital videomicroscopic evidence for regulation of metastasis by the hepatic microvasculutre: effect of interleukinla on metastasis and the location of melanoma cell arrest. Cancer Res. 1997, 57(15), 4105-4111.
[36]Barinaga, M. Cell suicide: by ICE, not fire. Science 1994, 263(5148), 754-756.
[37]Anuradha, C.D.; Kanno, S.; Hirano, S. RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation. Cell Biol. Toxicol. 2000,16 (5), 275-283.
[38]Chen, Y.; Xu, X.; Hong, S.; et al. RGD-Tachyplesin inhibits tumor growth. Cancer Res. 2001, 61 (6), 2434-2438.
[39]Buckley, C.D.; Pilling, D.; Henriquez, N.V.; et al. RGD peptides induce apoptosis by direct caspase-3 activation. Nature 1999, 397(6719), 534-539.
[40]Moreno, M. V.; Lucio, C. J.; Konta, T.; et al. Enhancement of TNF-alpha-induced apoptosis by immobilized arginine-glycine-aspartate: involvement of a tyrosine kinase-dependent,MAP kinase-independent mechanism. Biochem. Biophys. Res. Commun. 2000, 277(2), 293-298.
[41]Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 1971,285(21), 1182-1186.
[42] Folkman, J.; Shing, Y. Angiogenesis. J Biol Chem. 1992,267 (16), 10931-10934.
[43] Folkman, J. Angiogenesis-dependent diseases. Semin. Oncol. 2001, 28(6), 536-542.
[44] Hodivala-Dilke, K.M.; Reynolds, A.R.; Reynolds, L.E. Integrins in angiogenesis: multitalented molecules in abalancing act. Cell Tissue Res. 2003,314(1), 131-144.
[45] Kerbel, R.; Folkman, J. Clinical translation of angiogenesis inhibitors. Nat. Rev. Cancer. 2002,2 (10), 727—739.
[46] Hynes, R.O. A revaluation of integrins as regulators of angiogenesis. Nature Med. 2002, 8 (9), 918-921.
[47] Hynes, R.O. Integrins: bidirectional, allosteric signaling machines. Cell 2002, (110),673—687.
[48] Brooks, P.C.; Clark, R.A. Cheresh, D.A. Requirement of vascular integrin alpha v beta3 for angiogenesis. Science 1994,264(5158), 569-571.
[49] Maile, L.A.; Badley-Clarke, J.; Clemmons, D.R. Structural analysis of the ro le of the beta 3 subunit of the alpha V beta 3 integrin in IGF-I signaling.J Cell Sci. 2001,114(7), 1417-1425.
[50] Carlson, T.; Feng, Y.; Maisonpierre, P.; Mrksich, M.; Morla, A.O. Direct cell Adhesion to the angiopoietins mediated by integrins. J. Biol. Chem. 2001, 276(28), 26516—26525.
[51] Kim, S.; Bell, K.; Mousa, S.A.; Varner, J.A. Regulation of angiogenesis in vivo by ligation of integrin alpha 5 betal with the central cell-binding domain of fibronectin. Am. J. Pathol. 2000, 56(4), 1345 -1362.
[52]Huang, T.F.; Yeh, C.H.; Wu, W.B. Viper venom components affecting angiogenesis. Haemostasis 2001, 31 (3-6), 192-206.
[53]Fabio, P.; Chiara, B.; Danilo, M.; et al. Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposornal chemotherapy. Cancer Res. 2003, 63, 7400-7409.
[54]Zarovni, N.; Monaco, L.; Corti, A. Inhibition of Tumor Growth by Intramu scular Injection of cDNA Encoding Tumor Necrosis Factora Coupled to NGR and RGD Tumor-Homing Peptides. Hum. Gene Ther. 2004, 15,373-382.
[55]Curnis, F.; Gasparri, A.; Sacchi, A.; et al. Coupling Tumor Necrosis Factor-a with a_v Integrin Ligands Improves Its Antineoplastic Activity. Cancer Res. 2004, 64, 565-571.
[56]Pasqualini, R..; Koivunen, E.; Ruoslahti, E. a_v integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol. 1997, 15, 542-546.
[57]肖斌，朱永红。RGD肽在肿瘤治疗中的研究进展。中国肿瘤临床 2005，32(19)，1135-1137．
[58]Koizumi, N.; Mizuguchi, H.; Hosono, T.; et al. Efficient gene transfer by f iber-mutant adenoviral vectors containing RGD peptide. Biochim Biophys Acta 2001, 1568(1), 13-20.
[59]Witlox, A.M.; Van Beusechem, V.W.; Molenaar, B.; et al. Conditionally replicative adenovirus with tropism expanded towards integrins inhibits osteosarcoma tumor growth in vitro and in vivo. Clin Cancer Res. 2004, 10(1 Pt 1), 61-67.
[60]Grill, J.; van Beusechem, V.W.; van Der Valk, P.; et al. Combined targeting of adenoviruses to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroids. Clin Cancer Res. 2001, 7(3), 641-650
[61]Wu, H.; Han, T.; Lam, J.T.; et al. Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy. Gene Ther. 2004, 11(10), 874-878.
[62]Wesseling, J.G.; Bosma, P.J.; Krasnykh, V.; et al. Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin targeted adenoviral vectors. Gene Ther. 2001, 8(13), 969-976
[63]李前伟。肿瘤整合素αvβ3受体显像的研究现状。国外医学放射医学核医学分册，2003，27(5)，198-200．
[64]Su, Z.F.; Liu, G.; Gupta, S.; et al. In vitro and in vivo evaluation of a Technetium-99m-labeledcyclic RGD peptide as a specific marker of alpha (V) beta (3) integrin for tumor imaging. Bioconjug. Chem. 2002, 13(3), 561-570.
[65]Pytela, R.; Pierschbacher, M.D.; Ginsberg, M.H; et al. Platelet membrane glycoprotein Ⅱb/Ⅲa is a member of a family of Arg-Gly-Asp-specific adhesion receptors. Science, 1986, 231, 1559-1562.
[66]Goligorsky, M.S.; Noiri, E.; Kessler, H.; Romanov, V. Therapeutic potential of RGD peptides in acute renal injury. Kidney Int. 1997, 51, 1487-1492.
[67]Yi, Y.; Carderelli, P. M.; et al. LPAM-1 (integrin alpha 4 beta 7)-ligand binding: Overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen, a fibronectin-like polymer and RGD-like cyclic peptides. Eur. J. Immuno. 1998,28(3), 995-1004.
[68] Illana, B.; Zaballos &AACUTE.; Blanco, L.; Salas, M. The RGD sequence in phage 029 terminal protein is required for interaction with (?)29 DNA polymerase. Virology 1998,248(1), 12-19.
[69] Lin, N.; Zhao, M.; Wang, C.; et al. Synthesis and antithrombotic activity of carbolinecarboxyl RGD Sequence. Bioorg & Med Chem Lett. 2002, 12(4), 585-587.
[70] Zhang, L.H.; Pesti, J.A.; Costello, J.D. An efficient synthesis of cyclic RGD peptides as antithrombotic agents. J.Org.Chem. 1996, 61, 5180-5185.
[71] Meredith, J.E.; Fazeli, B.; Schwartz, M. A. The extracellular matrix as a cell survival factor. Mol Biol Cell, 1993,4(9), 953-961.
[72] Ouaissi, M.A.; Cornette, J.; Afchain, D. Trypanosoma Cruzi infection inhibited by peptides modeled from A fibronectin cell attachment domain. Science 1986,234, 603-607.
[73] Davis, D.H.; Giannoulis, C.S.; Johnson, R.W.; et al. Immobilization of RGD to <111>silicon surfaces for enhanced cell adhesion and proliferation. B iomaterials, 2002,23(19), 4019-4027.
[74] Lin, H.; Sun, W.; Mosher, D.F.; et al. Synthesis Surgace and cell-adhesion properties of polyurethanes containing covalently grafted RGD-Peptides. J. Biomed Mater Res. 1994,28, 329.
[75] Gill, L; Lopez-Fandino, R.; Jorba, X.; et al. Biologically Active Pep tides and Enzymatic Approaches to their Production. Enzyme Microb Technol. 1996,18,162-183.
[76] Erbeldinger, M.; Ni, X. W.; Hailing, P. J. Enzymatic Synthesis with Mainly Undissolved Substrates at Very H igh Concentrations. Enzyme Microb. Technol. 1998, 23, 141-148.
[77]Erbeldinger, M.; Hailing, P.J. Effect of Water and Enzyme Concentration on Thermolysin-Cataiyzed Solid to Solid Peptide Synthesis. Biotech. Bioeng. 1998, 59, 68-72.
[78]Lopez-Fandino, R.; Gill, I.; Vulfson, E. N. Enzymatic Catalysis in Heterogeous Mixtures of Subst rates: The Role of the Liqiud Phase and the Effects of"Adjuvants". Biotech. Bioeng. 1993, 43, 1016-1023.
[79]Erbeldinger, M.; Hailing, P.J. Thermodynamics of Solid to Solid Conversion and Application to Enzymic Peptide Synthesis. Enzyme Microb. Technol. 1995, 17, 601-606.
[80]黄惟德，陈常庆。多肽合成，科学出版社，第一版，1985．
[81]Valembois, C.; Mendre, C.; Cavadore, J.C.; Calas, B. Continuous flow synthesis of a biologically active small protein and a large peptide. Tetrahedron Lett. 1992, 33, 4005-4008.
[82]Sheehan, J.C.; Hess, G.P. A New Method of Forming Peptide Bonds. J. Am. Chem. Soc. 1955, 77(4), 1067-1068.
[83]Albertson, N.P. Synthesis of Peptides with mixed Anhydride in 'Organic Reagents'. 1962, 12, 157-355.
[84]Boissonnas, R.A. A new method of peptide synthesis. Helv Chim Acta. 1951, 34(3), 874-879.
[85]Vaughan, J. R.; Osato, J.R.L. The Preparation of Peptides Using Mixed Carbonic-Carboxylic Acid Anhydrides. J. Am. Chem. Soc. 1952, 74, 676-678.
[86]Wieland, T.; Schafer, W. Synthese von Oligopeptiden unter zellmoglichen Bedingungen. Angew. Chem. 1951, 63(6), 146-147.
[87]Schwyzer, R.; Iselin, B.; Feurer, M. Uber aktivierte Ester. I. Aktivierte Ester der Hippursaure und ihre Umsetzungen mit Benzylamin. Helv Chim Acta.1955, 38(1), 69-79.
[88]Bodanszky, M. Synthesis of peptides by aminolysis of nitrophenyl esters. Nature 1955, 175 (4459), 685-686.
[89]Curtius, T. Synthetische Versuche mit Hippurazid. Ber. Deutsch. Chem. Ges. 1902, 35, 3226-3228.
[90]Denkewalter, R.G.; Schwam, H.; Strachan, R.G.; et al. The Controlled Synthesis Of Peptides in Aqueous Medium I. The Use of α-Amino Acid N-Carboxyanhydrides. J. Am. Chem. Soc. 1966, 88, 3163-3164.
[91]Hirschmann, R.; et al. The controlled synthesis of peptides in aqueous medium. 3. Use of Leuchs' anhydrides in the synthesis of dipeptides. Mechanism and control of side reactions. J. Org. Chem. 1967, 32, 3415-3425.
[92]Veber, D.; Hirschmann, R.; Denkewalter, R.G. Synthesis of peptides in aqueous medium. Ⅵ. Synthesis of an unsymmetrical cystine peptide fragment of insulin. J. Org. Chem 1969, 34, 753-755.
[93]Iwakura, Y.; Uno, K.; Oya, M.; Katakai, R. Stepwise synthesis of oligopeptides with N-carboxy alpha-amino acid anhydrides. Biopolymers 1970, 9, 1419-1427.
[94]大屋正尚，片贝良一。有机合成化学协会志，1967，29，751．
[95]Merrifield, R.B. Peptide Synthesis on a Solid Polymer. Fed. Proc. 1962, 21(2), 412.
[96]Letsingerand, R.L.; Cornet, M.J.J. Am. Chem. Soc. 1963, 3045, 85.
[97]周云赟 吉林大学硕士论文 2002年
[98]Bashkin, J.K.; Mcbeath, R.J.; Modak, A.S.; et al. Synthesis and characterization of oligonucleotide peptides. J. Org. Chem. 1991, 56(9), 3168-3176.
