三磷酸腺苷（ATP）脂质体的制备研究
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摘要
研究背景：本课题旨在研究提高机体缺血缺氧组织对ATP的摄取,制备可方便临床应用的静脉使用或外用的的ATP新剂型；探索一种病灶靶向性高、不良反应少、作用稳定的载药系统。脂质体是目前应用最为广泛、治疗效果最为肯定的载药系统之一,其具有无毒,无免疫原性及良好组织相容性等诸多优点。目前在研究的各种药物载药系统中,只有脂质体已批准应用于临床,其他的纳米载药系统绝大多数仍处于临床前甚至实验室研究阶段。
研究方法：本课题采用大豆磷脂和胆固醇为主要膜材,以ATP为模型药物,通过十八胺来调节脂质体的Zeta电位,采用逆向蒸发法制备ATP脂质体。以包封率为评价指标,采用正交试验优化处方及制备工艺。考察了磷脂浓度、磷脂和胆固醇比、药脂比、水浴温度、旋蒸仪转速等对ATP脂质体包封率的影响,建立了ATP脂质体的含量测定方法及包封率测定方法,并初步研究了ATP脂质体的理化性质、体外释放性质、初步稳定性等,为脂质体制剂的开发应用提供了试验基础和理论依据。
研究结果：
1.ATP脂质体药物含量及包封率测定方法的建立
采用超低温超速离心法将载药脂质体与游离药物分离,并建立了HPLC法测定脂质体中ATP含量及包封率的测定方法。结果：ATP在20-640μg/ml浓度范围内线性关系良好,以峰面积(A)对浓度(C)作线性回归,得回归方程为A=0.1602C+7.3767,r=0.9997。日内、日间精密度RSD均小于2.0%,回收率中高浓度均在98%左右,但低浓度偏离较大,可能与仪器误差有关,日内及日间RSD均小于2%。
2.ATP脂质体的制备工艺、处方研究及脂质体的形态及理化性质
以ATP脂质体的包封率为主要参考指标,将制备温度、磷脂和胆固醇比、药脂比、有机相／水相比例、水浴温度及旋转蒸发仪转速等相关因素做单因素考察分析,在此基础上,采用正交优化设计法对ATP脂质体处方进行考察,选取磷脂浓度、药物脂质质量比、磷脂和胆固醇质量比、旋转蒸发仪转速等为影响因素,以包封率为主要考察指标进行评价,并最终得到了ATP脂质体的优化处方。结果采用优化处方制得的ATP脂质体经在透射电镜下为多囊状的多层的类圆球体,于处方中加入与磷脂质量比为0：1、0.05：1、0.10：1的十八胺,得到的脂质体的Zeta电位分别为2.1±0.4mv、12.3±1.2mv、20.4±2.6mv。
通过固定处方加十八胺(与磷脂质量比为0.05：1)逆向蒸发法制备ATP脂质体,得到脂质体的粒径为810.9nm,多分散系数为0.543。
最后以磷脂浓度为10mg/ml,磷脂与胆固醇质量比为4：1,药脂质量比为1：2,旋蒸仪转速为30r/min,十八胺与磷脂质量比为0.05：1,为最终优化处方制备脂质体,平均包封率分别为(56.99±4.91)%,载药率为(21.81±1.89)%。
3.ATP脂质体的体外释药动力学及初步稳定性研究
采用与人血浆等渗的pH=7.4的磷酸盐缓冲液作为释放介质,其中含137 mmol/L NaCl、3mmol/L KCl、8mmol/L Na2HPO4和1 mmol/LKH2PO4。采用动态透析膜法进行脂质体混悬液中药物的体外释放研究。结果：ATP脂质体的释放曲线方程为：Lnln(1/(1-Q))=0.5871nt-1.656,更为符合weibull释放数学模型。
初步稳定性考察结果表明ATP脂质体4℃贮存15天药物渗漏率达27.74%，因此,为了增加脂质体稳定性,课题计划下一步采用冷冻干燥法将ATP脂质体制备ATP前体脂质体增强稳定性。
结论：本课题成功研制了ATP脂质体制剂,所采用的制备工艺简便可行,重现性好,包封率较高；脂质体作为ATP的载体可以明显增加ATP在体外的稳定性,并极大地拓宽了使用范围；适于注射或外用给药,方便了临床应用等。但是课题研究发现ATP脂质体长时间放置后出现渗漏,而且随着时间的延长,渗漏率也会随之增加,我们计划下一步进行ATP脂质体冻干制剂的研究,以进一步提高ATP脂质体的稳定性。
Background:The project aims to study how to improve the intake of ATP in the state of tissue ischemia and hypoxia, and prepars the new formulation which is intravenous or external use for clinical application; explore a high bioavailability, low toxicity and stability drug delivery systems. Liposome is the most important and most widely used nano-drug delivery systems in the world, which is non-toxic, non-immunogenicity, and good tissue compatibility, it can improve the bioavailability of drugs, and enhance the organ targeting. At present, only the liposome dilivery system is used in clinical,and the vast majority of other delivery system are still in pre-clinical research stage.
