N-十六烷基乳糖酰胺介导葫芦素B肝靶向SLN的研究
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摘要
细胞表面存在的受体可特异性识别相应的配体,选择适当的配体修饰固体脂质纳米粒可实现受体介导的细胞靶向。肝实质细胞表面表达的去唾液酸糖蛋白受体(ASGPr)可特异性识别末端含半乳糖或乙酰氨基半乳糖的配体,ASGPr介导是近年来肝靶向制剂研究的热点。本研究合成了含半乳糖残基的十六烷基乳糖酰胺(N-HLBA);以其作为“自导向分子”制备了葫芦素B(Cuc B)半乳糖化固体脂质纳米粒(GalSLN),并用冷冻干燥技术制备葫芦素B固体脂质纳米粒(CucB-SLN)冻干制剂,提高其贮存稳定性；通过大鼠体内药动学和组织分布研究揭示了葫芦素B固体脂质纳米粒的体内行为的规律；考察了葫芦素B固体脂质纳米粒的体内体外抗肿瘤效果。
采用MTT法研究了葫芦素B对Bel-7402、Hela和SGC-7901细胞的体外增殖抑制作用,结果表明葫芦素B对体外培养的Bel-7402细胞与Hela细胞具有较强的体外增殖抑制作用,且呈现良好的浓度-效应依赖关系,对SGC-7901细胞有中等强度体外增值抑制作用,亦呈现浓度-效应依赖关系。从IC50结果看,葫芦素B对肝癌细胞Bel-7402的增殖抑制作用比5-Fu强。
以含半乳糖端基的乳糖酸和十六胺为起始物,通过酰化反应得到目标产物N-十六烷基乳糖酰胺。该靶向材料合成工艺简单、条件温和可控。
以山嵛酸甘油酯为脂质材料,卵磷脂和泊洛沙姆188为乳化剂,采用高压匀质法制备了葫芦素B普通固体脂质纳米粒(CSLN)和半乳糖化固体脂质纳米粒。通过单因素实验考察了工艺因素(如加入顺序、匀化压力和次数、超声功率和时间等)和处方因素(脂质相种类和用量、乳化剂浓度、乳化剂比例和葫芦素B用量)对SLN粒径的影响,并进一步通过正交设计优化了处方。考察了不同冻干保护剂在SLN冻干过程中的保护作用,最后确定采用7.5%海藻糖和5.0%甘露醇混合使用,并对预冻、干燥过程中的各工艺因素进行了考察,确定了最终的冻干工艺。
从粒子形态、粒径大小、药物分散状态、包封率和体外释放行为方面对SLN进行了研究。采用透射电镜观察了自制的CSLN和GalSLN的形态,结果表明所制得的纳米粒均为类球形粒子。采用激光粒度仪测定了两种SLN冻干前后的粒径分布,冻干前CSLN和GalSLN的粒径分别为123 nm和135 nm,冻干后两者的粒径分别为204 nm和218 nm；以电泳法测定了SLN的ζ电位,结果表明本研究制得的纳米粒表面带负电荷,ζ电位均在为-30 mv左右；DSC结果显示药物以无定形的形式分散于SLN中。采用低温超速离心法测定了Cuc B-SLN包封率,结果表明冻干前CSLN和GalSLN的包封率分别为93.1%和90.7%。体外释药结果表明,药物释放符合Higuchi释放模型。
建立了生物样品中Cuc B的HPLC分析方法,研究了大鼠静注Cuc B溶液、CSLN和GalSLN三种制剂后的体内药动学行为,结果显示,二组纳米制剂的AUC(0-t)和MRT(0-t)与对照溶液组相比均存在显著性差异,纳米粒组的体内滞留时间明显增加。研究了大鼠静注三种制剂后的组织分布特点,测定了血、心、肝、脾、肺、肾各组织中不同时间点的药物浓度,考察了纳米制剂的组织靶向作用,结果表明,纳米制剂明显的改变了药物的体内组织分布特点,GalSLN的肝靶向效率是CSLN的2.5倍,GalSLN能够较显著地提高肝脏靶向性。
采用MTT法比较了Cuc B溶液、CSLN和GalSLN三种制剂对人肝癌细胞HepG2的细胞毒性,结果表明,三种制剂均表现出明显的抗肿瘤活性,.其活性强度具有时间和剂量依赖性,且与溶液剂相比,两种微粒制剂均提高了药物的细胞毒性,其中GalSLN对肿瘤细胞的抑制活性最强。
建立异位皮下移植肝癌H22实体瘤及肉瘤S180小鼠肿瘤模型,考察葫芦素B固体脂质纳米粒的体内抗肿瘤效果。CSLN(0.11,0.055 mg／kg)、GalSLN(0.11,0.055 mg／kg)对小鼠肝癌H22、肉瘤S180生长均有显著抑制作用,且高剂量(0.11 mg／kg)的GalSLN对于小鼠肝癌H22的抑瘤作用优于CSLN。
Receptor-mediated cell targeting can be obtained by properly selection of a ligand that can be specifically recognized by the receptor over-expressed on the surface of target cells. Mammalian hepatocytes (parenchymal cells) exclusively possessing a large number of asialoglycoprotein receptors (ASGPr) can recognize terminalβ-D-galactose or,N-acetylgalactosamine residues. Recently, a great deal of concern was focused on ASGPr-mediated endocytosis to fulfill nanoparticles targeting to liver. In this paper, a galactosylated lipid, N-hexadecyl lactobionamide (N-HLBA) was synthesized. As a "homing molecule", N-HLBA was used to modify cucurbitacin B (Cue B) loaded solid lipid nanoparticles (SLN) and form galactosylated SLN (GalSLN). The freeze drying preparation of Cue B-SLN was prepared by lyophillization to enhance the storage stability. The pharmacokinetics and tissue distribution in rats was studied to reveal the fate in vivo of Cue B-SLN. The anti-tumor efficiency of Cue B-SLN in vitro and in vivo was investigated.
The inhibitory effect of Cue B in vitro was investigated using the Bel-7402, Hela and SGC-7901 cells with MTT method. Cue B greatly inhibited the growth of Bel-7402 and Hela cells, and moderately inhibited the growth of SGC-7901 cells, both demonstrating the clear dose-dependent manner. The results of IC50 showed that the inhibitory effect of Cue B on the Bel-7402 was better than that of 5-Fu.
