几种低温保护剂溶液玻璃化特性的分子模拟及机理分析
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摘要
玻璃化是保存大尺寸组织和器官的有效方法，但溶液的热应力和渗透应力等引起的问题限制了玻璃化保存方法的广泛应用。因此，低温玻璃化保存方法的成功很大程度上取决于玻璃化溶液的性质，如临界升降温速率、玻璃化转变温度等热及力学性质。
人们常用差示扫描量热法（DSC）、动态机械分析仪(DMA)和热机械分析仪（TMA）等仪器测量玻璃化溶液的性质，但要获得一种理想的玻璃化溶液，需要大量的实验。分子模拟不仅可以从微观上设计并控制各种条件，模拟玻璃化溶液的性质，而且可以分析宏观性质的分子机理，为选择合适的玻璃化溶液提供了一条捷径。
应用gromacs3.3和Material Studio软件，本文对几种低温保护剂溶液的玻璃化性质进行了分子模拟和机理分析，主要工作如下：
1、玻璃体的力学、热学性质与其结构有直接的关系，径向分布函数(radialdistribution function, RDF)反映了分子的聚集特性，是研究玻璃态结构的一个重要参数。本文研究了降温速率和压力对玻璃化转变温度和径向分布函数的影响，结果表明：降温速率对体系的RDF有影响，缩小温差并采取中间温度充分驰豫可减小这种影响；采用逐步冷却、直接冷却和两步法冷却时，Me_2SO溶液的RDF，RMSD（root mean squared diviation,均方根偏差）及氢键数量构成均表现不同的特征，但总氢键数目以及Me_2SO-H_2O的空间角位置没有明显变化，Me_2SO-H_2O的空间角位置关系有相似性和取向性和周期性的特征；对于30%、50%、70%Me_2SO溶液，在慢速降温时，RDF、RMSD及氢键数量构成表现不同的特征，其Me_2SO-H_2O空间角度分布也具有相似性，取向性和周期性的特点。
2、氢键是影响很多物质的结构、热力学、力学及其他性质的关键因素。在溶液的冷却过程中，无论结冰，还是玻璃化，都与分子内、外的氢键网有密切关系。研究了降温速率（1×107K/s，1×1012K/s）对70%Me_2SO溶液玻璃化过程氢键的角度、距离、生命期和数量的影响，结果表明：玻璃化过程中系统氢键、Me_2SO-H_2O氢键以及H_2O-H_2O氢键的键长分布均呈现泊松分布，不同的冷却速度对氢键键长分布影响不显著；玻璃化过程的系统氢键角度、Me_2SO-H_2O氢键角度以及H_2O-H_2O氢键角度的分布均呈现泊松分布，降温速度对氢键角度分布曲线的影响显著。
3、氢键生命期特性是氢键动力学重要特征之一，对保存在溶液中的细胞和组织的热及力的环境有较大的影响。研究了降温速率（1×107K/s，1×1012K/s）对70%Me_2SO溶液的玻璃化过程的氢键生命期分布的影响，结果表明：两种冷速下的系统氢键、Me_2SO-H_2O氢键、H_2O-H_2O氢键都表现出振荡变剧烈的特性；在时间大于大约3ps时，Me_2SO-H_2O氢键生命期分布在数值上要大于H_2O-H_2O的；氢键生命期以类似自变量系数为负的幂函数或者指数函数形式分布；不管是系统氢键、Me_2SO-H_2O氢键、还是H_2O-H_2O氢键的生命期分布，快速降温系统比慢速降温系统的生命期振幅要小得多，振幅变化也小很多。
4、首次用分子模拟方法研究和预测低温保护液玻璃化转变温度。对70%甘油水溶液玻璃化过程中的内聚能密度、溶解度参数和体积模量进行了计算，结果表明：这些参数在玻璃化转变温度附近出现明确的拐点，用它们来预测玻璃化温度要比用比热、密度、容积和径向分布函数更准确，得到的玻璃化转变温度与实验值吻合较好。
Vitrification is assumed to be a potential method for the cryopreservation of largetissues and organs. But the thermal stress and osmotic stress may damage the cells,which limit the practical use of vitrification. The properties of vitrification solution,such as critical cooling rate, glass transition temperature, are key factors that affect itssuccessful application in cryopreservation.