Methods:In the project we choose soybean Phophatidylcholine and cholesterol as the main membrane materials and ATP as a model drug, to regulate Zeta potential of liposomes by octadecylamine.We prepared ATP liposomes in the way of reverse phase evaporation, and optimize the formulation though the orthogonal test in the index of entrapment efficiency. The factors contain phospholipid concentration,phospholipids and cholesterol ratio, drug and lipids ratio, water temperature, spin speed and and so on, we establish the ATP assay method in the liposomes and the method of entrapment efficiency determination, and we also study the physical and chemical properties of ATP liposomes, in vitro release properties, the short-time stability, which provide the experimental and theoretical basis for the development and application of liposomes.
Results:
1. Establishment of the ATP assay method and liposome encapsulation efficency method.
The liposomes and free drug are separated by high speed centrifugation.and the entrapment effieieney and drug concentration were determined by the method of HPLC. Results:There was a good linearity of the drug concentration with in the range of 20-640ug/ml and the linear equation was A= 0.1602C+7.3767, r=0.9997. The recovery rate was more than 98% in the middle and high concentration,but there is large deviations in the low concentration, which may be related to instrumental error, and all of the RSD (intra-and inter-day) were less than 2.0%.
2. Preparation of ATP liposomes, and the physical and chemical properties
With the encapsulation efficiency as judgment index. The effects of various factors on entrapment efficiency were investigated by single factor and orthogonal experients. The factors contain phospholipid concentration,phospholipids and cholesterol ratio, drug and lipids ratio, water temperature, spin speed and and so on.The ATP liposome obtained after optimized prescription was multi-layer balls cystic body in the transmission electron microscopy.We regulate the zeta potential of liposomes by octadecylamine,and phospholipids and cholesterol in a fixed prescription,change the amount of octadecylamine added in the formulation at a ratio of 0:1,0.05:1,0.10:1 to lipids,the zeta potential was 2.1±0.4mv,12.3±L2mv,20.4±2.6mv.At a ratio of 0.05to l,the ATP liposome diameter we obtained is 810.9nm,and the polydispersity is 0.543.
The optimized fotmulation is as follows:the phospholipid concentration of 10mg/ml, phospholipids and cholesterol mass ratio of 4:1, lipid and drug ratio is 1:2, the speed of rotary evaporator is 30r/min, octadecylamine and phospholipid mass ratio is 0.05:1, the average encapsulation efficiency in this formulation is (56.99±4.91)%, drug loading rate is (21.81±1.89)%.
3. The release kinetics of ATP liposomes in vitro and the short-time stability.
We use the pH= 7.4 phosphate buffer isotonic with human plasma as the release medium, which contains 137 mmol/L NaCl,3 mmol/L KC1,8 mmol/L Na2HPO4, and 1 mmol/L KH2PO4. The in vitro release of ATP from the medium were evaluated using the dialysis bag diffusion technique.ATP-loaded liposomes all presented controlled release properties, and nonlinear fits of ATP release from liposomes was modeled. The in vitro release behavior of ATP from liposomes could be described by Weibull kineties model and expressed by the following equations:Lnln (1/(1-Q))= 0.5871nt-1.656.
The stability results show that the leakage rate of ATP liposomes is 27.74% at 4℃storage for 15 days, therefore, in order to increase the stability of liposome we can transfer ATP liposome into ATP proliposomes by using freeze-drying method next step.