Lactobionic acid (bearing a galactosyl group) and hexadecylamine were chemically attached via the amidation reactions to produce N-hexadecyl lactobionamide (N-HLBA) with a mono-galactoside moiety. The chemical structure was characterized by IR, NMR and ESI-MS. The synthesis process of N-HLBA is simple and easy-controlled.
Cue B-SLN was prepared by high pressure homogenization, which Compritol 888 ATO was lipid material and lecithin and poloxamer 188 as emulsifier. The single factor experiments were investigated the effects of technology factors (adding order, homogenization pressure and cycles, ultrasound power and time, et al) and formulation factors (the kinds and content of lipid phase, the kind and ratio of emulsifier and the content of Cue B) on particle size and distribution of Cue B-SLN.
The orthogonal design was carried to optimize the formulation further. Cue B-SLN was prepared into freeze drying preparation by lyophillization after the process and formulation parameters were optimized. At last, the optimal formulation with 7.5% of trehalose and 5.0% of mannitol presented good appearance and reconstitution.
The pharmaceutical properties of Cue B-SLN including the shape, particle size, drug status in SLN, encapsulation efficiency and release in vitro were studied. The micrographs of transmission electron microscope (TEM) of CSLN and GalSLN showed that the particles had quasi-spherical physical shapes and uniform size. The particle size was measured by laser diffraction, and the mean diameter before and after lyophilization was 123 nm and 204 nm for CSLN,135 nm and 218 nm for GalSLN respectively.ξpotential of Cue B-SLN was determined by electrophoretic method, which was negative.ξpotential of the two types of SLNs was about-30 mV. DSC analysis showed that Cue B was amorphously dispersed within the SLN. The drug entrapment efficiency (EE) was measured by HPLC after ultracentrifugation. The EE was 93.1% for CSLN and 90.7% for GalSLN. The in vitro drug release behavior revealed that drug release was fitted well by Higuchi equation.
HPLC method was developed for the determination of Cue B in biological specimen. The rat intravenous pharmacokinetic behaviors of three formulations (solution, CSLN, GalSLN) were investigated. The results showed that t the two types of SLNs had higher AUC and MRT than drug solution. Cue B-SLN showed the most obvious effect in prolonging the drug circulation time. The rat tissue distribution of the three formulations were examined after intravenous. The drug concentrations in the plasma, heart, liver, spleen, lung, kidney were determined at different time. The results suggested that compared with drug solution, the two types of SLNs changed the tissue distribution character obviously, The (Te) liver values suggested that the liver targetability of GalSLN was 2.5 times greater than that of CSLN. The biodistribution confirmed that the GalSLN could produce more efficient delivery into the target organ-liver.