[99]De Bont, D.B.A.; Moree, W.J.; Van Boom J.H.; et al. Solid-phase synthesis of o-phasphorathioylserine and threonine-Containing peptides as well as of o-phosphoserine-and-threonine-containing peptides. J.Org.Chem. 1993, 58(6), 1309-1317.
[100]Cohen-Auifieid, S.T.; Lansbury, P.T. A practical convergent method for glycopeptide synthesis. J. Am. Chem. Soc. 1993, 115(23), 10531-10537.
[101]叶蕴华，范崇旭，张德仪等，四个新型有机磷缩合试剂的合成及其在生物活性肽合成中的应用。高等学校化学学报，1997，18，1086—1092．
[102]Bodzansky, M. Peptide Chemistry: A Practical Textbook[M]. 2nd Ed., Springer-verlag, New York, 1993.
[103]罗九甫。酶和酶工程[M]。上海交通大学出版社，1996．314
[104]Fersht，A．酶的结构和作用机制(杜锦珠等译自Enzyme Structure and Mechanism)，北京大学出版社，1991，26．
[105]Schechter, I.; Berger, A. On the size of the active site in proteases. Ⅰ. Papain. Biochem. Biophy. Res. Commun., 1967, 27 (2), 157-162.
[106]Wong, C.H.; Wang, K. T. New developments in enzymatic peptide synthesis. Experientia, 1991, 47, 1123—1129.
[107]闫爱新等。蛋白酶在有机合成中应用的新进展。化学进展，2001，13(3)，203-208．
[108]彭师奇。多肽药物化学[M]。北京科学出版社，1993
[109]Van't Hoff, J.H. Uber die zunehmende Dedeutung der anorganischen Chemic. Z.Anorg. Allg. Chem. 1898, 18, 1-13.
[110]Bergmann, M., Fruton, J.S. On proteolytic enzymes. Synthetic substrates for chymotrypsin. J. Biol. Chem. 1937, 118, 405-415.
[111]Kullmann, W. Synthesis of Leu-and Met-enkephalin. Biochem.Biophys. Res.Commun. 1979, 91: 693-698.
[112]Moriharra, K.; Oka, T. α-chymotrypsin as the catalyst for peptide synthesis. Biochem. J. 1977, 163, 531-542.
[113]Chen, Y.X.; Zhang, X.Z,; et al. Kinetically controlled synthesis catalyzed by proteases in reverse micelles and separation of precursor dipeptide of RGD. Enzyme Microb. Technol, 1999, 25, 310-315.
[114]Ye, Y.H., Tian, G.L.; Xing, G.W.; et al. Enzymatic synthesis of N-protected Leu-enkephalin and some oligo-peptides in organic solvents. Tetrahedron 1998, 54, 12585-12596.
[115]So, J.E.; Kang, H.S.;Kim, B.G.; Lipase-Catalyzed synthesis of peptides containing D-Amino acid. Enzyme Microb.Technol. 1998, 23, 211-215.
[116]Oyama, K.; Niishimura, S.; et al. Synthesis of aspartotance precursor by immobilized thermolysin. J. Org. Chem. 1981, 46, 5241-5242.
[117]Moriharra, K. Using protease in peptide synthesis. Trend. Biotechnol. 1987, 5, 164-170.
[118]Fornanelez, M.M.; Margot, A.O.; Falender, C.A., et al. Enzymatic synthesis of peptides containing unnatural amino acids. Enzyme Microb. Technol. 1995, 17: 964-971.
[119]Krix, G.; Eichhorn, U.H.; Jakubke, H.D.; Kula, M.R., Protease-catalyzed synthesis of new hydrophobic amino acids. Enzyme Microb.Technol. 1997, 21,252-257.
[120]Ke, T.; et al. Enhancing Enzymatic enantio-selectivity in organic solvents by forming substrate salts. J. Am. Chem. Soc. 1999, 121, 3334-3340.
[121]Kullmann, W. Enzyntatic peptides Synthesis. CRC press. Boca Raton, 1987.
[122]Wang, B.K.; Gu, Q.M.; Sih, C.J. Enzyme-Catalyzed peptide segment condensation using 5(4H)-Oxazolones as donors. J. Am. Chem. Soc. 1993, 115: 7912-7913.
[123]张学忠，在有机介质中酶法合成小肽的研究进展，高技术通讯1998，7：55-58．
[124]Laane, C.; Boeren, S.; Vos, K. On optimizing organic solvents in multi-liquid phase biocatalysis. Trends Biotechnol. 1985, 3(10), 251-252.
[125]Reslow, M.; Adlercreutz, P.; Mattiassion B. Organic solvents for bioorganic synthesis. 1. Optimization of parameters for a chymotrypsin catalyzed process. Appl. Microbiol. Biotechnol. 1987, 26, 1-8.
[126]Simatos, D.; Multon, J. K. Properties of Water in Food, Dordrecht (Netherlands): Martinus Nijhoft, 1985, 111.
[127]Noritomi, H. et al. Enzymatic synthesis of peptide in acetonitrile/supercritical carbon dioxide. Biotechnol. Lett. 1995, 17 (12),1323-1328.
[128]Zhang, X. Z.; Wang, X.; Chen, S.; et al. Protease-catalyzed small peptide synthesis in organic media. Enzyme.Microb.Technol, 1996, 19, 538—544
[129]Zaks, A.; Klibanov, A.M. Enzyme-catalyzed process in organic solvents Pro. Natl. Acad.Sci. 1985, 82, 3192-3196.
[130]Yennawar, H.P.; Yennawar N.H.; Farber, G.K. A Structural Explanation for Enzyme Memory in Nonaqueous Solvents. J. Am. Chem. Soc. 1995,177 (2):577-585
[131]Stahl, M.; Jeppsson-Wistrand, U.;. Mansson, M.; et al. Induced stereoselectivity and substrate selectivity of bio-imprinted 2-chymotrypsin in anhydrous organic media. J. Am. Chem. Soc., 1991, 113: 9366-9369.
[132]Nilsson, K.; Skuladottir I, Mattiasson, B. Cryo-bioorganic synthesistemperature effects on enzymatic peptide synthesis. Biotechnol. Appl.Biochem, 1992, 16, i82
[133]邹国林，朱汝璠．酶学[M]．武汉：武汉大学出版社，1997
[134]Mitin, Y.V.; Zapevalova, N.P.; Gorbunova, E.Y. Peptide synthesis catalyzed by papain at alkaline pH values.Int J Peptide Protein Res May 1984; 23(5): 528-534.
[135]Stehle, P.; Bahsitta, H.P.; Monter, B.; Fürst, P. Papain-catalysed synthesis of dipeptides: A novel approach using free amino acids as nucleophiles. Enzyme Microb Technol 1990;12:56-60.
[136]赵明，彭师奇，迪丽努尔等。含RGD四肽的合成与功能。药学学报．1997，32(4)，271-277
[137]刘丽艳，崔凤侠，赵桂琴，王宏伟。活性肽RGD的液相合成化学试剂，2006，28(1)，55-56。
[138]张倩，张明智，张成海，丁伟。RGD三肽液相合成的研究。四川大学学报(工程科学版)，2001，33(6)，78-81．
[139]宋明媚，鞠培培，曹建军，沈树宝。化学法合成RGD三肽的研究生物加工过程2005，3(2)：23-26．
[140]杨大成，范莉，钟裕国．全保护RGD三肽的合成方法研究。有机化学，2003，23(5)：493-498．
[141]Hirano, Y.; Kando, Y.; Hayaahi, T.; Iwai, Y.; Hama, T.; Tsutsumi, Y.; Yamamoto, S.; Nakagawa, S.; Mayumi, T. Synthesis and cell attachment activity of bioactive oligopeptides:RGD RGDS RGDV and RGDT. J. Biomed. Mater. Res. 1991, 25, 1523-1534.
[142]Kawasaki, K.; Namikawa, M.; Yamashiro, Y. et al. Amino acid and peptides ⅩⅩⅤ preparation of fibronectin-related petide poly (ethylene gloycol) hybrid and their inhibitory effect on experimental metastasis. Chem. Pharm. Bull. 1995, 43(12), 2133-2137.
[143]Sofukku, S.; Hoshida, A.; Shimada, T. Synthesis of cyclic oigopeptides containing an Arg-Gly-Asp sequence. Chem. Pharm. Bull, 1999, 47(12), 1799-1801.
[144]Nicolaou, K. C., Trujillo, J. Chibate, K. Design, synthesis and biological evalution of carbohydrate-based mimetics. Tetrahedon, 1997, 53(26), 8751-8778.
[145]Dal-Pozzo, A.; Muzi, L.; Rondanin, R., et al. Synthesis of RGD analogues containing α-Tfm-Arginine as potential fibrinogen receptor antagonisis. Tetrahedon, 1998, 54(22), 6019-6028.
[146]Quirk, R.A.; Chan, W. C.; Davies, M. C.; et al. Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lacticacid). Biomaterials, 2001, 22(3), 865-872.
[147]Yamamoto, Y.; Sofuku, S. Synthesis of RGDF-containing cystine peptides by disulfide bond formation on a solid support. J Chem. Reseach. 1996, 12, 520-521.
[148]刘丽艳，陈治宇，崔凤侠，卢玉荣。RGD肽的固相合成。承德医学院学报，2005，22(1)，53-54．
[149]So, J.E.; Shin, J.S.; Kim, B.G. Protease-catalyzed tripeptide (RGD) synthesis. Enzyme Micro.Technol. 2000, 26, 108-114.
[150]陈育新，张学忠，陈松明，吴晓霞。在AOT／异辛烷反相胶束中酶法合成RGD前体二肽。中国生物化学与分子生物学报，1999，15(1)：146-150．
[151]Zhang, L.Q.; Xu, L.; Yang, X.C.; Wu, X.X.; Zhang, X.X. Lipase-Catalyzed Synthesis of Precursor Dipeptides of RGD in Aqueous Water-Miscible Organic Solvents. Prep. Biochem. Biotechnol. 2003, 33(1), 1-12.
[152]Bangham, A.D.; Standish, M.M.; Watkins, J.C. Diffusion of univalent ions across the lamellae of swollen phospholipid. J. Mol. Boil. 1965, 13, 238-252.
[153]Gregoriadis,G.; Swain, C.P.; Wills, E.J., et. al. Drag cartier potential of liposomes in cancer chemotherapy. Lancet, 1974, 1 (7870), 1313.
[154]刘昂，吴梧桐。制剂新技术在多肽、蛋白质类药物给药系统研究中的应用。中国现代应用药学杂志，2003，20(2)，110-114．
[155]艾国，杜丽娜，梅兴国。蛋白质、多肽类药物新剂型与新技术发展 动态。中国生化药物杂志，2004，25(6)，366-369．
[156]李国栋，王捷频。蛋白质、多肽类药物制剂的研究现状。药学实践杂志，2001，19(6)，344-349．
[157]顾学裘。中国多相脂质体研究。中国医药科技出版社，1991．116．
[158]李彦超，蔡宝昌。脂质体在抗肿瘤药物研究中的应用。南京中医药大学学报，2003，19(4)，250-253．
[159]崔福德。药剂学。人民卫生出版社，2003．7
[160]李彦超，蔡宝昌。脂质体在抗肿瘤药物研究中的应用。南京中医药大学学报，2003，19(4)，250-253．
[161]宫亮，杨和平。脂质体载药系统与抗肿瘤药物。药学专论，2005，14(3)，27-29．
[162]陈涛，王昭，傅经国，商鹏。靶向脂质体的研究。世界最新医学信息文摘，2003，2(6)，884-890．
[163]于洁，王振纲。脂质体在治疗肿瘤及感染性疾病中的前景。中国药理学通报，1998，14(3)，193-195．
[164]赵荣生，严宝霞。空间稳定脂质体与药物的靶向释放。中国药学杂志，1998，33(4)，452-455．
[165]符民桂，刘乃奎，唐朝枢。脂质体靶向性的研究进展。中国药理学通报，1999，15(1)，18-21．
[166]陈建明，张仰眉，高申。受脂质体作为药物载体的研究进展。药学实践杂志，2001，19(4)，214-218．
[167]Martin, C.; Danilo, W.; Lasic, D. Sterically stabilized liposomes. Biochimica et Biophysica Acta 1992, 1113, 171-199.