Conclutions:This project developes a liposome with high encapsulation efficiency successfully, and the processing technology is simple, feasible and reproducible; liposomes as carrier of ATP can significantly increase the half-life of ATP in vitro; are conducive to injection or topical administration and facilitate the clinical application.At the same time, this study finds that with the time goes, the leakage rate of ATP liposome increases, the next step we must solve is to further improve the stability of liposomes, so the lyophilized ATP liposomes study is needed.
研究方法：本课题采用大豆磷脂和胆固醇为主要膜材,以ATP为模型药物,通过十八胺来调节脂质体的Zeta电位,采用逆向蒸发法制备ATP脂质体。以包封率为评价指标,采用正交试验优化处方及制备工艺。考察了磷脂浓度、磷脂和胆固醇比、药脂比、水浴温度、旋蒸仪转速等对ATP脂质体包封率的影响,建立了ATP脂质体的含量测定方法及包封率测定方法,并初步研究了ATP脂质体的理化性质、体外释放性质、初步稳定性等,为脂质体制剂的开发应用提供了试验基础和理论依据。
研究结果：
1.ATP脂质体药物含量及包封率测定方法的建立
采用超低温超速离心法将载药脂质体与游离药物分离,并建立了HPLC法测定脂质体中ATP含量及包封率的测定方法。结果：ATP在20-640μg/ml浓度范围内线性关系良好,以峰面积(A)对浓度(C)作线性回归,得回归方程为A=0.1602C+7.3767,r=0.9997。日内、日间精密度RSD均小于2.0%,回收率中高浓度均在98%左右,但低浓度偏离较大,可能与仪器误差有关,日内及日间RSD均小于2%。
2.ATP脂质体的制备工艺、处方研究及脂质体的形态及理化性质
以ATP脂质体的包封率为主要参考指标,将制备温度、磷脂和胆固醇比、药脂比、有机相／水相比例、水浴温度及旋转蒸发仪转速等相关因素做单因素考察分析,在此基础上,采用正交优化设计法对ATP脂质体处方进行考察,选取磷脂浓度、药物脂质质量比、磷脂和胆固醇质量比、旋转蒸发仪转速等为影响因素,以包封率为主要考察指标进行评价,并最终得到了ATP脂质体的优化处方。结果采用优化处方制得的ATP脂质体经在透射电镜下为多囊状的多层的类圆球体,于处方中加入与磷脂质量比为0：1、0.05：1、0.10：1的十八胺,得到的脂质体的Zeta电位分别为2.1±0.4mv、12.3±1.2mv、20.4±2.6mv。
通过固定处方加十八胺(与磷脂质量比为0.05：1)逆向蒸发法制备ATP脂质体,得到脂质体的粒径为810.9nm,多分散系数为0.543。
最后以磷脂浓度为10mg/ml,磷脂与胆固醇质量比为4：1,药脂质量比为1：2,旋蒸仪转速为30r/min,十八胺与磷脂质量比为0.05：1,为最终优化处方制备脂质体,平均包封率分别为(56.99±4.91)%,载药率为(21.81±1.89)%。
3.ATP脂质体的体外释药动力学及初步稳定性研究
采用与人血浆等渗的pH=7.4的磷酸盐缓冲液作为释放介质,其中含137 mmol/L NaCl、3mmol/L KCl、8mmol/L Na2HPO4和1 mmol/LKH2PO4。采用动态透析膜法进行脂质体混悬液中药物的体外释放研究。结果：ATP脂质体的释放曲线方程为：Lnln(1/(1-Q))=0.5871nt-1.656,更为符合weibull释放数学模型。
初步稳定性考察结果表明ATP脂质体4℃贮存15天药物渗漏率达27.74%，因此,为了增加脂质体稳定性,课题计划下一步采用冷冻干燥法将ATP脂质体制备ATP前体脂质体增强稳定性。
结论：本课题成功研制了ATP脂质体制剂,所采用的制备工艺简便可行,重现性好,包封率较高；脂质体作为ATP的载体可以明显增加ATP在体外的稳定性,并极大地拓宽了使用范围；适于注射或外用给药,方便了临床应用等。但是课题研究发现ATP脂质体长时间放置后出现渗漏,而且随着时间的延长,渗漏率也会随之增加,我们计划下一步进行ATP脂质体冻干制剂的研究,以进一步提高ATP脂质体的稳定性。
Background:The project aims to study how to improve the intake of ATP in the state of tissue ischemia and hypoxia, and prepars the new formulation which is intravenous or external use for clinical application; explore a high bioavailability, low toxicity and stability drug delivery systems. Liposome is the most important and most widely used nano-drug delivery systems in the world, which is non-toxic, non-immunogenicity, and good tissue compatibility, it can improve the bioavailability of drugs, and enhance the organ targeting. At present, only the liposome dilivery system is used in clinical,and the vast majority of other delivery system are still in pre-clinical research stage.