The cytotoxicity of drug solution, CSLN and GalSLN was investigated in the HepG2 cell line with MTT method. The three formulations all displayed obvious cytotoxicity, and had time-dependent and dose-dependent manner. Besides, SLNs had more potent cytotoxicity compared with drug solution and GalSLN had the most potent cytotoxicity.
The efficiency of tumor inhibition after intravenous administration of different Cuc B-SLN were evaluated in KM mice bearing subcutaneous engrafted H22 murine hepatoma or S180 murine sarcopma. The result shows that GalSLN (0.11,0.055 mg/kg) and CSLN (0.11,0.055 mg/kg) exhibited siginificant anti-tumor effect in vivo against H22 murine hepatoma or S180 murine sarcopma. The effect of antitumor growth of GalSLN (0.11 mg/kg) was better compared with the CSLN (0.11 mg/kg).
采用MTT法研究了葫芦素B对Bel-7402、Hela和SGC-7901细胞的体外增殖抑制作用,结果表明葫芦素B对体外培养的Bel-7402细胞与Hela细胞具有较强的体外增殖抑制作用,且呈现良好的浓度-效应依赖关系,对SGC-7901细胞有中等强度体外增值抑制作用,亦呈现浓度-效应依赖关系。从IC50结果看,葫芦素B对肝癌细胞Bel-7402的增殖抑制作用比5-Fu强。
以含半乳糖端基的乳糖酸和十六胺为起始物,通过酰化反应得到目标产物N-十六烷基乳糖酰胺。该靶向材料合成工艺简单、条件温和可控。
以山嵛酸甘油酯为脂质材料,卵磷脂和泊洛沙姆188为乳化剂,采用高压匀质法制备了葫芦素B普通固体脂质纳米粒(CSLN)和半乳糖化固体脂质纳米粒。通过单因素实验考察了工艺因素(如加入顺序、匀化压力和次数、超声功率和时间等)和处方因素(脂质相种类和用量、乳化剂浓度、乳化剂比例和葫芦素B用量)对SLN粒径的影响,并进一步通过正交设计优化了处方。考察了不同冻干保护剂在SLN冻干过程中的保护作用,最后确定采用7.5%海藻糖和5.0%甘露醇混合使用,并对预冻、干燥过程中的各工艺因素进行了考察,确定了最终的冻干工艺。
从粒子形态、粒径大小、药物分散状态、包封率和体外释放行为方面对SLN进行了研究。采用透射电镜观察了自制的CSLN和GalSLN的形态,结果表明所制得的纳米粒均为类球形粒子。采用激光粒度仪测定了两种SLN冻干前后的粒径分布,冻干前CSLN和GalSLN的粒径分别为123 nm和135 nm,冻干后两者的粒径分别为204 nm和218 nm；以电泳法测定了SLN的ζ电位,结果表明本研究制得的纳米粒表面带负电荷,ζ电位均在为-30 mv左右；DSC结果显示药物以无定形的形式分散于SLN中。采用低温超速离心法测定了Cuc B-SLN包封率,结果表明冻干前CSLN和GalSLN的包封率分别为93.1%和90.7%。体外释药结果表明,药物释放符合Higuchi释放模型。
建立了生物样品中Cuc B的HPLC分析方法,研究了大鼠静注Cuc B溶液、CSLN和GalSLN三种制剂后的体内药动学行为,结果显示,二组纳米制剂的AUC(0-t)和MRT(0-t)与对照溶液组相比均存在显著性差异,纳米粒组的体内滞留时间明显增加。研究了大鼠静注三种制剂后的组织分布特点,测定了血、心、肝、脾、肺、肾各组织中不同时间点的药物浓度,考察了纳米制剂的组织靶向作用,结果表明,纳米制剂明显的改变了药物的体内组织分布特点,GalSLN的肝靶向效率是CSLN的2.5倍,GalSLN能够较显著地提高肝脏靶向性。
采用MTT法比较了Cuc B溶液、CSLN和GalSLN三种制剂对人肝癌细胞HepG2的细胞毒性,结果表明,三种制剂均表现出明显的抗肿瘤活性,.其活性强度具有时间和剂量依赖性,且与溶液剂相比,两种微粒制剂均提高了药物的细胞毒性,其中GalSLN对肿瘤细胞的抑制活性最强。
建立异位皮下移植肝癌H22实体瘤及肉瘤S180小鼠肿瘤模型,考察葫芦素B固体脂质纳米粒的体内抗肿瘤效果。CSLN(0.11,0.055 mg／kg)、GalSLN(0.11,0.055 mg／kg)对小鼠肝癌H22、肉瘤S180生长均有显著抑制作用,且高剂量(0.11 mg／kg)的GalSLN对于小鼠肝癌H22的抑瘤作用优于CSLN。
Receptor-mediated cell targeting can be obtained by properly selection of a ligand that can be specifically recognized by the receptor over-expressed on the surface of target cells. Mammalian hepatocytes (parenchymal cells) exclusively possessing a large number of asialoglycoprotein receptors (ASGPr) can recognize terminalβ-D-galactose or,N-acetylgalactosamine residues. Recently, a great deal of concern was focused on ASGPr-mediated endocytosis to fulfill nanoparticles targeting to liver. In this paper, a galactosylated lipid, N-hexadecyl lactobionamide (N-HLBA) was synthesized. As a "homing molecule", N-HLBA was used to modify cucurbitacin B (Cue B) loaded solid lipid nanoparticles (SLN) and form galactosylated SLN (GalSLN). The freeze drying preparation of Cue B-SLN was prepared by lyophillization to enhance the storage stability. The pharmacokinetics and tissue distribution in rats was studied to reveal the fate in vivo of Cue B-SLN. The anti-tumor efficiency of Cue B-SLN in vitro and in vivo was investigated.