[168]王闻珠，邓英杰。脂质体肺部给药研究进展。沈阳药科大学学报，2000，17(3)，226-229．
[169]纪俊敏，谢文磊。脂质体作为药物载体的研究进展。郑州工程学院学报，2002，23(4)，68-72．
[170]张激，程艳菊，吕浩，应汉杰。脂质体的研究新进展。南京工业大学学报，2003，25(4)，102-106．
[171]罗云敬，刘海涛，李天剑等。头孢菌素类抗生素脂质体的包封率测定与渗漏研究。中国生化药物杂志1999，20(1)：4-6．
[172]朱希强，翟光喜。低分子肝素类脂质体的研制中国生化药物杂志2001，22(5)，232-234．
[173]任进余，陈义，吴斌等。病毒性肝炎的药物靶向治疗研究：抗HBV脂质体的研制及临床观察。中西医结合肝病杂志1997，7(1)，1-4．
[174]阎家麟，童岩，王九一。紫杉醇脂质体的制备即其抑瘤作用的研究。药物生物技术1996，3(3)，1-5．
[175]丁玉玲，马疏贤，卢秀荣等。人参皂甙脂质体(GSL)的研制。中国药学杂志，1995，30(7)，414-418．
[176]肖旭。5-氟脲嘧啶温度敏感性脂质体制备方法的优化。药学实践杂志1998，16(6)，344-346．
[177]李国锋，周日红，曾抗等。维生素E脂质体的制备。中国应用药学 1997，14(4)，18-20．
[178]张今，秦德安，陈百先等。包有蓖麻毒蛋白的脂质体的制备、性质及毒性研究。生物化学和生物物理学报，1999，31(4)，472-474．
[179]隋因，刘宝庆，夏东亚。冷冻干燥法制备阿糖胞苷冻干脂质体粉针研究。中国医院药学杂志，1999，15(11)，487-490．
[180]王弘，吴梧桐，顾学裘。重组L-前体脂质体的研制及包封率测定。沈阳药科大学学报，1999，16(4)，235-238．
[181]裴瑾，杨成君，杨翰仪等。白细胞介素2脂质体的制备及抗肿瘤作 用的研究。中国生化药物杂志，1997，18，166-169．
[182]李志宇，何生，薛华等。葡萄球菌肠毒素A脂质体的制备及其体内分布试验。华西医科大学学报2002，33(2)，299-301．
[183]储茂泉，古宏晨，刘国杰。丹参酮脂质体的制备及其稳定性。中国医药工业杂志2001，32(9)，400-404．
[184]翟光喜，赵焰。低分子肝素前体脂质体制剂的研究。山东医科大学学报2001，39(3)，218-220．
[185]全东琴，苏德森，顾学裘。药物载体空白脂质体前体的制备及性质的研究。沈阳药科大学学报1999，16(4)，239-241．
[186]邓英杰，刘素芹，韩丽梅等。黄芪多糖脂质体的制备和稳定性研究。沈阳药科大学学报1996，13(1)，1-4．
[187]Hwang S H, Maitani Y, Qi X R,et al.Remote loading of diclofenac, insulin and fluorscein isothiocyanate labeled insulin in to liposomes by pH and acetate gradient methods[J].Int J Pharm,1999, 179(1): 85-91.
[188]储茂泉，古宏晨，刘国杰。喷雾干燥法制备丹参酮前体脂质体的研究。中国药学杂志2002，37(1)：32-35．
[189]瞿文，陈庆华．黄体激素释放激素及其类似物的缓释给药系统。中国医药工业杂志1999，30(4)：181-188．
[190]Trehan A, Ali A. Recent approaches in insulin delivery[J]Drug Dev Ind Pharm. 1998, 24(7): 589-597.
[191]Yang, J.; Cleland, J.L.; Factors affecting the in vitro release of recombinant human interfemnT-(rhIFN-7)from PLGA microspheres. J Pharm Sci. 1997, 86(8): 908-914.
[192]Chen, L.; Apte, R.; Cohen, S. Characterization of PLGA microspheres for the controlled delivery of IL-1 for tumor immunotherapy. J Controlled Release. 1997, 43, 261-272.
[193]Johnson, O.L.; Jaworowicz, W.; Cleland, J.L.; et al. The stabilization and encapsulation of human growth hormone into biodegradable Microspheres. Pharm. Res. 1997, 14(6), 730-735.
[194]Hanes, J.; Cleland, J.L.; Langer, R. New advances in microsphere based single-dose vaccines. Adv. Drug Deliv. Rev. 1997, 28, 97-119.
[195]杨正管。美国药物开发公司开发口服降钙素。药学进展1996，20(1)，58．
[196]张海松，陈阳述，陈文吟等。口服促肝细胞生长素微球的研究。中国生化药物杂志1998，19(4)，180．
[197]Pontiroli, A.E. Peptide hormones: Review of current and emerging uses by nasal delivery. Adv. Drug. Deliv. Rev 1998, 29(1-2), 81-87.
[198]Takenaga, M.; Serizawa, Y.; Azechi, Y.; et al. Microparticle resins as a potential nasal drug delivery system for insulin. J. Controlled Release 1998, 52(1-2),81-87.
[199]Cole, M.L.; Whateley, T.L. Release rate profiles of theophylline and insulin from stable multiple w/o/w emulsions. J Controlled Release, 1997, 49(1), 51-58.
[200]Li, J.K.; Wang, N.; Wu, X.S. A novel biodegradable system based on gelatin nanoparticles and poly(lactide-co-glycolic acid) microspheres for protein and peptide drug delivery. J. Pharm. Sci. 1997, 86 (8), 891-895.
[201]Chen, S.T.; Chen, S.Y.; Wang, K.T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease Alcalase. J. Org. Chem. 1992, 57, 6960-6965.
[202]Klein, J.U.; Prykhodzka, A.; Cerovsky, V. The Applicability of Subtilisin Carlsberg in Peptide Synthesis. J. Peptide Sci. 2000, 6, 541-549.
[1]Chen, S.T.; Chen, S.Y.; Wang, K.T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease alcalase. J. Org. Chem. 1992, 57, 6960-6965.
[2]Chen, S.T.; Kao, C.L.; Wang, K.T. Alkaline protease catalysis of a secondary amine to form a peptide bond. Int. J. Pept Protein Res. 1995, 46, 314-319.
[3]Chen, Y.X.; Zhang, X.Z. Protease-catalyzed synthesis of precursor dipeptides of RGD with reverse micelles. Enzyme Microbl&Technol. 1998, 23,243-248.
[4]Chen, S.T.; Chen, S.Y.; Hsiao, S.C.; Wang, K.T. Application of industrial protease "Alcalase" in peptide synthesis. Biomed Biochim Acta 1991, 50, 181-186.
[5]So, J.E.; Kang, H.S.; Kim, B.G.; Lipase-Catalyzed synthesis of peptides containing D-Amino acid. Enzyme Microb. Technol. 1998, 23, 211-215.
[6]Zhou, Y.Y.; Yang, T.; Chemo-enzymatic synthesis of tripeptide RGD in organic solvents. Enzyme Microb.Technol. 2003, 33, 55-61.
[7]Ren, G.P.; Gu, W.Y. Production of a Bioactive Gin-enriched oligopeptide Nutrient solution From corn Protein by(Ⅰ) Enzyme Screening for Alcohol-water System. Journal of the Chinese Cereals and Oils Association. 2000, 15, 18-22.
[8]Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rule of optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987; 30, 81-87.
[9]Okazaki, S.; Goto, M.; Furusaki, S. Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb. Technol. 2000, 26, 159-164.
[10]Lozano, P.; Cano, J.; Iborra, J.L.; Manjon, A. Glycylglycylphenylalanin amide synthesis catalysed by papain in a medium containing polyols. Biotechnol.Appl.Biochem. 1993, 18, 67-74.
[11]Hailing, P.J. High-affinity binding of water by proteins is similar in air and in organic solvents. Biochem.Biophys. Acta 1990, 1040, 225-228.
[12]Klibanov, A.M. Enzymes that work in organic solvents. Chemtech. 1986, 16, 354-359.
[1]Chen, S.T.; Chen, S.Y.; Wang, K.T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease Alcalase. J. Org. Chem. 1992, 57, 6960-6965.
[2]Klein, J.U.; Prykhodzka, A.; Cerovsky, V. The Applicability of Subtilisin Carlsberg in Peptide Synthesis. J. Peptide Sci. 2000, 6, 541-549.
[3]Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rule of optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987, 30, 81-87.
[4]Okazaki, S.; Goto, M.; Furusald, S. Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb. Technol. 2000, 26, 159-164.
[5]Affleck, R.; Xu, Z.F.; Suzawa, V.; et al. Enzymatic catalysis and dynamics in lower ater environment. Proc. Natl. Acad. Sci. USA, 1992, 89, 1100-1104.
[6]Xu, Z.F.; Affleck, R.; Wangikar, P.; et al. Transition state stabilization of subtilisins in organic media. Biotechnol. Bioeng. 1994, 43, 515-520.
[7]Zaks, A.; Klibanov, A.M. The effect of water on enzyme action in organic media. J. Biol. Chem. 1988, 263(17), 8017-8021.
[8]Bone, S.; Pethig, R. Dielectric studies of protein hydration and hydration-induced flexibility. J. Mol. Biol. 1985, 181,323-326.
[9]Maes, D.; Bouckaert, K.J.; Poortmans, F.; Wyns, L.; Looze, Y. Structure of chymopapain at 1.7 A resolution. Biochemistry 1996, 35, 16292-16298.
[10]Monter, B.; Herzog, B.; Stehle, P.; Furst, P. Kinietically controlled synthesis of dipeptides using ficin as biocatalyst. Biotechnol. Appl. Bioc. 1991, 14, 183-191.
[11]Mitin, Yu.V.; Zapevalova, N.P.; Gorbunova, E.Yu. Pepetide synthesis catalyzed by papain at alkaline pH values. Int. J. Peptide protein Res 1984, 23,528-534.
[12]Iqbal, G.; Rosina, L.F.; Evgeny, V. Enzymatic oligopeptide synthesis using a minimal protection strategy: sequential assemble of a growing oligopeptide chain. J. Am. Chem. Soc. 1995, 117, 6175-6181.
[13]Joseph, O.R.; Jonathan, S.D. Controlling subtilisin activity and selectivity in organic media by imprinting with nucleophilic substrates. J. Am. Chem.Soc. 1997, 119, 3245-3252.
[14]Marianne, S.; Ulla, J.W.; Mates, O.M.; Klaus, M. Induced stereoselectivity and substrate selectivity of bio-imprinted α-chymotrypsin in anhydrous organic media. J. Am. Chem. Soc. 1991, 113, 9366-9368.
[15]Isono, Y.; Nakajima, M. Enzymic peptide synthesis using s microaqueous highly concentrated amino acid mixture. Process Biochem. 2000, 26, 275-278.
[16]Erbeldinger, M.; Ni, X.; Halling, P.J. Enzymatic synthesis with mainly undissoved substrates at very high concentrations. Enzyme Microb. Tech. 2000, 23,141-148.
[1]So, J.E.; Kang, H.S.; Kim, B.G. Lipase-Catalyzed synthesis of peptides containing D-Amino acid. Enzyme Microb.Technol. 1998, 23, 211-215.
[2]Mitin, Y.V.; Zapevalova, N.P.; Gorbunova, E.Y. Peptide synthesis catalyzed by papain at alkaline pH values. Int J Peptide Protein Res. 1984, 23(5), 528-534.
[3]Schechter, I.; Berger, A. On the size of the active size in proteases. Ⅰ. Papaln Biochem Biophys Res Commun. 1967, 27, 157-162.
[4]Isowa, Y.; Ohmori,M.; Sato, M.; Moil, K. Bull Chem Soc Japan. The Enzymatic Synthesis of Protected Valine-5 Angiotensin Ⅱ Amide-1 1977, 50, 2766-2772.
[5]Kullmann, W. Enzymatic synthesis of Leu-and Met-enkephalin. Biochem Biophys Res Commun. 1979, 91,693-698.
[6]Kullmann W. Proteases as catalysts for enzymic syntheses of opioid peptides. J Biol Chem. 1980, 255, 8234-8238.