Methods:In the project we choose soybean Phophatidylcholine and cholesterol as the main membrane materials and ATP as a model drug, to regulate Zeta potential of liposomes by octadecylamine.We prepared ATP liposomes in the way of reverse phase evaporation, and optimize the formulation though the orthogonal test in the index of entrapment efficiency. The factors contain phospholipid concentration,phospholipids and cholesterol ratio, drug and lipids ratio, water temperature, spin speed and and so on, we establish the ATP assay method in the liposomes and the method of entrapment efficiency determination, and we also study the physical and chemical properties of ATP liposomes, in vitro release properties, the short-time stability, which provide the experimental and theoretical basis for the development and application of liposomes.
Results:
1. Establishment of the ATP assay method and liposome encapsulation efficency method.
The liposomes and free drug are separated by high speed centrifugation.and the entrapment effieieney and drug concentration were determined by the method of HPLC. Results:There was a good linearity of the drug concentration with in the range of 20-640ug/ml and the linear equation was A= 0.1602C+7.3767, r=0.9997. The recovery rate was more than 98% in the middle and high concentration,but there is large deviations in the low concentration, which may be related to instrumental error, and all of the RSD (intra-and inter-day) were less than 2.0%.
2. Preparation of ATP liposomes, and the physical and chemical properties
With the encapsulation efficiency as judgment index. The effects of various factors on entrapment efficiency were investigated by single factor and orthogonal experients. The factors contain phospholipid concentration,phospholipids and cholesterol ratio, drug and lipids ratio, water temperature, spin speed and and so on.The ATP liposome obtained after optimized prescription was multi-layer balls cystic body in the transmission electron microscopy.We regulate the zeta potential of liposomes by octadecylamine,and phospholipids and cholesterol in a fixed prescription,change the amount of octadecylamine added in the formulation at a ratio of 0:1,0.05:1,0.10:1 to lipids,the zeta potential was 2.1±0.4mv,12.3±L2mv,20.4±2.6mv.At a ratio of 0.05to l,the ATP liposome diameter we obtained is 810.9nm,and the polydispersity is 0.543.
The optimized fotmulation is as follows:the phospholipid concentration of 10mg/ml, phospholipids and cholesterol mass ratio of 4:1, lipid and drug ratio is 1:2, the speed of rotary evaporator is 30r/min, octadecylamine and phospholipid mass ratio is 0.05:1, the average encapsulation efficiency in this formulation is (56.99±4.91)%, drug loading rate is (21.81±1.89)%.
3. The release kinetics of ATP liposomes in vitro and the short-time stability.
We use the pH= 7.4 phosphate buffer isotonic with human plasma as the release medium, which contains 137 mmol/L NaCl,3 mmol/L KC1,8 mmol/L Na2HPO4, and 1 mmol/L KH2PO4. The in vitro release of ATP from the medium were evaluated using the dialysis bag diffusion technique.ATP-loaded liposomes all presented controlled release properties, and nonlinear fits of ATP release from liposomes was modeled. The in vitro release behavior of ATP from liposomes could be described by Weibull kineties model and expressed by the following equations:Lnln (1/(1-Q))= 0.5871nt-1.656.
The stability results show that the leakage rate of ATP liposomes is 27.74% at 4℃storage for 15 days, therefore, in order to increase the stability of liposome we can transfer ATP liposome into ATP proliposomes by using freeze-drying method next step.
Conclutions:This project developes a liposome with high encapsulation efficiency successfully, and the processing technology is simple, feasible and reproducible; liposomes as carrier of ATP can significantly increase the half-life of ATP in vitro; are conducive to injection or topical administration and facilitate the clinical application.At the same time, this study finds that with the time goes, the leakage rate of ATP liposome increases, the next step we must solve is to further improve the stability of liposomes, so the lyophilized ATP liposomes study is needed.