The inhibitory effect of Cue B in vitro was investigated using the Bel-7402, Hela and SGC-7901 cells with MTT method. Cue B greatly inhibited the growth of Bel-7402 and Hela cells, and moderately inhibited the growth of SGC-7901 cells, both demonstrating the clear dose-dependent manner. The results of IC50 showed that the inhibitory effect of Cue B on the Bel-7402 was better than that of 5-Fu.
Lactobionic acid (bearing a galactosyl group) and hexadecylamine were chemically attached via the amidation reactions to produce N-hexadecyl lactobionamide (N-HLBA) with a mono-galactoside moiety. The chemical structure was characterized by IR, NMR and ESI-MS. The synthesis process of N-HLBA is simple and easy-controlled.
Cue B-SLN was prepared by high pressure homogenization, which Compritol 888 ATO was lipid material and lecithin and poloxamer 188 as emulsifier. The single factor experiments were investigated the effects of technology factors (adding order, homogenization pressure and cycles, ultrasound power and time, et al) and formulation factors (the kinds and content of lipid phase, the kind and ratio of emulsifier and the content of Cue B) on particle size and distribution of Cue B-SLN.
The orthogonal design was carried to optimize the formulation further. Cue B-SLN was prepared into freeze drying preparation by lyophillization after the process and formulation parameters were optimized. At last, the optimal formulation with 7.5% of trehalose and 5.0% of mannitol presented good appearance and reconstitution.
The pharmaceutical properties of Cue B-SLN including the shape, particle size, drug status in SLN, encapsulation efficiency and release in vitro were studied. The micrographs of transmission electron microscope (TEM) of CSLN and GalSLN showed that the particles had quasi-spherical physical shapes and uniform size. The particle size was measured by laser diffraction, and the mean diameter before and after lyophilization was 123 nm and 204 nm for CSLN,135 nm and 218 nm for GalSLN respectively.ξpotential of Cue B-SLN was determined by electrophoretic method, which was negative.ξpotential of the two types of SLNs was about-30 mV. DSC analysis showed that Cue B was amorphously dispersed within the SLN. The drug entrapment efficiency (EE) was measured by HPLC after ultracentrifugation. The EE was 93.1% for CSLN and 90.7% for GalSLN. The in vitro drug release behavior revealed that drug release was fitted well by Higuchi equation.
HPLC method was developed for the determination of Cue B in biological specimen. The rat intravenous pharmacokinetic behaviors of three formulations (solution, CSLN, GalSLN) were investigated. The results showed that t the two types of SLNs had higher AUC and MRT than drug solution. Cue B-SLN showed the most obvious effect in prolonging the drug circulation time. The rat tissue distribution of the three formulations were examined after intravenous. The drug concentrations in the plasma, heart, liver, spleen, lung, kidney were determined at different time. The results suggested that compared with drug solution, the two types of SLNs changed the tissue distribution character obviously, The (Te) liver values suggested that the liver targetability of GalSLN was 2.5 times greater than that of CSLN. The biodistribution confirmed that the GalSLN could produce more efficient delivery into the target organ-liver.