Differential scanning calorimetry (DSC), dynamic mechanical analyzer (DMA) andthermal mechanical analysis (TMA) are common equipments used to determine theproperties of vitrification solution. Lots of experiments must be peformed to obtain aoptimal solution. Molecular dynamic simulation can be used to predict the properties ofvitrification solution in molecular level. It provides a shortcut for selecting suitablevitrification solutions.
Applying gromacs3.3andMaterial Studio software, the propeties of severalvitrification solutions were simulated and the mechanism was analyzed in molecularlevel.
The thermal and dynamic properties of the material related to its structure. As theradial distribution functions(RDFs) reflects the collective property of molecules, it isregarded as an important factor affecting the structure of vitrification solutions. Theeffect of cooling rates and pressures on the glass transition temperature and RDF wereinvestigated in this study. The results show that cooling rate affects the RDFsof vitifiedwater. Three cooling methods were used to study the structure change of Me_2SOaqueous solution. The first is cooling to final temperature via a series of temperatureswith an interval of10K at a rate of1×1012K/s and sufficient relaxation time at eachtemperature. The second is cooling to final temperature directly at1×1012K/s. The thirdis a two-step cooling method using average between the intial and final temperature asmiddle temperature. The results show that RDF, root mean squared diviation(RMSDs)and the compostion of hydrogen bond show different patterns. Meanwhile, total numberof hydrogen bond and H_2O-Me_2SO space angles are characterized by similarity,directivity and periodicity without significant change during vitrification. Threeconcentrations (70%,50%and30%, wt%) of Me_2SO solutions are investigated toobserve their effects on RDFs, RMSDs, hydrogen bond number and H_2O-Me_2SO space angles. The results show that RDFs, RMSDsand hydrogen bond numbers at threeconcentrations also show different patterns.
Hydrogen bond angles, hydrogen bond distances, hydrogen bond numbers andhydrogen bond lifes under three pressures (1bar,10bar,100bar) are calculated forwater separately. Their distributions are obtained by statistical method. The results showthat:(1)hydrogen bond angles distribute in a way similar to Poisson distribution. Whentemperature drops, distributing range become narrower, peak become higher, curves ofdistribution become higher when hydrogen bond angles is in the region between0and13°, but lower when hydrogen bond angles>13°.(2) hydrogen bond distances alsodistribute in a way similar to Poisson distribution with distributing region is bwtween0.14and0.27nm.(3) both hydrogen bond angles and hydrogen bond distances underthree pressures have no significant differences.(4) both the total number of hydrogenbonds and average hydrogen bonds number of each H_2O increase as temperature drop.During vitrifying process, the portion of free water became lowerand lower. Portion ofwater molecules with four hydrogen bonds forming tetrahedron get high dramaticallyfrom290K to260K, then shows little change. Portions of those with one, two or threehydrogen bonds show a overall tendency to get higher as temperature decrease.70%Me_2SO aqueous solution was cooled at1×107K/s and1×1012K/s. Hydrogen bonddistance, angle, n~n+i number are calculated. The results show that (1) the distances ofsystem hydrogen bonds, Me_2SO-H_2O hydrogen bonds and H_2O-H_2O hydrogen bondsall distribute in a manner similar to Poisson distribution. With the temperaturedecreasing, the distributing range became narrower, the peak became higher. Distanceof H_2O-H_2O hydrogen bond is longer than that of Me_2SO-H_2O hydrogen bond at thesame temperature and distribution.(2) the angles of system hydrogen bonds,Me_2SO-H_2O hydrogen bonds and H_2O-H_2O hydrogen bonds all distribute in a mannersimilar to Poisson distribution. With the temperature decreasing, the distributing rangebecame narrower, the peak bacame higher. There are differences in angle distribution ofH_2O-H_2O hydrogen bond and that of Me_2SO-H_2O hydrogen bond. Cooling rate haveweak effects on angle distribution of hydrogen bonds.(3) the number of hydrogen bondrespected by n+1～n+5became stable as temperature decreases, but the higher coolingrate results in stronger ability to get stable.