引文
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[38]Vincourt V, Nguyen L, Chaumeil JC, et al. Freeze-drying of ATP entrapped in cationic, low lipid liposomes. Cryobiology.2010,60(3):262-70.
[1]Korb V. Tep K, Escriou V, et al. Current data on ATP-containing liposomes and potential prospects to enhance cellular energy status for hepatic applications. Crit Rev Ther Drug Carrier Syst.2008,25(4):305-45.
[2]Chaudry IH. Use of ATP following shock and ischemia. Ann N Y Acad Sci. 1990.603:130-40; discussion 140-1.
[3]皮凤梅,屠锡德,吴悦.三磷酸腺苷二钠脂质体的制备及其对心肌缺血小鼠组织能量状态的影响.药学学报.2010,45(10)：1322-1326.
[4]Verma DD, Levchenko TS, Bernstein EA, et al. ATP-loaded liposomes effectively protect mechanical functions of the myocardium from global ischemia in an isolated rat heart model. J Control Release.2005,108(2-3):460-71.
[5]Xu GX, Xie XH, Liu FY, et al. Adenosine triphosphate liposomes:encapsulation and distribution studies. Pharm Res.1990,7(5):553-7.
[6]Liang W, Levchenko TS, Torchilin VP. Encapsulation of ATP into liposomes by different methods:optimization of the procedure. J Microencapsul.2004,21(3):251-61.
[7]Verma DD, Levchenko TS, Bernstein EA, et al. ATP-loaded liposomes effectively protect mechanical functions of the myocardium from global ischemiain an isolated rat heart model. J Control Release.2005,108(2-3):460-71.
[8]Verma DD, Hartner WC, Levchenko TS, et al. ATP-loaded liposomes effectively protect the myocardium in rabbits with an acute experimental myocardial infarction. Pharm Res.2005,22(12):2115-20.
[9]Rensen PC, Sliedregt LA, Ferns M, et al. Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo. J Biol Chem.2001,276(40):37577-84.
[10]Tep K, Korb V, Richard C, et al. Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand. J Liposome Res.2009,19(4):287-300.
[11]Liang W, Levchenko T, Khaw BA, et al. ATP-containing immunoliposomes specific for cardiac myosin. Curr Drug Deliv.2004,1(1):1-7.
[12]Verma DD, Levchenko TS, Bernstein EA, et al. ATP-loaded immunoliposomes specific for cardiac myosin provide improved protection of the mechanical functions of myocar-dium from global ischemia in an isolated rat heart model. J Drug Target. 2006,14(5):273-80.
[13]Hofheinz RD, Gnad-Vogt SU, Beyer U, et al. Liposomal encapsulated anti-cancer drugs. Anticancer Drugs.2005,16(7):691-707.
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[15]Avantilipids. Available from:www.avantilipids.com. Accessed in:2010.
[16]Liang W, Levchenko TS, Torchilin VP. Encapsulation of ATP into liposomes by different methods:optimization of the procedure. J Microencapsul.2004,21 (3):251-61.
[17]Vincourt V, Nguyen L, Chaumeil JC, et al. Freeze-drying of ATP entrapped in cationic, low lipid liposomes. Cryobiology.2010,60(3):262-70.
[18]Cui J, Li C, Deng Y, et al.Freeze-drying of liposomes using tertiary butyl alcohol/water cosolvent systems. Int J Pharm.2006,312(1-2):131-6.
[19]Abraham AM, Walubo A. The effect of surface charge on the disposition of liposome encapsulated gentamicin to the rat liver, brain, lungs and kidneys after intraperitoneal administration. Int J Antimicrob Agents.2005,25(5):392-7.
[20]Torchilin VP, Weissig, V, editors. Liposomes:a practical approach. Oxford:Oxford University Press; 2003.
[21]陈妍,邓英杰,郝艳丽等.膜修饰脂质体的制备及对心肌细胞的靶向作用.沈阳药科大学学报.2005,22(3)：138-141.
[22]Han YY, Huang L, Jackson EK, et al. Liposomal atp or NAD+protects human endothelial cells from energy failure in a cell culture model of sepsis. Res Commun Mol Pathol Pharmacol.2001,110(1-2):107-16.