The cytotoxicity of drug solution, CSLN and GalSLN was investigated in the HepG2 cell line with MTT method. The three formulations all displayed obvious cytotoxicity, and had time-dependent and dose-dependent manner. Besides, SLNs had more potent cytotoxicity compared with drug solution and GalSLN had the most potent cytotoxicity.
The efficiency of tumor inhibition after intravenous administration of different Cuc B-SLN were evaluated in KM mice bearing subcutaneous engrafted H22 murine hepatoma or S180 murine sarcopma. The result shows that GalSLN (0.11,0.055 mg/kg) and CSLN (0.11,0.055 mg/kg) exhibited siginificant anti-tumor effect in vivo against H22 murine hepatoma or S180 murine sarcopma. The effect of antitumor growth of GalSLN (0.11 mg/kg) was better compared with the CSLN (0.11 mg/kg).
引文
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[18]中华人民共和国药典委员会.中华人民共和国药典2005版.北京：化学工业出版社,第二部附录Ⅰ B.
[19]崔福德.药剂学.北京：中国医药科技出版社,2002：397
[20]崔福德.药剂学.北京：中国医药科技出版社,2002：399
[21]Wang W. Lyophilization and development of solid protein pharmaceutical. Int. J Pharm., 2003,203(1-2):1-60
[22]武华丽,胡一桥.冷冻干燥制剂的稳定性研究进展.中国药学杂志,2001,36(7)：436-438.
[1]王思玲,李玉娟,张景海,等.电泳光散射法测定异丙酚微乳剂的动电电位.沈阳药科大学学报,2002,19(5)：313-316.
[2]DELSA 440SX user manual.Supplement for the coulter DELSA 440SX. pp4.
[3]Freitas C, Muller RH. Correlation between long-term stability of solid lipid nanoparticles (SLN) and crystallinity of the lipid phase. Eur. J. Pharm. Biopharm.,1999,47:125-132.
[4]Westesen K, Bunjes H, Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J. Controlled Rel.,1997,48:223-236.
[5]Gref R., Minamitake Y, Peracchia MT, Trebetskoy VS, et al. Biodegradable long-circulating polymeric nanospheres. Science,1994,263:1600-1603.
[6]Allen C., Maysinger D, Eisenberg A. Nano-engineering block copolymer aggregates for drug delivery. Colloids Surf. B Biointerfaces,1999,16:3-27.
[7]Alexandridis P, Hatton TA. Poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces:thermodynamics, structure, dynamics, and modeling. Colloids Surf., B Biointerfaces,1995,96:1-46.
[8]Komatsu H, Kitajima A, Okada S. Phamaceutical characterization of commercially available intravenous fat emulsions:estimation of average particle size, size distribution and surface potential using photon correlation spectroscopy. Chem. Pham. Bull.,1995,43:1412-1415.
[9]Mehnert W, Mader K. Solid lipid nanoparticles production, characterization and applications. Adv. Drug Deliv. Rev.,2001,47:165-196.
[10]韩静,唐星,巴德纯.降香挥发油固体脂质纳米粒的制备.中成药,2004,26(6)：434-437.
[11]王秀敏,邓英杰,郝艳丽,等.微柱离心-气相色谱法测定β-榄香烯脂质体包封率.沈阳药科大学学报,2004,21(6)：416-419.
[12]Muhlen A, Mehnert W. Drug release and release mechanism of prednisolone loaded solid lipid nanoparticles. Pharmazie,1998,53:552.
[13]胡连栋,唐星,崔福德.维甲酸固体脂质纳米粒的制备及体内外评价.药学学报,2005,40(1)：71-75.
[14]Radomska A, Dobrucki R, Muller RH. Chemical stability of the lipid matrices of solid lipid nanoparticles (SLN)-development of an analytical method and determination of long-term stability. Pharmazie,1999,54:903-909.
[15]陆彬,熊素彬.米托蒽醌类脂质纳米球包封率和包封产率的测定.中国药品标准,2004,5(5)：32-36.
[16]Westesen K, Bunjes H,Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J. Controlled Rel.,1997,48:223-236.
[17]李喆,邓英杰,王秀敏.尼群地平脂质体的制备及包封率的测定.中国药剂学杂志,2005,3(4)：180-183.
[18]Westesen K, Siekmann B, Koch MHJ. Investigations on the Technology:physical state of lipid nanoparticles by synchrotron radiation X-ray diffraction. Int. J. Pharm.,1993, 93:189-199.
[19]Muller RH, Mader K, Cuc Bhla S. Solid lipid nanoparticles (SLN) for controlled drug delivery-a review of the state of the art. Eur. J. Pharm. Biopharm.,2000,50:161-177.