Hydrogen bond life distributions and their average lifes are calculated for70%Me_2SO aqueous solution at two cooling rates. The results show that with temperature dropping,(1)life distributions of system, Me_2SO-H_2O and H_2O-H_2O hydrogen bonds alloscillate by higher and higher magnitude,(2) life distributions of Me_2SO-H_2O hydrogenbonds angle are greater than those of H_2O-H_2O when time>3ps，(3) life distribute in amanner similar to power or exponential functions in which independent variable ismultiplied by a negative factor,(4) average life distributes of Me_2SO-H_2O hydrogenbonds are bigger than those of H_2O-H_2O,(5) vibrating amplitudes of life distributionsof system, Me_2SO-H_2O and H_2O-H_2O hydrogen bonds in the system cooled by highercooling rate are lower than those by lower high cooling rate, so do the changes ofamplitudes. Hydrogen bond life distributions of water are calculated under threepressures. Average lifes of water are also calculted basing on the resules of lifedistribution. Average lifes under three pressures are compared. The results show that:(1)hydrogen bond life distribute in a way similar to power or exponential functions inwhich independent variable is multiplied by a negative factor, and with temperaturesdropping, both the range and the values of life distribution show significant differences,(2) pressure has weak effects on the life distributions, but oscillations of life distributioncurves show differences when temperatures is below a temperature, leading tosignificant differences of average life at different pressures.
An isothermal-isobaric molecular simulation (NPT-MD) is employed to investigatethe vitrification transition and Tgof such vitrification solution. The cohesive energydensity (CED), solubility parameter (δ) and bulk modulus of the solution during theprocess of the glass transition are investigated as well. The results indicate that theseproperties as functions of temperature can give a definite inflexion, thus, theseproperties can be used to predict Tg more accurately than the heat capacity (Cp), density(ρ), volume (V) and radial distribution function (rdf). At the same time, the predictedvalues of Tg agree well with the experimental results. Therefore, molecular dynamicsimulation is a potential method for investigating the glass transition and (Tg) of thevitrification solutions.
人们常用差示扫描量热法（DSC）、动态机械分析仪(DMA)和热机械分析仪（TMA）等仪器测量玻璃化溶液的性质，但要获得一种理想的玻璃化溶液，需要大量的实验。分子模拟不仅可以从微观上设计并控制各种条件，模拟玻璃化溶液的性质，而且可以分析宏观性质的分子机理，为选择合适的玻璃化溶液提供了一条捷径。
应用gromacs3.3和Material Studio软件，本文对几种低温保护剂溶液的玻璃化性质进行了分子模拟和机理分析，主要工作如下：
1、玻璃体的力学、热学性质与其结构有直接的关系，径向分布函数(radialdistribution function, RDF)反映了分子的聚集特性，是研究玻璃态结构的一个重要参数。本文研究了降温速率和压力对玻璃化转变温度和径向分布函数的影响，结果表明：降温速率对体系的RDF有影响，缩小温差并采取中间温度充分驰豫可减小这种影响；采用逐步冷却、直接冷却和两步法冷却时，Me_2SO溶液的RDF，RMSD（root mean squared diviation,均方根偏差）及氢键数量构成均表现不同的特征，但总氢键数目以及Me_2SO-H_2O的空间角位置没有明显变化，Me_2SO-H_2O的空间角位置关系有相似性和取向性和周期性的特征；对于30%、50%、70%Me_2SO溶液，在慢速降温时，RDF、RMSD及氢键数量构成表现不同的特征，其Me_2SO-H_2O空间角度分布也具有相似性，取向性和周期性的特点。