[23]Skiba-Lahiani M, Auger J, Terribile J, et al. Stimulation of movement and acrosome reaction of human spermatozoa by PC 12 liposomes encapsulating ATP. Int J Androl. 1995,18(6):287-94.
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[25]Galina Dvoriantchikova, David J. Barakat, Eleut Hernandez, el al. Liposome-delivered ATP effectively protects the retina against ischemia-reperfusion injury. Mol Vision. 2010,16:2882-2890.
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[32]Chien S. Intracellular ATP delivery using highly fusogenic liposomes. Meth Molecular Biol.2009,605:387-391.
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[37]Chiang B, Essick E, Ehringer W, et al. Enhancing skin wound healing by direct delivery of intracellular adenosine triphosphate. Am J Surg.2007,193(2):213-8.
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[39]Wang J, Wan R, Mo Y, et al. Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits.Am J Surg.2010, 199(6):823-32.
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[1]Korb V. Tep K, Escriou V, et al. Current data on ATP-containing liposomes and potential prospects to enhance cellular energy status for hepatic applications. Crit Rev Ther Drug Carrier Syst.2008,25(4):305-45.
[2]Chaudry IH. Use of ATP following shock and ischemia. Ann N Y Acad Sci. 1990.603:130-40; discussion 140-1.
[3]皮凤梅,屠锡德,吴悦.三磷酸腺苷二钠脂质体的制备及其对心肌缺血小鼠组织能量状态的影响.药学学报.2010,45(10)：1322-1326.
[4]Verma DD, Levchenko TS, Bernstein EA, et al. ATP-loaded liposomes effectively protect mechanical functions of the myocardium from global ischemia in an isolated rat heart model. J Control Release.2005,108(2-3):460-71.
[5]Xu GX, Xie XH, Liu FY, et al. Adenosine triphosphate liposomes:encapsulation and distribution studies. Pharm Res.1990,7(5):553-7.
[6]Liang W, Levchenko TS, Torchilin VP. Encapsulation of ATP into liposomes by different methods:optimization of the procedure. J Microencapsul.2004,21(3):251-61.
[7]Verma DD, Levchenko TS, Bernstein EA, et al. ATP-loaded liposomes effectively protect mechanical functions of the myocardium from global ischemiain an isolated rat heart model. J Control Release.2005,108(2-3):460-71.
[8]Verma DD, Hartner WC, Levchenko TS, et al. ATP-loaded liposomes effectively protect the myocardium in rabbits with an acute experimental myocardial infarction. Pharm Res.2005,22(12):2115-20.
[9]Rensen PC, Sliedregt LA, Ferns M, et al. Determination of the upper size limit for uptake and processing of ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in vivo. J Biol Chem.2001,276(40):37577-84.
[10]Tep K, Korb V, Richard C, et al. Formulation and evaluation of ATP-containing liposomes including lactosylated ASGPr ligand. J Liposome Res.2009,19(4):287-300.
[11]Liang W, Levchenko T, Khaw BA, et al. ATP-containing immunoliposomes specific for cardiac myosin. Curr Drug Deliv.2004,1(1):1-7.
[12]Verma DD, Levchenko TS, Bernstein EA, et al. ATP-loaded immunoliposomes specific for cardiac myosin provide improved protection of the mechanical functions of myocar-dium from global ischemia in an isolated rat heart model. J Drug Target. 2006,14(5):273-80.
[13]Hofheinz RD, Gnad-Vogt SU, Beyer U, et al. Liposomal encapsulated anti-cancer drugs. Anticancer Drugs.2005,16(7):691-707.
[14]Rieder H, Meyer zum Buschenfelde KH, Ramadori G. Functional spectrum of sinusoidal endo-thelial liver cells. Filtration, endocytosis, synthetic capacities and intercellular communication. J Hepatol.1992,15(1-2):237-50.
[15]Avantilipids. Available from:www.avantilipids.com. Accessed in:2010.
[16]Liang W, Levchenko TS, Torchilin VP. Encapsulation of ATP into liposomes by different methods:optimization of the procedure. J Microencapsul.2004,21 (3):251-61.
[17]Vincourt V, Nguyen L, Chaumeil JC, et al. Freeze-drying of ATP entrapped in cationic, low lipid liposomes. Cryobiology.2010,60(3):262-70.
[18]Cui J, Li C, Deng Y, et al.Freeze-drying of liposomes using tertiary butyl alcohol/water cosolvent systems. Int J Pharm.2006,312(1-2):131-6.
