蛋白纳米脂质体制备过程中的活性保护研究
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摘要
为研究蛋白和多肽类药物在制剂过程中的活性变化及其机理,减少药物在制剂过程中的活性损失,本文以胰蛋白酶为模型药物制备纳米脂质体,研究制备过程中胰蛋白酶的活性变化,并添加四种活性保护剂保护蛋白活性,通过检测蛋白的结构变化和分子动力学模拟过程来考察保护剂保护蛋白活性的机制。
1,采用逆向蒸发法制备胰蛋白酶纳米脂质体,粒径160nm,包封率为47%,Zeta电位-58mv;胰蛋白酶在脂质体制备过程中活性损失为原始蛋白的37%,四种保护剂中PEG对活性的保护效果最好,可使活性保留为原始蛋白的46%,是一种良好的保护剂。
2,采用圆二色谱和傅里叶红外光谱研究胰蛋白酶在制剂过程中的二级结构变化,应用荧光光谱研究蛋白的三级结构变化。圆二色谱显示胰蛋白酶在纳米脂质体制备过程中其二级结构中的α-螺旋含量由18.7%降为13.2%,β-折叠和β-转角含量分别由15.6%和27.0%增加到20.3%和31.9%,红外结果显示相同趋势。荧光数据表明有机溶剂处理后蛋白荧光性增加,制备成脂质体后荧光性减小,分子更加致密。四种保护剂的加入可以使蛋白的二级结构得到保留,其中PEG的保护效果最佳,但对三级结构的变化影响不大。
3,采用flexX对接和amber软件对保护剂中最简单的分子精氨酸和胰蛋白酶活性中心进行了分子对接和分子模拟,研究精氨酸与胰蛋白酶的作用机制。结果显示精氨酸可进入到胰蛋白酶的活性腔道中,并与之可形成7对氢键,两者结合力很强,理论上可以维持蛋白活性腔的三维结构,保护活性腔不被外界因素的影响而发生变化。
结果表明,保护剂可能是通过防止胰蛋白酶在脂质体制备过程中的二级结构变化,达到减少蛋白活性在制剂过程中的损失。
To study the activity lost of protein and peptide drugs in the nano-liposome preparation process and mechanism of activity lost, in this paper, trypsin was token as a model drug, encapsulated in nano-liposome. And four protective agents were added in to prevent the protein activity lost, by detecting the changes in protein structure and molecular dynamics simulations to investigate the protective agent protection mechanism for protein activity.
1 . Prepared trypsin nano-liposome by reverse evaporation, particle size was160nm, encapsulation efficiency was 47%, Zeta potential was -58mv; trypsin liposome activity loss to 37% of the original protein during prepare process, PEG can help to reserve protein activity, PEG as the best protective agent could help the activity reserved 46%.
2. Investigated the secondary structural changes of trypsin in the preparation process using circular dichroism and Fourier transform infrared spectra, and investigated the tertiary structure changes using fluorescence spectroscopy. Circular dichroism data showed that in the preparation process the protein secondary structure changed, the ofα-helix content decreased from 18.7% to 13.2%,β-fold andβ-turn content increased from 15.6% and 27.0% to 20.3% and 31.9%, IR data showed the same trend. Fluorescence data show that the the whole liposome process decreased fluorescence. Four protective agents can stable the protein secondary structure, but less help with the changes in the tertiary structure loss, PEG best protection.
3. The simplest protective gent, arginine and trypsin active sites were simulated molecular docking and molecular modeling using flexX docking and amber software, to study the mechanism of arginine and trypsin interact. The results showed that arginine can enter into the activity cavity of trypsin, and 7 pairs of hydrogen bonds can be formed, in theory, arginine can maintain the cavity of the three-dimensional structure of protein activity, prevent external factors change the activity cavity.
The results showed that the protective agent may prevent trypsin active loss in nano-liposome prepraring process, by maintaining the secondary structure.