[20]陈宗淇,王光信,徐桂英.胶体与界面化学.北京：高等教育出版社.1991：150.
[21]王思玲,苏德森.胶体分散药物制剂.北京：人民卫生出版社,2006,12：368
[22]Vistnes AI, Puskin JSA.Spin label method for measuring internal volumes in liposomes or cells, applied to Ca dependent fusion of negatively charged vesicles. Biochim Biophs Acta, 1981,644:244-250.
[23]Zhang XM, Patel AB, Graaf RA,et al. Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. Chem.Phys.lipids,2004,127:113-120.
[24]Muhlen A, Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery-Drug release and release mechanism. Eur. J. Pharm. Biopharm.,1998,45:149-155.
[1]李超英,侯世祥,杨凯,等.血浆中葫芦素B的高效液相色谱测定方法研究.中国药学杂志,2001,36(8)：557-559.
[2]Wissing SA, Kayser O, Muller RH. Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev.,2004,56(9):1257-1272.
[3]Fundaro A, Cavalli R, Bargoni A, Vighetto D, Zara GP, Gasco MR. Non-stealth and stealth solid lipid nanoparticles (SLN) carrying doxorubicin:pharmacokinetics and tissue distribution after i.v. administration to rats. Pharmacol Res.,2000,42(4):337-343.
[4]Manjunath K, Venkateswarlu V. Pharmacokinetics, tissue distribution and bioavailability of nitrendipine solid lipid nanoparticles after intravenous and intraduodenal administration. J Drug Target,2006,14(9):632-645.
[5]Chen DB,Yang TZ, Lu WL, et al. In vitro and in vivo study of two types of long-circulating solid lipid nanoparticles containing paclitaxel. Chem Pharm Bull,2001,49(11):1444-1447.
[6]Gupta PK and Hung T, Quantitative evaluation of targeted drug delivery systems. Int. J. Pharm.,1989,56:217-226.
[7]Yuda T,Maruyama K,Iwatsuru M. Prolongation of liposome circulation time by various derivatives of polyethyleneglycols.Biol Pharm Bull,1996,19(10):1347-1351
[8]袁东锋,易以木.肝靶向纳米粒的研究进展.医药导报,2003,22(2)：113-114
[9]Brawn JR, Willnow TE, Ishibashi S, et al. The major subunit of the asialoglycoprotein receptor is expressed on the hepatocellularsur face in mice lacking the minor receptor or subunit. J Biol Chem,1996,270:21160-21169
[10]秦伯益.新药评价概论(第二版).北京：人民卫生出版社,1999：135
[11]毕殿洲.药剂学(第四版).北京：人民卫生出版社,2000：450
[12]蒋学华,廖工铁.肝靶向阿柔比星A聚氰基丙烯酸异丁酯毫微粒药物动力学研究.中国抗生素杂志,1999,24(1)：35-38
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[3]Cababnes A, Briggs KE, Gokhale PC, et al. Camparative in vivo studies with paclitaxel and liposome-encapsulated paclitaxel. Int J Oncol,1998,12:1035-1040.
[4]Miwa A, Ishibe A, Nakano M, et al. Development of novel chitosan derivatives as micellar carriers of taxol. Pharm Res,1998,15:1844-1850.
[5]Kang BK, Chon SK, Kim SH, et al. Controlled release of paclitaxel from micronemulsion containing PLGA and evaluation of antitumor activity in vitro and in vivo. Int J Pharm, 2004,286:147-156.
[6]Serpe L, Catalano MG, Cavalli R, et al. Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line. Eur J Pharm Biopharm, 2004,58:673-680.
[7]Miglietta A, Cavalli R, Bocca C, et al. Cellular uptake and cytotoxicity of solid lipid nanospheres (SLN) incorporating doxorubicin or paclitaxel. Int J Pharm,2000,210: 61-67.
[8]Couvreur P, Vauthier C. Polyalkylcyanoacrylate nanoparticles as drug carrier:present state and perspectives. J Controlled Release,1991,17:187-198.
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[1]Muller RH, Karsten M, Sven G. Solid lipid nanoparticles (SLN) for controlled drug delivery-a review of the state or the art. Eur J Pharm Biopharm,2000,50:161-177.
[2]S.A. Wissinga, O. Kayserb, R.H. Muller. Solid lipid nanoparticles for parenteral drug delivery. Advanced Drug Delivery Reviews,2004,56:1257-1272.