[7]Stehle, P.; Bahsitta, H.P.; Monter, B.; Fürst, P. Papain-catalysed synthesis of dipeptides: A novel approach using free amino acids as nucleophiles. Enzyme Microb Technol. 1990, 12, 56-60.
[8]Lowe, G. The cysteine proteinases.Tetrahedron 1976, 32, 291-302.
[9]Barbas, C.F.; Wong, C.H. One-pot tripeptide syntheses from three single amino acid derivatives catalyzed by papain. Tetrahedron Lett. 1988, 24, 2907-2910.
[10]Hou, R.Z.; Zhang, N. Synthesis of tripeptide RGD amide by a combination of chemical and enzymatic methodsv. Journal of Molecular Catalysis B: Enzymatic 2005, 37, 9-15.
[11]Okazaki, S.; Goto, M.; Furusaki, S. Surfactant-protease complex as a novel bio-catalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb Technol. 2000, 26, 159-164.
[12]Lozano, P.; Cano, J.; Iborra, J.L.; Manjón, A. Glycylglycylphenylalan inamide synthesis cataiysed by papain in a medium containing polyols. Biotechnol.Appl. Biochem. 1993, 18, 67-74.
[13]Whitaker, J. R.; Bender, A. J.Am.Chem.Soc. 1965, 87, 2728
[14]Isono, Y.; Nakajima, M. Enzymic peptide synthesis using a microaqueous highly concentrated amino acid mixture. Process Biochem. 2000, 36; 275-278.
[15]Erbeldinger, M.; Ni, X.; Hailing, P.J. Enzymatic synthesis with mainly undissolved substrates at very high concentrations. Enzyme Microb Technol. 2000, 23, c141-c148.
[1]黄惟德，陈长庆。《多肽合成》科学出版社。1985，P102—140．
[2]Hirschmann, R.; Strachan, R.G.; Schwam, H.; Schoenewaldt, E.F.; Joshua, H.; Barkemeyer, B.; Veber, D.F.; Paleveda, W.J.; Jacob,T.A.; Beesley, T.E.The Controlled Synthesis of Peptides in Aqueous Medium.Ⅲ.Use of Leuchs'Anhydrides in the Synthesis of Dipeptides. Mechanism and Control of Side Reactions, J.Org. Chem. 1967, 32, 3415-3425.
[3]Veber, D.F.; Hirschmann, R.; Denkenwalter, R.G. The Synthesis of Peptides in Aqueous Medium.Ⅵ. The Synthesis of an Unsymmetrical Cystine Peptide Fragment of Insulin. J.Org. Chem. 1969, 34,753-755.
[4]Patchornik, A.; Berger, A.; Katchalski, E. Poly-L-histidine, J. Am. Chem. Soc. 1957,79, 5227-5230.
[5]Denkewalter, R.G.; Schwam, H.; Strachan, R.G.; Beesley, T.E.; Veber, D.F.; Schoenewaldt, E.F.; Barkemeyer, H.; Paleveda, W.J.; Jacob, T.A.; Hirschmann, R. The Controlled Synthesis of Peptides in Aqueous Medium. Ⅰ The use of α-Amino Acid N-Carboxyanhydrides J. Am. Chem. Soc. 1966, 88, 3163-3164.
[6]Kawasaki, K.; Namikawa, M.; Yamashiro, Y.; Iwai, Y.; Hama, T,; Tsutsumi, Y.; Yamamoto, S.; Nakagawa, S.; Mayumi, T. Amino Acids and Peptides.ⅩⅩⅤ.~(1)) Preparation of Fibronectin-Related Peptide Poly (ethylene glycol) Hybrids and Their Inhibitory Effect on Experimental Metastasis. Chem. Pharm. Bull. 1995, 43, 2133-2138.
[7]Zhao, M.; Peng, S.Q.; Dilinuer, S.; Wang, Y.Y.; Zhou, Q.L.; Zhao,W.Z. Studies on the synthesis and activities of RGD related peptides. Acta Pharmaceutica Sinica 1997, 32, 271-277.
[8]Maeda, M.; Izuno, Y.; Kawasaki, K.; Kaneda, Y.; Mu, Y.; Tsutsmi, Y.; Lem, K.W.; Mayumi,T, Amino Acids and.Peptides ⅩⅩⅫ: A Bifunctional Poly(Ethylene Glycol) Hybrid of Fibronectin-Related Peptides, Biochem. Biophys. Res. Commun. 1997, 241,595-598.
[9]赵明，彭师奇，迪丽努尔，等．含RGD四肽的合成与功能[J]．药学，1997，32(4)：271-277．
[10]张倩，张明智，张成海，丁伟。RGD三肽液相合成的研究。[J]四川大学学报(工程科学板)，2001，33(6)：78-81
[11]杨大成，范莉，钟裕国。全保护RGD三肽的合成方法研究[J]有机化学，2003，23(5)：493-498．
[12]Quirk R A, Chan W C,Davies M C,et al.Poly(L-lysine)-GRGDS as a biominmetic surface modifier for poly(lacticacid)[J].Biomaterials, 2001,22(3):865-872.
[13]Marion Zhang, Michael N. Solid-phase synthesis of Arginine-containing peptide by guanidine attachment to a sulfony linker[J]. J Org. Chem. 1997, 62(26): 9326-9330.
[14]Yamamoto Y, Sofuku S. Synthesis of RGDF-containing cystine peptides by disulfide bond formation on a solid support[J]. J Chem. Reseach. 1996, 12: 520-521.
[15]Gibson C,Goodman SL,Hahn D,etal. Novel solid-phase synthesis of azapeptides and azapeptoides via strategy and its application in the synthesis of RGD-mimetics.J Org.Chem. 1999, 64(20): 7388-7394.
[1]崔福德主编，药剂学[M]。人民卫生出版社，2003．7
[2]栾军主编，现代试验设计优化方法[M]。上海交通大学出版社，1995，3 第1版。
[3]曹宁宁，羡菲，刘金鹏。脂质体的制备方法及研究进展。天津理工学院学报，2003，19(1)，30-34．
[4]余旭亚，赵声兰，李涛等。仙人掌SOD脂质体的制备研究。昆明理工大学学报，2002，27(1)，103-105．
[5]胥传来，乐国伟，姚惠源。冻干PST脂质体的制备及物理稳定性的研究。中国油脂，2001，26(6)，54-57．
[6]Cansell, M.; Parisel, C.; Jozefonvicz, J.; et al. Liposomes coated with chemically modified dextran interaet with human endothelial cells. J Biomed Mater Res.1999, 44(2), 140-148.
[7]胡俊，张康，王筱榕。一种新型脂质体的制作方法。第一军医大学学报，2001，16(3)，167-169．
[8]丁玉玲，马淑贤，卢秀荣。人参皂甙脂质体的研制。中国药学杂志，1995，30(7)，414-415．
[9]张奇，邓英杰。冻融法制备5-氟尿嘧啶脂质体及其稳定性考察。沈阳药科大学学报，2002，17(2)，64-67．
[10]赵荣生，侯新朴，严宝霞。两性霉素B脂质体的制备及稳定性研究。中国医药工业杂志，2001，32(4)，160-162．
[11]刘辉，汤韧，何小霞等。脂质体处方和制备方法对阿昔洛韦棕榈酸酯脂质体稳定性的影响。药学学报，2002，37(7)，563-566．
[12]胥传来，乐国伟，姚惠源等，生长激素脂质体的制备。云南农业大学学报，2001，16(3)，167-169．
[13]李晓燕，陈卫，侯新朴。癸烷基葡萄糖对载药脂质体的渗漏保护作用。北京医科大学学报，1995，27(1)，75．
[1]Ruoslahti, E.; Pierschbacher, M.D. New perspectives in cells adhesion RGD and integrins. Science 1987, 218, 491-499.
[2]顾玲，杨连甲，董绍忠等。RGDS对脑转移舌癌细胞系Tb侵袭转移行为的影响。临床口腔医学杂志，2004，20(1)，14-16．
[3]Hou, R.Z.; Liu, Y.J.; Zhang, N.; Huang, Y.B.; Wang, H.; Yang, Y.; Xu, L.; Zhang, X.Z. New synthetic route for RGD tripeptide. Prep Biochem Biotechnol. 2006, 36(3), 243-252.
[4]司徒德强。细胞培养 北京世界图书出版公司，2004．3
[5]Anuradha, C.D.; Kanno, S.; Hirano, S. RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation. Cell Biol Toxicol. 2000, 16(5), 275-283.
[6]Buckley, C.D.; Pilling, D.; Henriquez, N.V.; et al. RGD peptides induce apoptosis by direct caspase-3 activation. Nature, 1999, 397(6719) 534-539.
[7]Yamamoto, Y.; Tsutsumi, Y.; Mayumi, T. Molecular design of bioconjugated cell adhesion peptide with a water-soluble polymeric modifier for enhancement of antimetastatic effect. Curr Drug Targets, 2002, 3(2), 123.
[8]Madeja, Z.; Sroka, J. Contact guidance of Walker carcinosarcoma cells by the underlying normal fibroblasts is inhibited by RGD-containing synthetic peptides. Folia Histochem Cytobiol. 2002, 40(3), 251
[2] Pierschbacher, M.D.; Hayman, E.G.; Ruoslahti E. Location of the Cell-attachment site in Fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell 1981,26, 259-267.
[3] Yamada, K.M.; Kennedy D.W. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. Cell Biol. 1984, 99,29-36.
[4] Hayman, E.G.; Pierschbacher, M.D.; Ruolslahti, E. Detachment of cells from culture substrate by soluble fibronectin peptides. J.Cell Biol. 1995, 100,1948-1954.
[5] Pierschbacher, M.D.; Ruolslahti, E. Variants of the cell recognition site of fibronectin that retain attachment-promoting activity.Proc. Nalt. Acad. Sci. USA 1984, 81,5985-5988.
[6] Ruolslahti, E.; Pierschbacher, M.D. New perspectives in cell adhesion: RGD and integrins. Science 1987, 238,491-497.
[7] Juliano, R.L.; Haskill, S. Signal transduction from the extracellular matrix. J. Cell Biol. 1993,120, 577-585.
[8] Plow, E.F.; Haas, T.A.; Zhang, L.; Loftusi, J.; Smith, J.W. Ligand Binding to Integrins. J. Biol. Chem. 2000, 275(29), 21785-21788.
[9] Ruoslahti, E. Integrins. J Clin Invest 1991, 87, 1-5.
[10] Larson, R.S.; Springer, T.A. Structure and function of leukocyte integrins. Immunol. Rev. 1990,114, 181-217.
[11] Luscinskas, F.W.; Lawler, J. Integrins as dynamic regulators of vascular function. FASEB J 1994, 8, 929-938.
[12] Hemler, M.E. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 1990, 8, 365-400.
[13]Spinger, T.A. Adhesion receptors of the immune syntem. Nature 1990, 346, 425-434.
[14]Elices, M.J.; Osborn, L.; Takada, Y.; et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 1990, 60, 577-584.
[15]Mould, A.P.; Wheldon, L.A.; Komoriya, A.; et al. Affinity chromatographic islation of the melanoma adhesion receptor for the ⅢCS region of fibronectin and its identification as the integrin α_4β_1. J. Biol. Chem. 1990, 265, 4020-4024.
[16]Ginsberg, M.H.; Loflus, J.C.; Plow, E.F. Cytoadhesions, integrins, and platelets. Thromb. Haemostasis 1988, 59, 1-6.
[17]Litta, L.A.; Steter Stevenson, W.G. Tumor invation and metastasis: an inbalance of positive and negative regulation. Cancer Res. 1991, 51 (supp 1), 5054-5061.
[18]汤钊猷主编，现代肿瘤学。上海医科大学出版社，1993年，1-5．
[19]细胞生物学教程，绍兴文理学院生命科学学院，田润刚博士主讲。http://www.cella.cn/jxck/index.htm.
[20]Liotta, L.A. Tumor Invasion and Metastases-Role of the Extracellullar Martrix: R.hoacls Memorial Award Lecture. Cancer Res. 1986, 46, 1-7.
[21]Aznavoorian, S.; Syetler-Stevenson W.G.; Liotta, L.A. Molecular aspects of tumor cell invasion and metastasis. Cancer, 1993, 71, 1368-1383.