1,采用逆向蒸发法制备胰蛋白酶纳米脂质体,粒径160nm,包封率为47%,Zeta电位-58mv;胰蛋白酶在脂质体制备过程中活性损失为原始蛋白的37%,四种保护剂中PEG对活性的保护效果最好,可使活性保留为原始蛋白的46%,是一种良好的保护剂。
2,采用圆二色谱和傅里叶红外光谱研究胰蛋白酶在制剂过程中的二级结构变化,应用荧光光谱研究蛋白的三级结构变化。圆二色谱显示胰蛋白酶在纳米脂质体制备过程中其二级结构中的α-螺旋含量由18.7%降为13.2%,β-折叠和β-转角含量分别由15.6%和27.0%增加到20.3%和31.9%,红外结果显示相同趋势。荧光数据表明有机溶剂处理后蛋白荧光性增加,制备成脂质体后荧光性减小,分子更加致密。四种保护剂的加入可以使蛋白的二级结构得到保留,其中PEG的保护效果最佳,但对三级结构的变化影响不大。
3,采用flexX对接和amber软件对保护剂中最简单的分子精氨酸和胰蛋白酶活性中心进行了分子对接和分子模拟,研究精氨酸与胰蛋白酶的作用机制。结果显示精氨酸可进入到胰蛋白酶的活性腔道中,并与之可形成7对氢键,两者结合力很强,理论上可以维持蛋白活性腔的三维结构,保护活性腔不被外界因素的影响而发生变化。
结果表明,保护剂可能是通过防止胰蛋白酶在脂质体制备过程中的二级结构变化,达到减少蛋白活性在制剂过程中的损失。
To study the activity lost of protein and peptide drugs in the nano-liposome preparation process and mechanism of activity lost, in this paper, trypsin was token as a model drug, encapsulated in nano-liposome. And four protective agents were added in to prevent the protein activity lost, by detecting the changes in protein structure and molecular dynamics simulations to investigate the protective agent protection mechanism for protein activity.
1 . Prepared trypsin nano-liposome by reverse evaporation, particle size was160nm, encapsulation efficiency was 47%, Zeta potential was -58mv; trypsin liposome activity loss to 37% of the original protein during prepare process, PEG can help to reserve protein activity, PEG as the best protective agent could help the activity reserved 46%.
2. Investigated the secondary structural changes of trypsin in the preparation process using circular dichroism and Fourier transform infrared spectra, and investigated the tertiary structure changes using fluorescence spectroscopy. Circular dichroism data showed that in the preparation process the protein secondary structure changed, the ofα-helix content decreased from 18.7% to 13.2%,β-fold andβ-turn content increased from 15.6% and 27.0% to 20.3% and 31.9%, IR data showed the same trend. Fluorescence data show that the the whole liposome process decreased fluorescence. Four protective agents can stable the protein secondary structure, but less help with the changes in the tertiary structure loss, PEG best protection.
3. The simplest protective gent, arginine and trypsin active sites were simulated molecular docking and molecular modeling using flexX docking and amber software, to study the mechanism of arginine and trypsin interact. The results showed that arginine can enter into the activity cavity of trypsin, and 7 pairs of hydrogen bonds can be formed, in theory, arginine can maintain the cavity of the three-dimensional structure of protein activity, prevent external factors change the activity cavity.
The results showed that the protective agent may prevent trypsin active loss in nano-liposome prepraring process, by maintaining the secondary structure.
引文
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[27] Zhao, H L , Xue C , Wang Y , et al. Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-α2b and HSA fusion protein increases its stability against mechanical and thermal stresses[J]. European Journal of Pharmaceutics and Biopharmaceutics, 2009, 72(2): 405~411.
[28] Chi E Y, Krishnan S, Randolph T W, et al. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation[J]. Pharmaceutical Research, 2003 ,9 (20) :1325～1336
[29] Lee C S, Kim B G. Improvement of protein stability in protein microarrays[J]. Biotechnology Letters , 2002 ,10 (24) :839～844
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[31] Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals[J]. Inter J Pharm, 1999 ,8 (20) :129～188
[32] Martin AH, Grolle K, Bos MA , et al . Network forming properties of various proteins adsorbed at the air/ water interface in relation to foam stability. J Col Inter Sci[J] , 2002 ,7(254) :175～183
[33] Perez C, Griebenow K. Effect of salts on lysozyme stability at the water-oil interface and upon encapsulation in poly(lacticco- glycolic) acid microspheres. Biotechnol Bioeng [J], 2003, 82( 7): 825-832.
[34] He J T, Su H B, Li G P, et al. Stabilization and encapsulation of a ataphylokinase variant (K35R) intopoly (lactic-co-glycolicacid) microspheres [J], International Journal of Pharmacuticals [J], 2006,309:101~108;
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[36] Sah H. Stabilization of proteins against methylene chloride/water interface-induced denaturation and aggregation[J]. J Controlled Release, 1999, 58(2): 143-51.