2、氢键是影响很多物质的结构、热力学、力学及其他性质的关键因素。在溶液的冷却过程中，无论结冰，还是玻璃化，都与分子内、外的氢键网有密切关系。研究了降温速率（1×107K/s，1×1012K/s）对70%Me_2SO溶液玻璃化过程氢键的角度、距离、生命期和数量的影响，结果表明：玻璃化过程中系统氢键、Me_2SO-H_2O氢键以及H_2O-H_2O氢键的键长分布均呈现泊松分布，不同的冷却速度对氢键键长分布影响不显著；玻璃化过程的系统氢键角度、Me_2SO-H_2O氢键角度以及H_2O-H_2O氢键角度的分布均呈现泊松分布，降温速度对氢键角度分布曲线的影响显著。
3、氢键生命期特性是氢键动力学重要特征之一，对保存在溶液中的细胞和组织的热及力的环境有较大的影响。研究了降温速率（1×107K/s，1×1012K/s）对70%Me_2SO溶液的玻璃化过程的氢键生命期分布的影响，结果表明：两种冷速下的系统氢键、Me_2SO-H_2O氢键、H_2O-H_2O氢键都表现出振荡变剧烈的特性；在时间大于大约3ps时，Me_2SO-H_2O氢键生命期分布在数值上要大于H_2O-H_2O的；氢键生命期以类似自变量系数为负的幂函数或者指数函数形式分布；不管是系统氢键、Me_2SO-H_2O氢键、还是H_2O-H_2O氢键的生命期分布，快速降温系统比慢速降温系统的生命期振幅要小得多，振幅变化也小很多。
4、首次用分子模拟方法研究和预测低温保护液玻璃化转变温度。对70%甘油水溶液玻璃化过程中的内聚能密度、溶解度参数和体积模量进行了计算，结果表明：这些参数在玻璃化转变温度附近出现明确的拐点，用它们来预测玻璃化温度要比用比热、密度、容积和径向分布函数更准确，得到的玻璃化转变温度与实验值吻合较好。
Vitrification is assumed to be a potential method for the cryopreservation of largetissues and organs. But the thermal stress and osmotic stress may damage the cells,which limit the practical use of vitrification. The properties of vitrification solution,such as critical cooling rate, glass transition temperature, are key factors that affect itssuccessful application in cryopreservation.
Differential scanning calorimetry (DSC), dynamic mechanical analyzer (DMA) andthermal mechanical analysis (TMA) are common equipments used to determine theproperties of vitrification solution. Lots of experiments must be peformed to obtain aoptimal solution. Molecular dynamic simulation can be used to predict the properties ofvitrification solution in molecular level. It provides a shortcut for selecting suitablevitrification solutions.
Applying gromacs3.3andMaterial Studio software, the propeties of severalvitrification solutions were simulated and the mechanism was analyzed in molecularlevel.
The thermal and dynamic properties of the material related to its structure. As theradial distribution functions(RDFs) reflects the collective property of molecules, it isregarded as an important factor affecting the structure of vitrification solutions. Theeffect of cooling rates and pressures on the glass transition temperature and RDF wereinvestigated in this study. The results show that cooling rate affects the RDFsof vitifiedwater. Three cooling methods were used to study the structure change of Me_2SOaqueous solution. The first is cooling to final temperature via a series of temperatureswith an interval of10K at a rate of1×1012K/s and sufficient relaxation time at eachtemperature. The second is cooling to final temperature directly at1×1012K/s. The thirdis a two-step cooling method using average between the intial and final temperature asmiddle temperature. The results show that RDF, root mean squared diviation(RMSDs)and the compostion of hydrogen bond show different patterns. Meanwhile, total numberof hydrogen bond and H_2O-Me_2SO space angles are characterized by similarity,directivity and periodicity without significant change during vitrification. Threeconcentrations (70%,50%and30%, wt%) of Me_2SO solutions are investigated toobserve their effects on RDFs, RMSDs, hydrogen bond number and H_2O-Me_2SO space angles. The results show that RDFs, RMSDsand hydrogen bond numbers at threeconcentrations also show different patterns.