[22]Honn, K.V.; Tang, D.G.; Crissman, J.D. Platelets and cancer metastasis: a causal relationship. Cancer Metastasis Rev. 1992, 11(3/4), 325-351.
[23]Mohri, H.; Ohkubo, T. The role of RGD peptides and the Y chain peptides of fibrinogen on fibrinogen binding to activated platelets. Peptides 1993, 14(2), 353-357.
[24]王树人。细胞粘附分子的生理和病理生理。四川生理科学杂志 1996，18(3)，24—28．
[25]Hillis, G.S.; MacLeod, A.M. Integrins and disease. Clin Sci (Lond). 1996(Dec), 91(6), 639-650.
[26]Price, J.T.; Bonovich, M: T.; Kohn, E. C. The biochemistry of cancer dissemination. Crit. Rev. Biochem. Mol. Biol. 1997, 32(3), 175-253.
[27]Naruo, K. Organ-specific adhesion of neuroblastoma cells in vitro: correlation with their hepatic metastasis potential. J. Ped. Surg. 1997, 32(4), 546-551.
[28]Humphries, M.J.; Olden, K.; Yamada, K.M. A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science 1986, 233(4762), 467-470.
[29]Hirasawa, M.; Shijubo, N.; Uede;.T.; et al. Integrin expression and ability to adhere to extracellular matrix protein and endothelials cells in human lung cancer lines. Br. J. Cancer 1994, 70(3), 466-472.
[30]Kang, I.C.; Kim, D.S.; Jang, Y.; et al. Suppressive mechanism of salmosin, a novel disintegrin in B16 melanoma cell metastasis. Biochem Biophys Res Commun. 2000, 275(1), 169-173.
[31]吴泽志，黄岂平，肖国辉等。肝癌细胞—胞外基质粘附性与粘附识别的相关性。生物物理学报，2000，16(4)，831-838．
[32]Cui, G.P.H.; Puddefoot, J.R.; Vision, G.P.; et al. Altered cell-matrix contact: a prerequisite for breast cancer metastasis. Br. J. Cancer 1997, 75(5), 623-630.
[33]Nierodzik, M.L.; Klepfish, A.; Karpatkin, S.; et al. Role of platelets, thrombin, integrin Ⅱb/Ⅲa, fibronectin, and von Willebrand factors on tumor adhesion in vitro and metastasis in vivo. Thromb Haemost 1995, 74(3), 282-489.
[34]赵明，彭师奇，迪丽努尔等。含RGD四肽的合成与功能。药学学报，1997，32(4)，271．
[35]Scherbarth, S.; Orr, F.W. Intravital videomicroscopic evidence for regulation of metastasis by the hepatic microvasculutre: effect of interleukinla on metastasis and the location of melanoma cell arrest. Cancer Res. 1997, 57(15), 4105-4111.
[36]Barinaga, M. Cell suicide: by ICE, not fire. Science 1994, 263(5148), 754-756.
[37]Anuradha, C.D.; Kanno, S.; Hirano, S. RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation. Cell Biol. Toxicol. 2000,16 (5), 275-283.
[38]Chen, Y.; Xu, X.; Hong, S.; et al. RGD-Tachyplesin inhibits tumor growth. Cancer Res. 2001, 61 (6), 2434-2438.
[39]Buckley, C.D.; Pilling, D.; Henriquez, N.V.; et al. RGD peptides induce apoptosis by direct caspase-3 activation. Nature 1999, 397(6719), 534-539.
[40]Moreno, M. V.; Lucio, C. J.; Konta, T.; et al. Enhancement of TNF-alpha-induced apoptosis by immobilized arginine-glycine-aspartate: involvement of a tyrosine kinase-dependent,MAP kinase-independent mechanism. Biochem. Biophys. Res. Commun. 2000, 277(2), 293-298.
[41]Folkman, J. Tumor angiogenesis: therapeutic implications. N. Engl. J. Med. 1971,285(21), 1182-1186.
[42] Folkman, J.; Shing, Y. Angiogenesis. J Biol Chem. 1992,267 (16), 10931-10934.
[43] Folkman, J. Angiogenesis-dependent diseases. Semin. Oncol. 2001, 28(6), 536-542.
[44] Hodivala-Dilke, K.M.; Reynolds, A.R.; Reynolds, L.E. Integrins in angiogenesis: multitalented molecules in abalancing act. Cell Tissue Res. 2003,314(1), 131-144.
[45] Kerbel, R.; Folkman, J. Clinical translation of angiogenesis inhibitors. Nat. Rev. Cancer. 2002,2 (10), 727—739.
[46] Hynes, R.O. A revaluation of integrins as regulators of angiogenesis. Nature Med. 2002, 8 (9), 918-921.
[47] Hynes, R.O. Integrins: bidirectional, allosteric signaling machines. Cell 2002, (110),673—687.
[48] Brooks, P.C.; Clark, R.A. Cheresh, D.A. Requirement of vascular integrin alpha v beta3 for angiogenesis. Science 1994,264(5158), 569-571.
[49] Maile, L.A.; Badley-Clarke, J.; Clemmons, D.R. Structural analysis of the ro le of the beta 3 subunit of the alpha V beta 3 integrin in IGF-I signaling.J Cell Sci. 2001,114(7), 1417-1425.
[50] Carlson, T.; Feng, Y.; Maisonpierre, P.; Mrksich, M.; Morla, A.O. Direct cell Adhesion to the angiopoietins mediated by integrins. J. Biol. Chem. 2001, 276(28), 26516—26525.
[51] Kim, S.; Bell, K.; Mousa, S.A.; Varner, J.A. Regulation of angiogenesis in vivo by ligation of integrin alpha 5 betal with the central cell-binding domain of fibronectin. Am. J. Pathol. 2000, 56(4), 1345 -1362.
[52]Huang, T.F.; Yeh, C.H.; Wu, W.B. Viper venom components affecting angiogenesis. Haemostasis 2001, 31 (3-6), 192-206.
[53]Fabio, P.; Chiara, B.; Danilo, M.; et al. Vascular damage and anti-angiogenic effects of tumor vessel-targeted liposornal chemotherapy. Cancer Res. 2003, 63, 7400-7409.
[54]Zarovni, N.; Monaco, L.; Corti, A. Inhibition of Tumor Growth by Intramu scular Injection of cDNA Encoding Tumor Necrosis Factora Coupled to NGR and RGD Tumor-Homing Peptides. Hum. Gene Ther. 2004, 15,373-382.
[55]Curnis, F.; Gasparri, A.; Sacchi, A.; et al. Coupling Tumor Necrosis Factor-a with a_v Integrin Ligands Improves Its Antineoplastic Activity. Cancer Res. 2004, 64, 565-571.
[56]Pasqualini, R..; Koivunen, E.; Ruoslahti, E. a_v integrins as receptors for tumor targeting by circulating ligands. Nat. Biotechnol. 1997, 15, 542-546.
[57]肖斌，朱永红。RGD肽在肿瘤治疗中的研究进展。中国肿瘤临床 2005，32(19)，1135-1137．
[58]Koizumi, N.; Mizuguchi, H.; Hosono, T.; et al. Efficient gene transfer by f iber-mutant adenoviral vectors containing RGD peptide. Biochim Biophys Acta 2001, 1568(1), 13-20.
[59]Witlox, A.M.; Van Beusechem, V.W.; Molenaar, B.; et al. Conditionally replicative adenovirus with tropism expanded towards integrins inhibits osteosarcoma tumor growth in vitro and in vivo. Clin Cancer Res. 2004, 10(1 Pt 1), 61-67.
[60]Grill, J.; van Beusechem, V.W.; van Der Valk, P.; et al. Combined targeting of adenoviruses to integrins and epidermal growth factor receptors increases gene transfer into primary glioma cells and spheroids. Clin Cancer Res. 2001, 7(3), 641-650
[61]Wu, H.; Han, T.; Lam, J.T.; et al. Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy. Gene Ther. 2004, 11(10), 874-878.
[62]Wesseling, J.G.; Bosma, P.J.; Krasnykh, V.; et al. Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin targeted adenoviral vectors. Gene Ther. 2001, 8(13), 969-976
[63]李前伟。肿瘤整合素αvβ3受体显像的研究现状。国外医学放射医学核医学分册，2003，27(5)，198-200．
[64]Su, Z.F.; Liu, G.; Gupta, S.; et al. In vitro and in vivo evaluation of a Technetium-99m-labeledcyclic RGD peptide as a specific marker of alpha (V) beta (3) integrin for tumor imaging. Bioconjug. Chem. 2002, 13(3), 561-570.
[65]Pytela, R.; Pierschbacher, M.D.; Ginsberg, M.H; et al. Platelet membrane glycoprotein Ⅱb/Ⅲa is a member of a family of Arg-Gly-Asp-specific adhesion receptors. Science, 1986, 231, 1559-1562.
[66]Goligorsky, M.S.; Noiri, E.; Kessler, H.; Romanov, V. Therapeutic potential of RGD peptides in acute renal injury. Kidney Int. 1997, 51, 1487-1492.
[67]Yi, Y.; Carderelli, P. M.; et al. LPAM-1 (integrin alpha 4 beta 7)-ligand binding: Overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen, a fibronectin-like polymer and RGD-like cyclic peptides. Eur. J. Immuno. 1998,28(3), 995-1004.
[68] Illana, B.; Zaballos &AACUTE.; Blanco, L.; Salas, M. The RGD sequence in phage 029 terminal protein is required for interaction with (?)29 DNA polymerase. Virology 1998,248(1), 12-19.
[69] Lin, N.; Zhao, M.; Wang, C.; et al. Synthesis and antithrombotic activity of carbolinecarboxyl RGD Sequence. Bioorg & Med Chem Lett. 2002, 12(4), 585-587.
[70] Zhang, L.H.; Pesti, J.A.; Costello, J.D. An efficient synthesis of cyclic RGD peptides as antithrombotic agents. J.Org.Chem. 1996, 61, 5180-5185.
[71] Meredith, J.E.; Fazeli, B.; Schwartz, M. A. The extracellular matrix as a cell survival factor. Mol Biol Cell, 1993,4(9), 953-961.
[72] Ouaissi, M.A.; Cornette, J.; Afchain, D. Trypanosoma Cruzi infection inhibited by peptides modeled from A fibronectin cell attachment domain. Science 1986,234, 603-607.
[73] Davis, D.H.; Giannoulis, C.S.; Johnson, R.W.; et al. Immobilization of RGD to <111>silicon surfaces for enhanced cell adhesion and proliferation. B iomaterials, 2002,23(19), 4019-4027.
[74] Lin, H.; Sun, W.; Mosher, D.F.; et al. Synthesis Surgace and cell-adhesion properties of polyurethanes containing covalently grafted RGD-Peptides. J. Biomed Mater Res. 1994,28, 329.
[75] Gill, L; Lopez-Fandino, R.; Jorba, X.; et al. Biologically Active Pep tides and Enzymatic Approaches to their Production. Enzyme Microb Technol. 1996,18,162-183.
[76] Erbeldinger, M.; Ni, X. W.; Hailing, P. J. Enzymatic Synthesis with Mainly Undissolved Substrates at Very H igh Concentrations. Enzyme Microb. Technol. 1998, 23, 141-148.
[77]Erbeldinger, M.; Hailing, P.J. Effect of Water and Enzyme Concentration on Thermolysin-Cataiyzed Solid to Solid Peptide Synthesis. Biotech. Bioeng. 1998, 59, 68-72.
[78]Lopez-Fandino, R.; Gill, I.; Vulfson, E. N. Enzymatic Catalysis in Heterogeous Mixtures of Subst rates: The Role of the Liqiud Phase and the Effects of"Adjuvants". Biotech. Bioeng. 1993, 43, 1016-1023.
[79]Erbeldinger, M.; Hailing, P.J. Thermodynamics of Solid to Solid Conversion and Application to Enzymic Peptide Synthesis. Enzyme Microb. Technol. 1995, 17, 601-606.
[80]黄惟德，陈常庆。多肽合成，科学出版社，第一版，1985．
[81]Valembois, C.; Mendre, C.; Cavadore, J.C.; Calas, B. Continuous flow synthesis of a biologically active small protein and a large peptide. Tetrahedron Lett. 1992, 33, 4005-4008.