[37] Morlock M, Koll H, Winter G, et al. Microencapsulation of rh-erythropoietin, using biodegradable poly(D, L-lactide-coglycolide). Protein stability and the effects of stabilizing excipients[J]. European Journal of Pharmaceuticals and Biopharmaceuticals, 1997, 43(1): 29-36.
[38] Kang F, Jiang G, Hinderliter A, et al. Lysozyme stability in primary emulsion for PLGA microsphere preparation: effect of recovery methods and stabilizing excipients. Pharmaceutical Research [J], 2002, 19(5): 629-633.
[39] Lam X F, Duenas ET, Cleland JL. Encapsulation and stabilization of nerve growth factor into poly(lactic-co-glycolic) acid microspheres[J]. Journal of Pharmacy Science, 2001, 90(9): 1356-1365.
[40] Pean J M , Boury F , Venier M C , et al. Why does PEG 400 co-encapsulation improve NGF stability and release from PLGA biodegradable microspheres?[J]. Pharm Res, 1999, 16 (8): 1294-1299.
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[2]姚祖华,李亦德.生态制品的冻干及保护剂的应用[J].中国微生态学杂志,1997,9(6):48.
[3] Hsu C C. Determining the optimum residual moisture in lyophilized protein pharmaceuticals[J]. Development Biology Standard, 1991, 74(5):255.
[4] Shan J, Steven L N. Effect of process condition on recovery of protein activity after freezing and freezedrying[J]. European Journal of Pharmaceuticals and Biopharmaceuticals. 1998, 45:249~257.
[5] Tsvetkov TD, Tsonev LI, Tsvetkova NM, et al. Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers[J]. Cryobiology, 1989, 26: 162.
[6] Carpenter J F, Crowe J H. The mechanism of cryoprotection of proteins by solutes[J]. Cryobiology, 1988, 25:244.
[7] Labrude P, Chaillot B, Vigneron C. Problems of haemoglobin freezedrying: evidence that water removal is the key to Iron oxidation[J]. Journal of Pharmaceutical Pharmacology, 1987, 39:344.
[8] Hanafusa N. The behavior of hydration water of protein with the protectant in the view of HNMR[J]. Development Biology Standard, 1991, 74(3): 241.
[9] Tian Z M, Wall M X, Wang S P, et a1.Efects of ultrasound and additives on the function and structure of trypsin.Ultrason Sonochem,2004,l1:399~404
[10]朱建华,杨晓泉,熊犍.超声处理对大豆蛋白表面性质的影响,中国生物工程杂志[J],2005,31(3):16~20
[11]罗昭峰,瞿鑫,沐万孟等.超声和高压处理对牛血清白蛋白结构的影响,中国生物工程杂志[J], 2006,26(1)46~49
[12] Mett H, Schacher B, Wegmann L. Ultrasonic disintegration of bacteria may lead to irreversible inactivation of beta~lactamase[J]. Journal of Antimicrobial Chemotherapy, 1988, 22:293~298
[13]Griebenow AS, Theodore W. Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration induced damage and its inhibition[J]. Archives of Biochemistry and Biophysics, 1998, 46(1): 171.
[14] Pérez C R, Montano N, Gonzalez K. Stabilization ofα-chymotrypsin at the CH2Cl2/water interface and upon water-in-oil-in-water encapsulation in PLGA microspheres[J]. Journal of Controlled Release, 2003, 89(1):75~89
[15]阎微.高压和热处理对蛋黄体系中蛋白质的影响,[硕士学位论文],江苏,江南大, 2009
[16]肖和兰,孙素琴,周群等.温度对胶原蛋白二级结构影响的二维红外相关光谱的研究[J],原子与分子物理学报. 2003, 20(2):211~220.
[17] OlivaA,SantovenaA,Farina J,et a1.EffectofhJ gh shear rate on stability of proteins:kinetic study [J]. Journal of Pharmaceutical Biomed Ana1.2003, 33:145~155.
[18]张杰,郭妮妮,张代佳等.复想乳化法制备海藻酸钠微球及其释放行为,过程工程学报[J]. 2006, 6(6):964~969.
[19]李乐军.荧光光谱与激光拉曼光谱研究脉冲电场对蛋白质构象的影响[硕士学位论文],上海,,华东师范大学, 2005.