[3]王建新,张志荣.固体脂质纳米粒的研究进展.中国药学杂志,2001,36(2)：73-76.
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[10]Wolfgang Mehnert, Karsten Mader. Solid lipid nanoparticles:Production, characterization and applications. Advanced Drug Delivery Reviews,2001,47:165-196.
[11]应晓英,胡富强,袁弘.卡马西平硬脂酸固体脂质纳米粒的制备与理化性质研究.中国医药工业杂志,2002,33(11)：543—545
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[15]Patrick Shahgaldian, JerOme Gualbert, Kais Aissa, et al. A study of the freeze-drying conditions of calixarene based solid lipid nanoparticles. Euro. J Pharm. Biopharm.,2003,55: 181-184
[16]于志江,陈旭,欧阳藩.冷冻干燥技术在基因工程药物中的应用.生物加工过程,2005,3(2)：58-63
[17]崔福德主编.药剂学.北京：中国医药科技出版社,2002：74.
[18]中华人民共和国药典委员会.中华人民共和国药典2005版.北京：化学工业出版社,第二部附录Ⅰ B.
[19]崔福德.药剂学.北京：中国医药科技出版社,2002：397
[20]崔福德.药剂学.北京：中国医药科技出版社,2002：399
[21]Wang W. Lyophilization and development of solid protein pharmaceutical. Int. J Pharm., 2003,203(1-2):1-60
[22]武华丽,胡一桥.冷冻干燥制剂的稳定性研究进展.中国药学杂志,2001,36(7)：436-438.
[1]王思玲,李玉娟,张景海,等.电泳光散射法测定异丙酚微乳剂的动电电位.沈阳药科大学学报,2002,19(5)：313-316.
[2]DELSA 440SX user manual.Supplement for the coulter DELSA 440SX. pp4.
[3]Freitas C, Muller RH. Correlation between long-term stability of solid lipid nanoparticles (SLN) and crystallinity of the lipid phase. Eur. J. Pharm. Biopharm.,1999,47:125-132.
[4]Westesen K, Bunjes H, Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J. Controlled Rel.,1997,48:223-236.
[5]Gref R., Minamitake Y, Peracchia MT, Trebetskoy VS, et al. Biodegradable long-circulating polymeric nanospheres. Science,1994,263:1600-1603.
[6]Allen C., Maysinger D, Eisenberg A. Nano-engineering block copolymer aggregates for drug delivery. Colloids Surf. B Biointerfaces,1999,16:3-27.
[7]Alexandridis P, Hatton TA. Poly(ethylene oxide)-poly(propyleneoxide)-poly(ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces:thermodynamics, structure, dynamics, and modeling. Colloids Surf., B Biointerfaces,1995,96:1-46.
[8]Komatsu H, Kitajima A, Okada S. Phamaceutical characterization of commercially available intravenous fat emulsions:estimation of average particle size, size distribution and surface potential using photon correlation spectroscopy. Chem. Pham. Bull.,1995,43:1412-1415.
[9]Mehnert W, Mader K. Solid lipid nanoparticles production, characterization and applications. Adv. Drug Deliv. Rev.,2001,47:165-196.
[10]韩静,唐星,巴德纯.降香挥发油固体脂质纳米粒的制备.中成药,2004,26(6)：434-437.
[11]王秀敏,邓英杰,郝艳丽,等.微柱离心-气相色谱法测定β-榄香烯脂质体包封率.沈阳药科大学学报,2004,21(6)：416-419.
[12]Muhlen A, Mehnert W. Drug release and release mechanism of prednisolone loaded solid lipid nanoparticles. Pharmazie,1998,53:552.
[13]胡连栋,唐星,崔福德.维甲酸固体脂质纳米粒的制备及体内外评价.药学学报,2005,40(1)：71-75.
[14]Radomska A, Dobrucki R, Muller RH. Chemical stability of the lipid matrices of solid lipid nanoparticles (SLN)-development of an analytical method and determination of long-term stability. Pharmazie,1999,54:903-909.
[15]陆彬,熊素彬.米托蒽醌类脂质纳米球包封率和包封产率的测定.中国药品标准,2004,5(5)：32-36.
[16]Westesen K, Bunjes H,Koch MHJ. Physicochemical characterization of lipid nanoparticles and evaluation of their drug loading capacity and sustained release potential. J. Controlled Rel.,1997,48:223-236.
[17]李喆,邓英杰,王秀敏.尼群地平脂质体的制备及包封率的测定.中国药剂学杂志,2005,3(4)：180-183.