[82]Sheehan, J.C.; Hess, G.P. A New Method of Forming Peptide Bonds. J. Am. Chem. Soc. 1955, 77(4), 1067-1068.
[83]Albertson, N.P. Synthesis of Peptides with mixed Anhydride in 'Organic Reagents'. 1962, 12, 157-355.
[84]Boissonnas, R.A. A new method of peptide synthesis. Helv Chim Acta. 1951, 34(3), 874-879.
[85]Vaughan, J. R.; Osato, J.R.L. The Preparation of Peptides Using Mixed Carbonic-Carboxylic Acid Anhydrides. J. Am. Chem. Soc. 1952, 74, 676-678.
[86]Wieland, T.; Schafer, W. Synthese von Oligopeptiden unter zellmoglichen Bedingungen. Angew. Chem. 1951, 63(6), 146-147.
[87]Schwyzer, R.; Iselin, B.; Feurer, M. Uber aktivierte Ester. I. Aktivierte Ester der Hippursaure und ihre Umsetzungen mit Benzylamin. Helv Chim Acta.1955, 38(1), 69-79.
[88]Bodanszky, M. Synthesis of peptides by aminolysis of nitrophenyl esters. Nature 1955, 175 (4459), 685-686.
[89]Curtius, T. Synthetische Versuche mit Hippurazid. Ber. Deutsch. Chem. Ges. 1902, 35, 3226-3228.
[90]Denkewalter, R.G.; Schwam, H.; Strachan, R.G.; et al. The Controlled Synthesis Of Peptides in Aqueous Medium I. The Use of α-Amino Acid N-Carboxyanhydrides. J. Am. Chem. Soc. 1966, 88, 3163-3164.
[91]Hirschmann, R.; et al. The controlled synthesis of peptides in aqueous medium. 3. Use of Leuchs' anhydrides in the synthesis of dipeptides. Mechanism and control of side reactions. J. Org. Chem. 1967, 32, 3415-3425.
[92]Veber, D.; Hirschmann, R.; Denkewalter, R.G. Synthesis of peptides in aqueous medium. Ⅵ. Synthesis of an unsymmetrical cystine peptide fragment of insulin. J. Org. Chem 1969, 34, 753-755.
[93]Iwakura, Y.; Uno, K.; Oya, M.; Katakai, R. Stepwise synthesis of oligopeptides with N-carboxy alpha-amino acid anhydrides. Biopolymers 1970, 9, 1419-1427.
[94]大屋正尚，片贝良一。有机合成化学协会志，1967，29，751．
[95]Merrifield, R.B. Peptide Synthesis on a Solid Polymer. Fed. Proc. 1962, 21(2), 412.
[96]Letsingerand, R.L.; Cornet, M.J.J. Am. Chem. Soc. 1963, 3045, 85.
[97]周云赟 吉林大学硕士论文 2002年
[98]Bashkin, J.K.; Mcbeath, R.J.; Modak, A.S.; et al. Synthesis and characterization of oligonucleotide peptides. J. Org. Chem. 1991, 56(9), 3168-3176.
[99]De Bont, D.B.A.; Moree, W.J.; Van Boom J.H.; et al. Solid-phase synthesis of o-phasphorathioylserine and threonine-Containing peptides as well as of o-phosphoserine-and-threonine-containing peptides. J.Org.Chem. 1993, 58(6), 1309-1317.
[100]Cohen-Auifieid, S.T.; Lansbury, P.T. A practical convergent method for glycopeptide synthesis. J. Am. Chem. Soc. 1993, 115(23), 10531-10537.
[101]叶蕴华，范崇旭，张德仪等，四个新型有机磷缩合试剂的合成及其在生物活性肽合成中的应用。高等学校化学学报，1997，18，1086—1092．
[102]Bodzansky, M. Peptide Chemistry: A Practical Textbook[M]. 2nd Ed., Springer-verlag, New York, 1993.
[103]罗九甫。酶和酶工程[M]。上海交通大学出版社，1996．314
[104]Fersht，A．酶的结构和作用机制(杜锦珠等译自Enzyme Structure and Mechanism)，北京大学出版社，1991，26．
[105]Schechter, I.; Berger, A. On the size of the active site in proteases. Ⅰ. Papain. Biochem. Biophy. Res. Commun., 1967, 27 (2), 157-162.
[106]Wong, C.H.; Wang, K. T. New developments in enzymatic peptide synthesis. Experientia, 1991, 47, 1123—1129.
[107]闫爱新等。蛋白酶在有机合成中应用的新进展。化学进展，2001，13(3)，203-208．
[108]彭师奇。多肽药物化学[M]。北京科学出版社，1993
[109]Van't Hoff, J.H. Uber die zunehmende Dedeutung der anorganischen Chemic. Z.Anorg. Allg. Chem. 1898, 18, 1-13.
[110]Bergmann, M., Fruton, J.S. On proteolytic enzymes. Synthetic substrates for chymotrypsin. J. Biol. Chem. 1937, 118, 405-415.
[111]Kullmann, W. Synthesis of Leu-and Met-enkephalin. Biochem.Biophys. Res.Commun. 1979, 91: 693-698.
[112]Moriharra, K.; Oka, T. α-chymotrypsin as the catalyst for peptide synthesis. Biochem. J. 1977, 163, 531-542.
[113]Chen, Y.X.; Zhang, X.Z,; et al. Kinetically controlled synthesis catalyzed by proteases in reverse micelles and separation of precursor dipeptide of RGD. Enzyme Microb. Technol, 1999, 25, 310-315.
[114]Ye, Y.H., Tian, G.L.; Xing, G.W.; et al. Enzymatic synthesis of N-protected Leu-enkephalin and some oligo-peptides in organic solvents. Tetrahedron 1998, 54, 12585-12596.
[115]So, J.E.; Kang, H.S.;Kim, B.G.; Lipase-Catalyzed synthesis of peptides containing D-Amino acid. Enzyme Microb.Technol. 1998, 23, 211-215.
[116]Oyama, K.; Niishimura, S.; et al. Synthesis of aspartotance precursor by immobilized thermolysin. J. Org. Chem. 1981, 46, 5241-5242.
[117]Moriharra, K. Using protease in peptide synthesis. Trend. Biotechnol. 1987, 5, 164-170.
[118]Fornanelez, M.M.; Margot, A.O.; Falender, C.A., et al. Enzymatic synthesis of peptides containing unnatural amino acids. Enzyme Microb. Technol. 1995, 17: 964-971.
[119]Krix, G.; Eichhorn, U.H.; Jakubke, H.D.; Kula, M.R., Protease-catalyzed synthesis of new hydrophobic amino acids. Enzyme Microb.Technol. 1997, 21,252-257.
[120]Ke, T.; et al. Enhancing Enzymatic enantio-selectivity in organic solvents by forming substrate salts. J. Am. Chem. Soc. 1999, 121, 3334-3340.
[121]Kullmann, W. Enzyntatic peptides Synthesis. CRC press. Boca Raton, 1987.
[122]Wang, B.K.; Gu, Q.M.; Sih, C.J. Enzyme-Catalyzed peptide segment condensation using 5(4H)-Oxazolones as donors. J. Am. Chem. Soc. 1993, 115: 7912-7913.
[123]张学忠，在有机介质中酶法合成小肽的研究进展，高技术通讯1998，7：55-58．
[124]Laane, C.; Boeren, S.; Vos, K. On optimizing organic solvents in multi-liquid phase biocatalysis. Trends Biotechnol. 1985, 3(10), 251-252.
[125]Reslow, M.; Adlercreutz, P.; Mattiassion B. Organic solvents for bioorganic synthesis. 1. Optimization of parameters for a chymotrypsin catalyzed process. Appl. Microbiol. Biotechnol. 1987, 26, 1-8.
[126]Simatos, D.; Multon, J. K. Properties of Water in Food, Dordrecht (Netherlands): Martinus Nijhoft, 1985, 111.
[127]Noritomi, H. et al. Enzymatic synthesis of peptide in acetonitrile/supercritical carbon dioxide. Biotechnol. Lett. 1995, 17 (12),1323-1328.
[128]Zhang, X. Z.; Wang, X.; Chen, S.; et al. Protease-catalyzed small peptide synthesis in organic media. Enzyme.Microb.Technol, 1996, 19, 538—544
[129]Zaks, A.; Klibanov, A.M. Enzyme-catalyzed process in organic solvents Pro. Natl. Acad.Sci. 1985, 82, 3192-3196.
[130]Yennawar, H.P.; Yennawar N.H.; Farber, G.K. A Structural Explanation for Enzyme Memory in Nonaqueous Solvents. J. Am. Chem. Soc. 1995,177 (2):577-585
[131]Stahl, M.; Jeppsson-Wistrand, U.;. Mansson, M.; et al. Induced stereoselectivity and substrate selectivity of bio-imprinted 2-chymotrypsin in anhydrous organic media. J. Am. Chem. Soc., 1991, 113: 9366-9369.
[132]Nilsson, K.; Skuladottir I, Mattiasson, B. Cryo-bioorganic synthesistemperature effects on enzymatic peptide synthesis. Biotechnol. Appl.Biochem, 1992, 16, i82
[133]邹国林，朱汝璠．酶学[M]．武汉：武汉大学出版社，1997
[134]Mitin, Y.V.; Zapevalova, N.P.; Gorbunova, E.Y. Peptide synthesis catalyzed by papain at alkaline pH values.Int J Peptide Protein Res May 1984; 23(5): 528-534.
[135]Stehle, P.; Bahsitta, H.P.; Monter, B.; Fürst, P. Papain-catalysed synthesis of dipeptides: A novel approach using free amino acids as nucleophiles. Enzyme Microb Technol 1990;12:56-60.
[136]赵明，彭师奇，迪丽努尔等。含RGD四肽的合成与功能。药学学报．1997，32(4)，271-277
[137]刘丽艳，崔凤侠，赵桂琴，王宏伟。活性肽RGD的液相合成化学试剂，2006，28(1)，55-56。
[138]张倩，张明智，张成海，丁伟。RGD三肽液相合成的研究。四川大学学报(工程科学版)，2001，33(6)，78-81．
[139]宋明媚，鞠培培，曹建军，沈树宝。化学法合成RGD三肽的研究生物加工过程2005，3(2)：23-26．
[140]杨大成，范莉，钟裕国．全保护RGD三肽的合成方法研究。有机化学，2003，23(5)：493-498．
[141]Hirano, Y.; Kando, Y.; Hayaahi, T.; Iwai, Y.; Hama, T.; Tsutsumi, Y.; Yamamoto, S.; Nakagawa, S.; Mayumi, T. Synthesis and cell attachment activity of bioactive oligopeptides:RGD RGDS RGDV and RGDT. J. Biomed. Mater. Res. 1991, 25, 1523-1534.
[142]Kawasaki, K.; Namikawa, M.; Yamashiro, Y. et al. Amino acid and peptides ⅩⅩⅤ preparation of fibronectin-related petide poly (ethylene gloycol) hybrid and their inhibitory effect on experimental metastasis. Chem. Pharm. Bull. 1995, 43(12), 2133-2137.
[143]Sofukku, S.; Hoshida, A.; Shimada, T. Synthesis of cyclic oigopeptides containing an Arg-Gly-Asp sequence. Chem. Pharm. Bull, 1999, 47(12), 1799-1801.
[144]Nicolaou, K. C., Trujillo, J. Chibate, K. Design, synthesis and biological evalution of carbohydrate-based mimetics. Tetrahedon, 1997, 53(26), 8751-8778.
[145]Dal-Pozzo, A.; Muzi, L.; Rondanin, R., et al. Synthesis of RGD analogues containing α-Tfm-Arginine as potential fibrinogen receptor antagonisis. Tetrahedon, 1998, 54(22), 6019-6028.
[146]Quirk, R.A.; Chan, W. C.; Davies, M. C.; et al. Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lacticacid). Biomaterials, 2001, 22(3), 865-872.
[147]Yamamoto, Y.; Sofuku, S. Synthesis of RGDF-containing cystine peptides by disulfide bond formation on a solid support. J Chem. Reseach. 1996, 12, 520-521.