[20] Masson P L, Cléry C, Renault F, et al. Pressure-induced molten globule state of cholinesterase[J]. Febs letters, 1995, 370: 212~214.
[21] Steven L N , Michael J A. Development and Manufacture of Protein Pharmaceuticals[M],北京:化学工业出版社,2006,40~45.
[22]樊莉,鲁莹,吴诚等.干扰素α-2b复合物PLGA微球的制备及影响因素[J],中国医药工业杂志, 2006, 37(10):674~678.
[23] Carrascosa C., ITorresA., Lopez L.C., et al.. Microspheres containing insulin-like growth factor I for treatment of chronic neurodegeneration, Biomaterials[J]. 2004, 25(4):707~714 .
[24] Zhu G Z, Schwendeman S P. Stabilization of proteins encapsulated in cylindricalpoly(lactied-co-glycolide)implants: mechanism of stabilization by basic additives[J]. Pharmaceutical Reseach, 2000, 17(3):351~357.
[25] Strub C, Alies C , Lougarre A , et al . Mutation of exposed hydrophobic amino acids to arginine to increase protein stability[J]. BMC Biochem, 2004,1 (5) :9～15.
[26] Gerald R G, Kevinl S K, Lanetter F, et al . Increasing protein stability by altering long-range coulombic interactions[J]. Protein Science, 1999, 8(9):1843~1849
[27] Zhao, H L , Xue C , Wang Y , et al. Elimination of the free sulfhydryl group in the human serum albumin (HSA) moiety of human interferon-α2b and HSA fusion protein increases its stability against mechanical and thermal stresses[J]. European Journal of Pharmaceutics and Biopharmaceutics, 2009, 72(2): 405~411.
[28] Chi E Y, Krishnan S, Randolph T W, et al. Physical stability of proteins in aqueous solution: mechanism and driving forces in nonnative protein aggregation[J]. Pharmaceutical Research, 2003 ,9 (20) :1325～1336
[29] Lee C S, Kim B G. Improvement of protein stability in protein microarrays[J]. Biotechnology Letters , 2002 ,10 (24) :839～844
[30] Cloos AC, Christgau S. Non - enzymatic covalent modifications of proteins : mechanisms, physiological consequences and clinical applications. Mat rix Bio[J] , 2002 ,1 (21) :39～53
[31] Wang W. Instability, stabilization, and formulation of liquid protein pharmaceuticals[J]. Inter J Pharm, 1999 ,8 (20) :129～188
[32] Martin AH, Grolle K, Bos MA , et al . Network forming properties of various proteins adsorbed at the air/ water interface in relation to foam stability. J Col Inter Sci[J] , 2002 ,7(254) :175～183
[33] Perez C, Griebenow K. Effect of salts on lysozyme stability at the water-oil interface and upon encapsulation in poly(lacticco- glycolic) acid microspheres. Biotechnol Bioeng [J], 2003, 82( 7): 825-832.
[34] He J T, Su H B, Li G P, et al. Stabilization and encapsulation of a ataphylokinase variant (K35R) intopoly (lactic-co-glycolicacid) microspheres [J], International Journal of Pharmacuticals [J], 2006,309:101~108;
[35]约翰逊OL,坎姆金MM,伯斯滕H,等.人生长激素的延续释放组合物[P].CN: 1102854C, 2003-03-12.
[36] Sah H. Stabilization of proteins against methylene chloride/water interface-induced denaturation and aggregation[J]. J Controlled Release, 1999, 58(2): 143-51.
[37] Morlock M, Koll H, Winter G, et al. Microencapsulation of rh-erythropoietin, using biodegradable poly(D, L-lactide-coglycolide). Protein stability and the effects of stabilizing excipients[J]. European Journal of Pharmaceuticals and Biopharmaceuticals, 1997, 43(1): 29-36.
[38] Kang F, Jiang G, Hinderliter A, et al. Lysozyme stability in primary emulsion for PLGA microsphere preparation: effect of recovery methods and stabilizing excipients. Pharmaceutical Research [J], 2002, 19(5): 629-633.
[39] Lam X F, Duenas ET, Cleland JL. Encapsulation and stabilization of nerve growth factor into poly(lactic-co-glycolic) acid microspheres[J]. Journal of Pharmacy Science, 2001, 90(9): 1356-1365.
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