[18]Westesen K, Siekmann B, Koch MHJ. Investigations on the Technology:physical state of lipid nanoparticles by synchrotron radiation X-ray diffraction. Int. J. Pharm.,1993, 93:189-199.
[19]Muller RH, Mader K, Cuc Bhla S. Solid lipid nanoparticles (SLN) for controlled drug delivery-a review of the state of the art. Eur. J. Pharm. Biopharm.,2000,50:161-177.
[20]陈宗淇,王光信,徐桂英.胶体与界面化学.北京：高等教育出版社.1991：150.
[21]王思玲,苏德森.胶体分散药物制剂.北京：人民卫生出版社,2006,12：368
[22]Vistnes AI, Puskin JSA.Spin label method for measuring internal volumes in liposomes or cells, applied to Ca dependent fusion of negatively charged vesicles. Biochim Biophs Acta, 1981,644:244-250.
[23]Zhang XM, Patel AB, Graaf RA,et al. Determination of liposomal encapsulation efficiency using proton NMR spectroscopy. Chem.Phys.lipids,2004,127:113-120.
[24]Muhlen A, Schwarz C, Mehnert W. Solid lipid nanoparticles (SLN) for controlled drug delivery-Drug release and release mechanism. Eur. J. Pharm. Biopharm.,1998,45:149-155.
[1]李超英,侯世祥,杨凯,等.血浆中葫芦素B的高效液相色谱测定方法研究.中国药学杂志,2001,36(8)：557-559.
[2]Wissing SA, Kayser O, Muller RH. Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev.,2004,56(9):1257-1272.
[3]Fundaro A, Cavalli R, Bargoni A, Vighetto D, Zara GP, Gasco MR. Non-stealth and stealth solid lipid nanoparticles (SLN) carrying doxorubicin:pharmacokinetics and tissue distribution after i.v. administration to rats. Pharmacol Res.,2000,42(4):337-343.
[4]Manjunath K, Venkateswarlu V. Pharmacokinetics, tissue distribution and bioavailability of nitrendipine solid lipid nanoparticles after intravenous and intraduodenal administration. J Drug Target,2006,14(9):632-645.
[5]Chen DB,Yang TZ, Lu WL, et al. In vitro and in vivo study of two types of long-circulating solid lipid nanoparticles containing paclitaxel. Chem Pharm Bull,2001,49(11):1444-1447.
[6]Gupta PK and Hung T, Quantitative evaluation of targeted drug delivery systems. Int. J. Pharm.,1989,56:217-226.
[7]Yuda T,Maruyama K,Iwatsuru M. Prolongation of liposome circulation time by various derivatives of polyethyleneglycols.Biol Pharm Bull,1996,19(10):1347-1351
[8]袁东锋,易以木.肝靶向纳米粒的研究进展.医药导报,2003,22(2)：113-114
[9]Brawn JR, Willnow TE, Ishibashi S, et al. The major subunit of the asialoglycoprotein receptor is expressed on the hepatocellularsur face in mice lacking the minor receptor or subunit. J Biol Chem,1996,270:21160-21169
[10]秦伯益.新药评价概论(第二版).北京：人民卫生出版社,1999：135
[11]毕殿洲.药剂学(第四版).北京：人民卫生出版社,2000：450
[12]蒋学华,廖工铁.肝靶向阿柔比星A聚氰基丙烯酸异丁酯毫微粒药物动力学研究.中国抗生素杂志,1999,24(1)：35-38
[1]Maeda H. The tumor blood vessel as an ideal target for macromolecular anticancer agents. J Control Release,1992,19:315-324.
[2]Gokhale PC, Radhakrishnan B, Husain SR, et al. An improved method of encapsulation of doxorubicin in liposomes:pharmacological, toxicological and therapeutic evaluation. Br J Cancer,1996,34:45-48.
[3]Cababnes A, Briggs KE, Gokhale PC, et al. Camparative in vivo studies with paclitaxel and liposome-encapsulated paclitaxel. Int J Oncol,1998,12:1035-1040.
[4]Miwa A, Ishibe A, Nakano M, et al. Development of novel chitosan derivatives as micellar carriers of taxol. Pharm Res,1998,15:1844-1850.
[5]Kang BK, Chon SK, Kim SH, et al. Controlled release of paclitaxel from micronemulsion containing PLGA and evaluation of antitumor activity in vitro and in vivo. Int J Pharm, 2004,286:147-156.
[6]Serpe L, Catalano MG, Cavalli R, et al. Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line. Eur J Pharm Biopharm, 2004,58:673-680.
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