聚乙二醇介导RNase-BSA偶联物的制备及性质表征
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摘要
共价偶联是将一种蛋白偶联于另一种蛋白或者其他大分子物质，达到改善原蛋白溶解性或其他性质的化学交联或化学修饰的方法。白蛋白与药物可通过共价键相连形成偶联物，进入人体后，连接键将被体内酶催化断裂而释放药物，此过程不仅使药物溶解性提高，而且实现药物的缓释及靶向作用。聚乙二醇（PEG）由于具有良好的水溶性，与许多有机物组分有良好的相容性，已被广泛应用于食品、医药、化妆品及化学纤维等领域。PEG化可以通过延长蛋白质药物在体内的循环半衰期以及降低免疫原性和抗原性达到提高蛋白质药物的功效。研究表明，牛胰核糖核酸酶A（RNase A）的结构和功能清晰明确，近年来的研究还发现其超家族与结构类似物具有抗肿瘤活性，但其本身表现不明显。为此，本论文以牛胰RNase A为模型蛋白，采用PEG等交联剂与大分子载体BSA（牛血清白蛋白）的交联技术对其进行修饰偶联研究，以增强、稳定其生物学活性。
一、制备偶联产物。通过引入异型双功能交联剂Sulfo-SMCC、PEG3500及辅助交联剂HDA，运用3种修饰策略实现RNaseA与BSA的偶联。采用分析型Superdex200凝胶过滤柱分析(1cm×30cm)及SDS-PAGE电泳法检测偶联物，得出最佳偶联条件。结果表明，以4℃作为最适反应温度； BSA:NEM:SMCC:RNase:IT=1:5:20:8:80；BSA:IT:RNase:PEG3500=1:10:0.8:4；RNase:IT:PEG3500:HDA=1:3:2:2，每200μL体系中加入20μL0.2mmol/L的BSA作为最佳摩尔比条件。采用AKTA蛋白纯化系统与制备型Superdex200凝胶过滤柱(2.6cm×70cm)制备偶联产物。
二、对偶联产物进行结构和功能的表征。首先利用内源荧光光谱、动态光散射技术及超速离心分析技术对3种RNase-BSA偶联产物的结构进行表征。发现与RNase A相比，BSA的荧光强度占主导地位，并且在偶联产物中RNase A起到荧光猝灭作用；偶联后流体力学半径都明显增加；RNase-SMCC-BSA偶联物具有最大的沉降系数和最小的摩擦系数比。推测其与分子量大小，交联剂的性质及偶联物的空间结构特点有关。另外，体外活性结果表明，RNase-SMCC-BSA偶联物拥有最高的相对活性保留率，偶联产物过夜之后（19h以后），相对活性保持率都基本稳定在75%以上，实现了缓释作用。说明白蛋白偶联及PEG修饰在改良RNaseA药学性质方面具有很大的优势。
本论文的修饰策略可对其它蛋白质和多肽类药物的药学性质改善提供借鉴，并为食品、饲料、化妆品及医药等领域的应用提供参考。
Bioconjugation technology is the process of conjugate proteins with proteins or otherbiomolecules by chemical crosslinking and modification methods, to alter the solubility or otherproperties of the original protein. Conjugation of albumin to the protein drug is achieved bycovalent bond that can be broken by the enzyme in vivo, which imparts resistance to proteolysis,improves solubility and achieves targeting. Poly-ethylene glycol (PEG) has been widely used infood, medicine, cosmetics, chemical fiber and other fields, due to its good water-solubility andfavorable compatibility with many organic components. PEGylation can improve the therapeuticefficacy of proteins by enhancing persistence in circulation and reducing immunogenicity andantigenicity. The structure and function of bovine pancreatic ribonuclease A (RNase A) has beenclearly known. Recent studies have found that the super-family and structural analogues haveanti-tumor activity. However, its own performance is not obvious. To strengthen and stabilize thebiological activity, RNase A was chosen as the model protein in the present study. Theconjugation of RNase A with albumin (BSA) by crosslinkers like PEG was conducted.
First, the RNase-BSA conjugates were prepared. Heterobifunctional crosslinkersSulfo-SMCC, PEG3500and assistant crosslinker Hexadecylamine (HDA) were chosen tofacilitate conjugation of RNase to albumin. The optimal conjugation conditions were analyzedby size exclusion chromatography using analytical grade Superdex200column (1cm×30cm)and SDS-PAGE. The result showed that the optimal reactant ratios were:BSA:NEM:SMCC:RNase:IT=1:5:20:8:80; BSA:IT:RNase:PEG3500=1:10:0.8:4;RNase:IT:PEG3500:HDA=1:3:2:2,20μL of0.2mmol/L BSA was added to200μL reactionsystem, and all reactions were taken at4℃. AKTA Purification System and preparative gradeSuperdex200(2.6cm×70cm) were used for purification of the RNase-BSA conjugates.
Secondly, the structural and functional characterization of the RNase-BSA conjugates wascarried out. Structural characterization of the conjugates was carried out using intrinsicfluorescence spectra, dynamic light scattering (DLS) and sedimentation velocity analyticalultracentrifugation (AUC). The present study suggested that compared with the weak emissionfluorescence intensity of RNase A which was equivalent to a quencher, BSA was predominant. The hydrodynamic radius of RNase A was increased greatly after conjugation of albumin. Whilethe RNase-SMCC-BSA conjugate showed the largest sedimentation coefficients (S20, w) andlowest frictional ratios (f/f0). It was presumably related to the molecular weight, the properties ofcrosslinkers and the structural characteristic of the conjugates. Then, the result of vitro activityshowed that the bioactivity of RNase-SMCC-BSA conjugate was the highest, and the bioactivityof75%was remained overnight (above19h), delay releasing was realized. This indicated that thealbumin binding and PEGylation would be a better strategy to improve the therapeutic efficacyof RNase A.