[19]Abraham AM, Walubo A. The effect of surface charge on the disposition of liposome encapsulated gentamicin to the rat liver, brain, lungs and kidneys after intraperitoneal administration. Int J Antimicrob Agents.2005,25(5):392-7.
[20]Torchilin VP, Weissig, V, editors. Liposomes:a practical approach. Oxford:Oxford University Press; 2003.
[21]陈妍,邓英杰,郝艳丽等.膜修饰脂质体的制备及对心肌细胞的靶向作用.沈阳药科大学学报.2005,22(3)：138-141.
[22]Han YY, Huang L, Jackson EK, et al. Liposomal atp or NAD+protects human endothelial cells from energy failure in a cell culture model of sepsis. Res Commun Mol Pathol Pharmacol.2001,110(1-2):107-16.
[23]Skiba-Lahiani M, Auger J, Terribile J, et al. Stimulation of movement and acrosome reaction of human spermatozoa by PC 12 liposomes encapsulating ATP. Int J Androl. 1995,18(6):287-94.
[24]Skiba-Lahiani M, Skiba M, Nikas G, et al.Interaction between PC 12 liposomes encapsulating ATP and human spermatozoa. Int J Pharm.1998,172:147-59.
[25]Galina Dvoriantchikova, David J. Barakat, Eleut Hernandez, el al. Liposome-delivered ATP effectively protects the retina against ischemia-reperfusion injury. Mol Vision. 2010,16:2882-2890.
[26]Lanir A, Jenkins RL, Caldwell C, et al. Hepatic transplantation survival:correlation with adenine nucleotide level in donor liver. Hepatology.1988,8(3):471-5.
[27]Higashi H, Takenaka K, Fukuzawa K, et al. Restoration of ATP contents in the transplanted liver closely relates to graft viability in dogs.Eur Surg Res. 1989,21(2):76-82.
[28]Neveux N, De Bandt JP, Fattal E. et al. Cold preservation injury in rat liver:effect of liposomally-entrapped adenosine triphosphate. J Hepatol.2000,33(1):68-75.
[29]Neveux N, De Bandt JP, Chaumeil JC, et al. Hepatic preservation,liposomally entrapped adenosine triphosphate and nitric oxide production:a study of energy state and protein meta-bolism in the cold-stored rat liver. Scand J Gastroenterol. 2002,37(9):1057-63.
[30]Kingsley PB, Sako EY, Yang MQ, et al.Ischemic contracture begins when anaerobic glycolysis stops:a 31P-NMR study of isolated rat hearts. Am J Physiol.1991,261(2): 469-78.
[31]白军强,张权宇,裴建明.器官保存液及其分子机制的研究进展.中华医学研究杂志.2007,7(5)：419-424.
[32]Chien S. Intracellular ATP delivery using highly fusogenic liposomes. Meth Molecular Biol.2009,605:387-391.
[33]Laham A, Claperon N, Durussel JJ, et al. Intracarotidal administration of liposomally-entrapped ATP:improved efficiency against experimental brain ischemia. Pharmacol Res Commun.1988,20(8):699-705.
[34]Laham A, Claperon N, Durussel JJ, et al. Liposomally entrapped adenosine triphosphate. Improved efficiency against experimental brain ischaemia in the rat. J Chromatogr.1988,440:455-8.
[35]Laham A, Claperon N, Durussel JJ, et al. Liposomally-entrapped ATP:improved efficiency against experimental brain ischemia in the rat. Life Sci.1987,40(20):2011-6.
[36]Rossignol P, Laham A, Claperon N, et al. Is energy supply to the brain by ATP possible? Preliminary studies in rats subjected to repeated ischemia. Bull Acad Natl Med.1988, 172(8):1129-34.
[37]Chiang B, Essick E, Ehringer W, et al. Enhancing skin wound healing by direct delivery of intracellular adenosine triphosphate. Am J Surg.2007,193(2):213-8.
[38]Wang J, Zhang Q, Wan R, et al. Intracellular adenosine triphosphate delivery enhanced skin wound healing in rabbits. Ann Plast Surg.2009,62(2):180-6.
[39]Wang J, Wan R, Mo Y, et al. Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits.Am J Surg.2010, 199(6):823-32.