[148]刘丽艳，陈治宇，崔凤侠，卢玉荣。RGD肽的固相合成。承德医学院学报，2005，22(1)，53-54．
[149]So, J.E.; Shin, J.S.; Kim, B.G. Protease-catalyzed tripeptide (RGD) synthesis. Enzyme Micro.Technol. 2000, 26, 108-114.
[150]陈育新，张学忠，陈松明，吴晓霞。在AOT／异辛烷反相胶束中酶法合成RGD前体二肽。中国生物化学与分子生物学报，1999，15(1)：146-150．
[151]Zhang, L.Q.; Xu, L.; Yang, X.C.; Wu, X.X.; Zhang, X.X. Lipase-Catalyzed Synthesis of Precursor Dipeptides of RGD in Aqueous Water-Miscible Organic Solvents. Prep. Biochem. Biotechnol. 2003, 33(1), 1-12.
[152]Bangham, A.D.; Standish, M.M.; Watkins, J.C. Diffusion of univalent ions across the lamellae of swollen phospholipid. J. Mol. Boil. 1965, 13, 238-252.
[153]Gregoriadis,G.; Swain, C.P.; Wills, E.J., et. al. Drag cartier potential of liposomes in cancer chemotherapy. Lancet, 1974, 1 (7870), 1313.
[154]刘昂，吴梧桐。制剂新技术在多肽、蛋白质类药物给药系统研究中的应用。中国现代应用药学杂志，2003，20(2)，110-114．
[155]艾国，杜丽娜，梅兴国。蛋白质、多肽类药物新剂型与新技术发展 动态。中国生化药物杂志，2004，25(6)，366-369．
[156]李国栋，王捷频。蛋白质、多肽类药物制剂的研究现状。药学实践杂志，2001，19(6)，344-349．
[157]顾学裘。中国多相脂质体研究。中国医药科技出版社，1991．116．
[158]李彦超，蔡宝昌。脂质体在抗肿瘤药物研究中的应用。南京中医药大学学报，2003，19(4)，250-253．
[159]崔福德。药剂学。人民卫生出版社，2003．7
[160]李彦超，蔡宝昌。脂质体在抗肿瘤药物研究中的应用。南京中医药大学学报，2003，19(4)，250-253．
[161]宫亮，杨和平。脂质体载药系统与抗肿瘤药物。药学专论，2005，14(3)，27-29．
[162]陈涛，王昭，傅经国，商鹏。靶向脂质体的研究。世界最新医学信息文摘，2003，2(6)，884-890．
[163]于洁，王振纲。脂质体在治疗肿瘤及感染性疾病中的前景。中国药理学通报，1998，14(3)，193-195．
[164]赵荣生，严宝霞。空间稳定脂质体与药物的靶向释放。中国药学杂志，1998，33(4)，452-455．
[165]符民桂，刘乃奎，唐朝枢。脂质体靶向性的研究进展。中国药理学通报，1999，15(1)，18-21．
[166]陈建明，张仰眉，高申。受脂质体作为药物载体的研究进展。药学实践杂志，2001，19(4)，214-218．
[167]Martin, C.; Danilo, W.; Lasic, D. Sterically stabilized liposomes. Biochimica et Biophysica Acta 1992, 1113, 171-199.
[168]王闻珠，邓英杰。脂质体肺部给药研究进展。沈阳药科大学学报，2000，17(3)，226-229．
[169]纪俊敏，谢文磊。脂质体作为药物载体的研究进展。郑州工程学院学报，2002，23(4)，68-72．
[170]张激，程艳菊，吕浩，应汉杰。脂质体的研究新进展。南京工业大学学报，2003，25(4)，102-106．
[171]罗云敬，刘海涛，李天剑等。头孢菌素类抗生素脂质体的包封率测定与渗漏研究。中国生化药物杂志1999，20(1)：4-6．
[172]朱希强，翟光喜。低分子肝素类脂质体的研制中国生化药物杂志2001，22(5)，232-234．
[173]任进余，陈义，吴斌等。病毒性肝炎的药物靶向治疗研究：抗HBV脂质体的研制及临床观察。中西医结合肝病杂志1997，7(1)，1-4．
[174]阎家麟，童岩，王九一。紫杉醇脂质体的制备即其抑瘤作用的研究。药物生物技术1996，3(3)，1-5．
[175]丁玉玲，马疏贤，卢秀荣等。人参皂甙脂质体(GSL)的研制。中国药学杂志，1995，30(7)，414-418．
[176]肖旭。5-氟脲嘧啶温度敏感性脂质体制备方法的优化。药学实践杂志1998，16(6)，344-346．
[177]李国锋，周日红，曾抗等。维生素E脂质体的制备。中国应用药学 1997，14(4)，18-20．
[178]张今，秦德安，陈百先等。包有蓖麻毒蛋白的脂质体的制备、性质及毒性研究。生物化学和生物物理学报，1999，31(4)，472-474．
[179]隋因，刘宝庆，夏东亚。冷冻干燥法制备阿糖胞苷冻干脂质体粉针研究。中国医院药学杂志，1999，15(11)，487-490．
[180]王弘，吴梧桐，顾学裘。重组L-前体脂质体的研制及包封率测定。沈阳药科大学学报，1999，16(4)，235-238．
[181]裴瑾，杨成君，杨翰仪等。白细胞介素2脂质体的制备及抗肿瘤作 用的研究。中国生化药物杂志，1997，18，166-169．
[182]李志宇，何生，薛华等。葡萄球菌肠毒素A脂质体的制备及其体内分布试验。华西医科大学学报2002，33(2)，299-301．
[183]储茂泉，古宏晨，刘国杰。丹参酮脂质体的制备及其稳定性。中国医药工业杂志2001，32(9)，400-404．
[184]翟光喜，赵焰。低分子肝素前体脂质体制剂的研究。山东医科大学学报2001，39(3)，218-220．
[185]全东琴，苏德森，顾学裘。药物载体空白脂质体前体的制备及性质的研究。沈阳药科大学学报1999，16(4)，239-241．
[186]邓英杰，刘素芹，韩丽梅等。黄芪多糖脂质体的制备和稳定性研究。沈阳药科大学学报1996，13(1)，1-4．
[187]Hwang S H, Maitani Y, Qi X R,et al.Remote loading of diclofenac, insulin and fluorscein isothiocyanate labeled insulin in to liposomes by pH and acetate gradient methods[J].Int J Pharm,1999, 179(1): 85-91.
[188]储茂泉，古宏晨，刘国杰。喷雾干燥法制备丹参酮前体脂质体的研究。中国药学杂志2002，37(1)：32-35．
[189]瞿文，陈庆华．黄体激素释放激素及其类似物的缓释给药系统。中国医药工业杂志1999，30(4)：181-188．
[190]Trehan A, Ali A. Recent approaches in insulin delivery[J]Drug Dev Ind Pharm. 1998, 24(7): 589-597.
[191]Yang, J.; Cleland, J.L.; Factors affecting the in vitro release of recombinant human interfemnT-(rhIFN-7)from PLGA microspheres. J Pharm Sci. 1997, 86(8): 908-914.
[192]Chen, L.; Apte, R.; Cohen, S. Characterization of PLGA microspheres for the controlled delivery of IL-1 for tumor immunotherapy. J Controlled Release. 1997, 43, 261-272.
[193]Johnson, O.L.; Jaworowicz, W.; Cleland, J.L.; et al. The stabilization and encapsulation of human growth hormone into biodegradable Microspheres. Pharm. Res. 1997, 14(6), 730-735.
[194]Hanes, J.; Cleland, J.L.; Langer, R. New advances in microsphere based single-dose vaccines. Adv. Drug Deliv. Rev. 1997, 28, 97-119.
[195]杨正管。美国药物开发公司开发口服降钙素。药学进展1996，20(1)，58．
[196]张海松，陈阳述，陈文吟等。口服促肝细胞生长素微球的研究。中国生化药物杂志1998，19(4)，180．
[197]Pontiroli, A.E. Peptide hormones: Review of current and emerging uses by nasal delivery. Adv. Drug. Deliv. Rev 1998, 29(1-2), 81-87.
[198]Takenaga, M.; Serizawa, Y.; Azechi, Y.; et al. Microparticle resins as a potential nasal drug delivery system for insulin. J. Controlled Release 1998, 52(1-2),81-87.
[199]Cole, M.L.; Whateley, T.L. Release rate profiles of theophylline and insulin from stable multiple w/o/w emulsions. J Controlled Release, 1997, 49(1), 51-58.
[200]Li, J.K.; Wang, N.; Wu, X.S. A novel biodegradable system based on gelatin nanoparticles and poly(lactide-co-glycolic acid) microspheres for protein and peptide drug delivery. J. Pharm. Sci. 1997, 86 (8), 891-895.
[201]Chen, S.T.; Chen, S.Y.; Wang, K.T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease Alcalase. J. Org. Chem. 1992, 57, 6960-6965.
[202]Klein, J.U.; Prykhodzka, A.; Cerovsky, V. The Applicability of Subtilisin Carlsberg in Peptide Synthesis. J. Peptide Sci. 2000, 6, 541-549.
[1]Chen, S.T.; Chen, S.Y.; Wang, K.T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease alcalase. J. Org. Chem. 1992, 57, 6960-6965.
[2]Chen, S.T.; Kao, C.L.; Wang, K.T. Alkaline protease catalysis of a secondary amine to form a peptide bond. Int. J. Pept Protein Res. 1995, 46, 314-319.
[3]Chen, Y.X.; Zhang, X.Z. Protease-catalyzed synthesis of precursor dipeptides of RGD with reverse micelles. Enzyme Microbl&Technol. 1998, 23,243-248.
[4]Chen, S.T.; Chen, S.Y.; Hsiao, S.C.; Wang, K.T. Application of industrial protease "Alcalase" in peptide synthesis. Biomed Biochim Acta 1991, 50, 181-186.
[5]So, J.E.; Kang, H.S.; Kim, B.G.; Lipase-Catalyzed synthesis of peptides containing D-Amino acid. Enzyme Microb. Technol. 1998, 23, 211-215.
[6]Zhou, Y.Y.; Yang, T.; Chemo-enzymatic synthesis of tripeptide RGD in organic solvents. Enzyme Microb.Technol. 2003, 33, 55-61.
[7]Ren, G.P.; Gu, W.Y. Production of a Bioactive Gin-enriched oligopeptide Nutrient solution From corn Protein by(Ⅰ) Enzyme Screening for Alcohol-water System. Journal of the Chinese Cereals and Oils Association. 2000, 15, 18-22.
[8]Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rule of optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987; 30, 81-87.
[9]Okazaki, S.; Goto, M.; Furusaki, S. Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb. Technol. 2000, 26, 159-164.
[10]Lozano, P.; Cano, J.; Iborra, J.L.; Manjon, A. Glycylglycylphenylalanin amide synthesis catalysed by papain in a medium containing polyols. Biotechnol.Appl.Biochem. 1993, 18, 67-74.
[11]Hailing, P.J. High-affinity binding of water by proteins is similar in air and in organic solvents. Biochem.Biophys. Acta 1990, 1040, 225-228.
[12]Klibanov, A.M. Enzymes that work in organic solvents. Chemtech. 1986, 16, 354-359.
[1]Chen, S.T.; Chen, S.Y.; Wang, K.T. Kinetically controlled peptide bond formation in anhydrous alcohol catalyzed by the industrial protease Alcalase. J. Org. Chem. 1992, 57, 6960-6965.
[2]Klein, J.U.; Prykhodzka, A.; Cerovsky, V. The Applicability of Subtilisin Carlsberg in Peptide Synthesis. J. Peptide Sci. 2000, 6, 541-549.
[3]Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rule of optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 1987, 30, 81-87.
[4]Okazaki, S.; Goto, M.; Furusald, S. Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb. Technol. 2000, 26, 159-164.
[5]Affleck, R.; Xu, Z.F.; Suzawa, V.; et al. Enzymatic catalysis and dynamics in lower ater environment. Proc. Natl. Acad. Sci. USA, 1992, 89, 1100-1104.
[6]Xu, Z.F.; Affleck, R.; Wangikar, P.; et al. Transition state stabilization of subtilisins in organic media. Biotechnol. Bioeng. 1994, 43, 515-520.
[7]Zaks, A.; Klibanov, A.M. The effect of water on enzyme action in organic media. J. Biol. Chem. 1988, 263(17), 8017-8021.