Hydrogen bond angles, hydrogen bond distances, hydrogen bond numbers andhydrogen bond lifes under three pressures (1bar,10bar,100bar) are calculated forwater separately. Their distributions are obtained by statistical method. The results showthat:(1)hydrogen bond angles distribute in a way similar to Poisson distribution. Whentemperature drops, distributing range become narrower, peak become higher, curves ofdistribution become higher when hydrogen bond angles is in the region between0and13°, but lower when hydrogen bond angles>13°.(2) hydrogen bond distances alsodistribute in a way similar to Poisson distribution with distributing region is bwtween0.14and0.27nm.(3) both hydrogen bond angles and hydrogen bond distances underthree pressures have no significant differences.(4) both the total number of hydrogenbonds and average hydrogen bonds number of each H_2O increase as temperature drop.During vitrifying process, the portion of free water became lowerand lower. Portion ofwater molecules with four hydrogen bonds forming tetrahedron get high dramaticallyfrom290K to260K, then shows little change. Portions of those with one, two or threehydrogen bonds show a overall tendency to get higher as temperature decrease.70%Me_2SO aqueous solution was cooled at1×107K/s and1×1012K/s. Hydrogen bonddistance, angle, n~n+i number are calculated. The results show that (1) the distances ofsystem hydrogen bonds, Me_2SO-H_2O hydrogen bonds and H_2O-H_2O hydrogen bondsall distribute in a manner similar to Poisson distribution. With the temperaturedecreasing, the distributing range became narrower, the peak became higher. Distanceof H_2O-H_2O hydrogen bond is longer than that of Me_2SO-H_2O hydrogen bond at thesame temperature and distribution.(2) the angles of system hydrogen bonds,Me_2SO-H_2O hydrogen bonds and H_2O-H_2O hydrogen bonds all distribute in a mannersimilar to Poisson distribution. With the temperature decreasing, the distributing rangebecame narrower, the peak bacame higher. There are differences in angle distribution ofH_2O-H_2O hydrogen bond and that of Me_2SO-H_2O hydrogen bond. Cooling rate haveweak effects on angle distribution of hydrogen bonds.(3) the number of hydrogen bondrespected by n+1～n+5became stable as temperature decreases, but the higher coolingrate results in stronger ability to get stable.
Hydrogen bond life distributions and their average lifes are calculated for70%Me_2SO aqueous solution at two cooling rates. The results show that with temperature dropping,(1)life distributions of system, Me_2SO-H_2O and H_2O-H_2O hydrogen bonds alloscillate by higher and higher magnitude,(2) life distributions of Me_2SO-H_2O hydrogenbonds angle are greater than those of H_2O-H_2O when time>3ps，(3) life distribute in amanner similar to power or exponential functions in which independent variable ismultiplied by a negative factor,(4) average life distributes of Me_2SO-H_2O hydrogenbonds are bigger than those of H_2O-H_2O,(5) vibrating amplitudes of life distributionsof system, Me_2SO-H_2O and H_2O-H_2O hydrogen bonds in the system cooled by highercooling rate are lower than those by lower high cooling rate, so do the changes ofamplitudes. Hydrogen bond life distributions of water are calculated under threepressures. Average lifes of water are also calculted basing on the resules of lifedistribution. Average lifes under three pressures are compared. The results show that:(1)hydrogen bond life distribute in a way similar to power or exponential functions inwhich independent variable is multiplied by a negative factor, and with temperaturesdropping, both the range and the values of life distribution show significant differences,(2) pressure has weak effects on the life distributions, but oscillations of life distributioncurves show differences when temperatures is below a temperature, leading tosignificant differences of average life at different pressures.
An isothermal-isobaric molecular simulation (NPT-MD) is employed to investigatethe vitrification transition and Tgof such vitrification solution. The cohesive energydensity (CED), solubility parameter (δ) and bulk modulus of the solution during theprocess of the glass transition are investigated as well. The results indicate that theseproperties as functions of temperature can give a definite inflexion, thus, theseproperties can be used to predict Tg more accurately than the heat capacity (Cp), density(ρ), volume (V) and radial distribution function (rdf). At the same time, the predictedvalues of Tg agree well with the experimental results. Therefore, molecular dynamicsimulation is a potential method for investigating the glass transition and (Tg) of thevitrification solutions.
引文
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