The present strategy may rationally optimize and tailor the pharmacological properties ofpeptides and proteins. Moreover, it can also provide a reference for application in many fieldslike food, feed, cosmetics, medicine and so on.
一、制备偶联产物。通过引入异型双功能交联剂Sulfo-SMCC、PEG3500及辅助交联剂HDA，运用3种修饰策略实现RNaseA与BSA的偶联。采用分析型Superdex200凝胶过滤柱分析(1cm×30cm)及SDS-PAGE电泳法检测偶联物，得出最佳偶联条件。结果表明，以4℃作为最适反应温度； BSA:NEM:SMCC:RNase:IT=1:5:20:8:80；BSA:IT:RNase:PEG3500=1:10:0.8:4；RNase:IT:PEG3500:HDA=1:3:2:2，每200μL体系中加入20μL0.2mmol/L的BSA作为最佳摩尔比条件。采用AKTA蛋白纯化系统与制备型Superdex200凝胶过滤柱(2.6cm×70cm)制备偶联产物。
二、对偶联产物进行结构和功能的表征。首先利用内源荧光光谱、动态光散射技术及超速离心分析技术对3种RNase-BSA偶联产物的结构进行表征。发现与RNase A相比，BSA的荧光强度占主导地位，并且在偶联产物中RNase A起到荧光猝灭作用；偶联后流体力学半径都明显增加；RNase-SMCC-BSA偶联物具有最大的沉降系数和最小的摩擦系数比。推测其与分子量大小，交联剂的性质及偶联物的空间结构特点有关。另外，体外活性结果表明，RNase-SMCC-BSA偶联物拥有最高的相对活性保留率，偶联产物过夜之后（19h以后），相对活性保持率都基本稳定在75%以上，实现了缓释作用。说明白蛋白偶联及PEG修饰在改良RNaseA药学性质方面具有很大的优势。
本论文的修饰策略可对其它蛋白质和多肽类药物的药学性质改善提供借鉴，并为食品、饲料、化妆品及医药等领域的应用提供参考。
Bioconjugation technology is the process of conjugate proteins with proteins or otherbiomolecules by chemical crosslinking and modification methods, to alter the solubility or otherproperties of the original protein. Conjugation of albumin to the protein drug is achieved bycovalent bond that can be broken by the enzyme in vivo, which imparts resistance to proteolysis,improves solubility and achieves targeting. Poly-ethylene glycol (PEG) has been widely used infood, medicine, cosmetics, chemical fiber and other fields, due to its good water-solubility andfavorable compatibility with many organic components. PEGylation can improve the therapeuticefficacy of proteins by enhancing persistence in circulation and reducing immunogenicity andantigenicity. The structure and function of bovine pancreatic ribonuclease A (RNase A) has beenclearly known. Recent studies have found that the super-family and structural analogues haveanti-tumor activity. However, its own performance is not obvious. To strengthen and stabilize thebiological activity, RNase A was chosen as the model protein in the present study. Theconjugation of RNase A with albumin (BSA) by crosslinkers like PEG was conducted.
First, the RNase-BSA conjugates were prepared. Heterobifunctional crosslinkersSulfo-SMCC, PEG3500and assistant crosslinker Hexadecylamine (HDA) were chosen tofacilitate conjugation of RNase to albumin. The optimal conjugation conditions were analyzedby size exclusion chromatography using analytical grade Superdex200column (1cm×30cm)and SDS-PAGE. The result showed that the optimal reactant ratios were:BSA:NEM:SMCC:RNase:IT=1:5:20:8:80; BSA:IT:RNase:PEG3500=1:10:0.8:4;RNase:IT:PEG3500:HDA=1:3:2:2,20μL of0.2mmol/L BSA was added to200μL reactionsystem, and all reactions were taken at4℃. AKTA Purification System and preparative gradeSuperdex200(2.6cm×70cm) were used for purification of the RNase-BSA conjugates.
Secondly, the structural and functional characterization of the RNase-BSA conjugates wascarried out. Structural characterization of the conjugates was carried out using intrinsicfluorescence spectra, dynamic light scattering (DLS) and sedimentation velocity analyticalultracentrifugation (AUC). The present study suggested that compared with the weak emissionfluorescence intensity of RNase A which was equivalent to a quencher, BSA was predominant. The hydrodynamic radius of RNase A was increased greatly after conjugation of albumin. Whilethe RNase-SMCC-BSA conjugate showed the largest sedimentation coefficients (S20, w) andlowest frictional ratios (f/f0). It was presumably related to the molecular weight, the properties ofcrosslinkers and the structural characteristic of the conjugates. Then, the result of vitro activityshowed that the bioactivity of RNase-SMCC-BSA conjugate was the highest, and the bioactivityof75%was remained overnight (above19h), delay releasing was realized. This indicated that thealbumin binding and PEGylation would be a better strategy to improve the therapeutic efficacyof RNase A.
The present strategy may rationally optimize and tailor the pharmacological properties ofpeptides and proteins. Moreover, it can also provide a reference for application in many fieldslike food, feed, cosmetics, medicine and so on.
引文
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