[8]Bone, S.; Pethig, R. Dielectric studies of protein hydration and hydration-induced flexibility. J. Mol. Biol. 1985, 181,323-326.
[9]Maes, D.; Bouckaert, K.J.; Poortmans, F.; Wyns, L.; Looze, Y. Structure of chymopapain at 1.7 A resolution. Biochemistry 1996, 35, 16292-16298.
[10]Monter, B.; Herzog, B.; Stehle, P.; Furst, P. Kinietically controlled synthesis of dipeptides using ficin as biocatalyst. Biotechnol. Appl. Bioc. 1991, 14, 183-191.
[11]Mitin, Yu.V.; Zapevalova, N.P.; Gorbunova, E.Yu. Pepetide synthesis catalyzed by papain at alkaline pH values. Int. J. Peptide protein Res 1984, 23,528-534.
[12]Iqbal, G.; Rosina, L.F.; Evgeny, V. Enzymatic oligopeptide synthesis using a minimal protection strategy: sequential assemble of a growing oligopeptide chain. J. Am. Chem. Soc. 1995, 117, 6175-6181.
[13]Joseph, O.R.; Jonathan, S.D. Controlling subtilisin activity and selectivity in organic media by imprinting with nucleophilic substrates. J. Am. Chem.Soc. 1997, 119, 3245-3252.
[14]Marianne, S.; Ulla, J.W.; Mates, O.M.; Klaus, M. Induced stereoselectivity and substrate selectivity of bio-imprinted α-chymotrypsin in anhydrous organic media. J. Am. Chem. Soc. 1991, 113, 9366-9368.
[15]Isono, Y.; Nakajima, M. Enzymic peptide synthesis using s microaqueous highly concentrated amino acid mixture. Process Biochem. 2000, 26, 275-278.
[16]Erbeldinger, M.; Ni, X.; Halling, P.J. Enzymatic synthesis with mainly undissoved substrates at very high concentrations. Enzyme Microb. Tech. 2000, 23,141-148.
[1]So, J.E.; Kang, H.S.; Kim, B.G. Lipase-Catalyzed synthesis of peptides containing D-Amino acid. Enzyme Microb.Technol. 1998, 23, 211-215.
[2]Mitin, Y.V.; Zapevalova, N.P.; Gorbunova, E.Y. Peptide synthesis catalyzed by papain at alkaline pH values. Int J Peptide Protein Res. 1984, 23(5), 528-534.
[3]Schechter, I.; Berger, A. On the size of the active size in proteases. Ⅰ. Papaln Biochem Biophys Res Commun. 1967, 27, 157-162.
[4]Isowa, Y.; Ohmori,M.; Sato, M.; Moil, K. Bull Chem Soc Japan. The Enzymatic Synthesis of Protected Valine-5 Angiotensin Ⅱ Amide-1 1977, 50, 2766-2772.
[5]Kullmann, W. Enzymatic synthesis of Leu-and Met-enkephalin. Biochem Biophys Res Commun. 1979, 91,693-698.
[6]Kullmann W. Proteases as catalysts for enzymic syntheses of opioid peptides. J Biol Chem. 1980, 255, 8234-8238.
[7]Stehle, P.; Bahsitta, H.P.; Monter, B.; Fürst, P. Papain-catalysed synthesis of dipeptides: A novel approach using free amino acids as nucleophiles. Enzyme Microb Technol. 1990, 12, 56-60.
[8]Lowe, G. The cysteine proteinases.Tetrahedron 1976, 32, 291-302.
[9]Barbas, C.F.; Wong, C.H. One-pot tripeptide syntheses from three single amino acid derivatives catalyzed by papain. Tetrahedron Lett. 1988, 24, 2907-2910.
[10]Hou, R.Z.; Zhang, N. Synthesis of tripeptide RGD amide by a combination of chemical and enzymatic methodsv. Journal of Molecular Catalysis B: Enzymatic 2005, 37, 9-15.
[11]Okazaki, S.; Goto, M.; Furusaki, S. Surfactant-protease complex as a novel bio-catalyst for peptide synthesis in hydrophilic organic solvents. Enzyme Microb Technol. 2000, 26, 159-164.
[12]Lozano, P.; Cano, J.; Iborra, J.L.; Manjón, A. Glycylglycylphenylalan inamide synthesis cataiysed by papain in a medium containing polyols. Biotechnol.Appl. Biochem. 1993, 18, 67-74.
[13]Whitaker, J. R.; Bender, A. J.Am.Chem.Soc. 1965, 87, 2728
[14]Isono, Y.; Nakajima, M. Enzymic peptide synthesis using a microaqueous highly concentrated amino acid mixture. Process Biochem. 2000, 36; 275-278.
[15]Erbeldinger, M.; Ni, X.; Hailing, P.J. Enzymatic synthesis with mainly undissolved substrates at very high concentrations. Enzyme Microb Technol. 2000, 23, c141-c148.
[1]黄惟德，陈长庆。《多肽合成》科学出版社。1985，P102—140．
[2]Hirschmann, R.; Strachan, R.G.; Schwam, H.; Schoenewaldt, E.F.; Joshua, H.; Barkemeyer, B.; Veber, D.F.; Paleveda, W.J.; Jacob,T.A.; Beesley, T.E.The Controlled Synthesis of Peptides in Aqueous Medium.Ⅲ.Use of Leuchs'Anhydrides in the Synthesis of Dipeptides. Mechanism and Control of Side Reactions, J.Org. Chem. 1967, 32, 3415-3425.
[3]Veber, D.F.; Hirschmann, R.; Denkenwalter, R.G. The Synthesis of Peptides in Aqueous Medium.Ⅵ. The Synthesis of an Unsymmetrical Cystine Peptide Fragment of Insulin. J.Org. Chem. 1969, 34,753-755.
[4]Patchornik, A.; Berger, A.; Katchalski, E. Poly-L-histidine, J. Am. Chem. Soc. 1957,79, 5227-5230.
[5]Denkewalter, R.G.; Schwam, H.; Strachan, R.G.; Beesley, T.E.; Veber, D.F.; Schoenewaldt, E.F.; Barkemeyer, H.; Paleveda, W.J.; Jacob, T.A.; Hirschmann, R. The Controlled Synthesis of Peptides in Aqueous Medium. Ⅰ The use of α-Amino Acid N-Carboxyanhydrides J. Am. Chem. Soc. 1966, 88, 3163-3164.
[6]Kawasaki, K.; Namikawa, M.; Yamashiro, Y.; Iwai, Y.; Hama, T,; Tsutsumi, Y.; Yamamoto, S.; Nakagawa, S.; Mayumi, T. Amino Acids and Peptides.ⅩⅩⅤ.~(1)) Preparation of Fibronectin-Related Peptide Poly (ethylene glycol) Hybrids and Their Inhibitory Effect on Experimental Metastasis. Chem. Pharm. Bull. 1995, 43, 2133-2138.
[7]Zhao, M.; Peng, S.Q.; Dilinuer, S.; Wang, Y.Y.; Zhou, Q.L.; Zhao,W.Z. Studies on the synthesis and activities of RGD related peptides. Acta Pharmaceutica Sinica 1997, 32, 271-277.
[8]Maeda, M.; Izuno, Y.; Kawasaki, K.; Kaneda, Y.; Mu, Y.; Tsutsmi, Y.; Lem, K.W.; Mayumi,T, Amino Acids and.Peptides ⅩⅩⅫ: A Bifunctional Poly(Ethylene Glycol) Hybrid of Fibronectin-Related Peptides, Biochem. Biophys. Res. Commun. 1997, 241,595-598.
[9]赵明，彭师奇，迪丽努尔，等．含RGD四肽的合成与功能[J]．药学，1997，32(4)：271-277．
[10]张倩，张明智，张成海，丁伟。RGD三肽液相合成的研究。[J]四川大学学报(工程科学板)，2001，33(6)：78-81
[11]杨大成，范莉，钟裕国。全保护RGD三肽的合成方法研究[J]有机化学，2003，23(5)：493-498．
[12]Quirk R A, Chan W C,Davies M C,et al.Poly(L-lysine)-GRGDS as a biominmetic surface modifier for poly(lacticacid)[J].Biomaterials, 2001,22(3):865-872.
[13]Marion Zhang, Michael N. Solid-phase synthesis of Arginine-containing peptide by guanidine attachment to a sulfony linker[J]. J Org. Chem. 1997, 62(26): 9326-9330.
[14]Yamamoto Y, Sofuku S. Synthesis of RGDF-containing cystine peptides by disulfide bond formation on a solid support[J]. J Chem. Reseach. 1996, 12: 520-521.
[15]Gibson C,Goodman SL,Hahn D,etal. Novel solid-phase synthesis of azapeptides and azapeptoides via strategy and its application in the synthesis of RGD-mimetics.J Org.Chem. 1999, 64(20): 7388-7394.
[1]崔福德主编，药剂学[M]。人民卫生出版社，2003．7
[2]栾军主编，现代试验设计优化方法[M]。上海交通大学出版社，1995，3 第1版。
[3]曹宁宁，羡菲，刘金鹏。脂质体的制备方法及研究进展。天津理工学院学报，2003，19(1)，30-34．
[4]余旭亚，赵声兰，李涛等。仙人掌SOD脂质体的制备研究。昆明理工大学学报，2002，27(1)，103-105．
[5]胥传来，乐国伟，姚惠源。冻干PST脂质体的制备及物理稳定性的研究。中国油脂，2001，26(6)，54-57．
[6]Cansell, M.; Parisel, C.; Jozefonvicz, J.; et al. Liposomes coated with chemically modified dextran interaet with human endothelial cells. J Biomed Mater Res.1999, 44(2), 140-148.
[7]胡俊，张康，王筱榕。一种新型脂质体的制作方法。第一军医大学学报，2001，16(3)，167-169．
[8]丁玉玲，马淑贤，卢秀荣。人参皂甙脂质体的研制。中国药学杂志，1995，30(7)，414-415．
[9]张奇，邓英杰。冻融法制备5-氟尿嘧啶脂质体及其稳定性考察。沈阳药科大学学报，2002，17(2)，64-67．
[10]赵荣生，侯新朴，严宝霞。两性霉素B脂质体的制备及稳定性研究。中国医药工业杂志，2001，32(4)，160-162．
[11]刘辉，汤韧，何小霞等。脂质体处方和制备方法对阿昔洛韦棕榈酸酯脂质体稳定性的影响。药学学报，2002，37(7)，563-566．
[12]胥传来，乐国伟，姚惠源等，生长激素脂质体的制备。云南农业大学学报，2001，16(3)，167-169．
[13]李晓燕，陈卫，侯新朴。癸烷基葡萄糖对载药脂质体的渗漏保护作用。北京医科大学学报，1995，27(1)，75．
[1]Ruoslahti, E.; Pierschbacher, M.D. New perspectives in cells adhesion RGD and integrins. Science 1987, 218, 491-499.
[2]顾玲，杨连甲，董绍忠等。RGDS对脑转移舌癌细胞系Tb侵袭转移行为的影响。临床口腔医学杂志，2004，20(1)，14-16．
[3]Hou, R.Z.; Liu, Y.J.; Zhang, N.; Huang, Y.B.; Wang, H.; Yang, Y.; Xu, L.; Zhang, X.Z. New synthetic route for RGD tripeptide. Prep Biochem Biotechnol. 2006, 36(3), 243-252.
[4]司徒德强。细胞培养 北京世界图书出版公司，2004．3
[5]Anuradha, C.D.; Kanno, S.; Hirano, S. RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation. Cell Biol Toxicol. 2000, 16(5), 275-283.
[6]Buckley, C.D.; Pilling, D.; Henriquez, N.V.; et al. RGD peptides induce apoptosis by direct caspase-3 activation. Nature, 1999, 397(6719) 534-539.
[7]Yamamoto, Y.; Tsutsumi, Y.; Mayumi, T. Molecular design of bioconjugated cell adhesion peptide with a water-soluble polymeric modifier for enhancement of antimetastatic effect. Curr Drug Targets, 2002, 3(2), 123.
[8]Madeja, Z.; Sroka, J. Contact guidance of Walker carcinosarcoma cells by the underlying normal fibroblasts is inhibited by RGD-containing synthetic peptides. Folia Histochem Cytobiol. 2002, 40(3), 251