纳米载体介导柔红霉素穿透血脑屏障的研究
|
摘要
目的:本研究的目的旨在制备纳米载体,将柔红霉素纳米化,建立简便、易行的能够模拟在体血脑屏障的体外细胞模型。为柔红霉素纳米粒的临床应用提供实验依据。
方法:①首先选择聚氰基丙烯酸酯为载体材料,用乳化聚合法制备柔红霉素聚氰基丙烯酸正丁酯纳米粒DAM-PBCA-NP,通过单因素试验初选、均匀设计试验、并以柔红霉素纳米粒的载药量、包封率、平均球径等多个指标进行综合评分,优化柔红霉素纳米粒的制备工艺。②以SD大鼠为实验材料,采用两步滤过法获得微血管段,胶原酶消化,低分子右旋糖苷密度离心获得脑微血管内皮细胞,接种4小时换液使获得的细胞纯化。倒置显微镜下观察细胞的形态,细胞Ⅷ因子相关抗原(ⅧF-Ag)免疫组化法鉴定细胞及其纯度,MTT法检测细胞活性。将传至第2代的脑微血管内皮细胞接种于Transwell内侧,细胞培养一周。通过柔红霉素纳米化在体外模型中的分布情况,探讨柔红霉素纳米粒透过血脑屏障的体外模型通过率。
结果:①在优化条件下制备的DAM-PBCA-NP为乳红色胶体溶液,毫微粒外形圆整光滑、分布均匀、不粘连,平均粒径(82±3)nm,包封率为(92.65±1.87)%,载药量为(68.52±1.56)%.②用我们的分离培养方法获得的脑微血管内皮细胞在倒置显微镜下细胞呈多角形或“铺路石”形,单层贴壁生长。培养的细胞Ⅷ因子相关抗原免疫组化试验阳性,细胞纯度为90%.③柔红霉素纳米粒透过血脑屏障的体外模型通过率为(70.15±1.23)%。
讨论:①通过优选工艺所制得的柔红霉素纳米粒可以满足本实验研究的需要,该优化条件可作为DAM-PBCA-NP的最佳制备工艺。②我们实验采用的两步滤过获得脑微血管段,胶原酶消化,低分子量右旋糖昔密度离心可分离和培养大鼠脑微血管内皮细胞。这一方法较其他方法程序简单,获得细胞纯度高,适合用于体外BBB模型的建立。③柔红霉素纳米粒透过血脑屏障的体外模型通过率为(70.15±1.23)%,表明对进一步的体内试验及临床试验提供了实验依据。
Objective: To prepare nanoparticle drug carriers for daunorbic, and to simulate a simple in-vitro cell model of blood brain barrier, as well as to provide evidence on clinical application of daunorbic nanoparticles.
Methods:①Polybutylcyano acrylate was selected to be drug carrier, and daunorubicin polybutylcyano acrylate nanoparticles were prepared using emulsifi-polymerization. DAM-PBCA-NP, examined with single factor experiment and equality test, was analyzed and evaluated on multi factors, in terms of drug load of daunorubicin, coating rate, and average particle diameter, for optimizing preparation of daunorubicin nanoparticle.②SD rats were selected as the experimental material. Micro-blood vessels were attained using two-step filtration. Assimilated by collagen enzyme and centrifuged with low-molecular dextran, micro-blood vessel endothelium cells were gained. Cell culture base was changed 4 hours after inoculation. With inversion microscope, cellular purification was immunohistochemically determined byⅧF-Ag, and cellular activation was examined using MTT method. The second generation micro-blood vessel endothelium cells were inoculated inner Transwell and cultured for 1 week. Rate of permeation through blood brain barrier was determined by DAM-PBCA-NP distribution in the in-vitro model.
Results:①DAM-PBCA-NP solution presented red with optimized preparation. Particle shape was round and smooth, well distributed, with an average diameter of (82±3) nm. Its coating rate reached (92.65±1.87)%, and drug load was (68.52±1.56)%.②Micro-blood vessel endothelium cells gained with separated culture approach had multi-angle shape with monolayer growth. Immunohistochemical test showed positive, and cellular purification reached 90%.③Rate of permeation through blood brain barrier was (70.15±1.23) %.
Discussion:①The optimized preparation of DAM-PBCA-NP satis fied the needs in the present study, which could be considered the best preparation.②The two-step method for attaining rat micro-blood vess el endothelium cells is easy to approach, and the high cellular purification was suitable for BBB model.③Rate of permeation through blood brain barrier was (70.15±1.23) %, which has provided clinical evidence for further in-vivo experiments.
方法:①首先选择聚氰基丙烯酸酯为载体材料,用乳化聚合法制备柔红霉素聚氰基丙烯酸正丁酯纳米粒DAM-PBCA-NP,通过单因素试验初选、均匀设计试验、并以柔红霉素纳米粒的载药量、包封率、平均球径等多个指标进行综合评分,优化柔红霉素纳米粒的制备工艺。②以SD大鼠为实验材料,采用两步滤过法获得微血管段,胶原酶消化,低分子右旋糖苷密度离心获得脑微血管内皮细胞,接种4小时换液使获得的细胞纯化。倒置显微镜下观察细胞的形态,细胞Ⅷ因子相关抗原(ⅧF-Ag)免疫组化法鉴定细胞及其纯度,MTT法检测细胞活性。将传至第2代的脑微血管内皮细胞接种于Transwell内侧,细胞培养一周。通过柔红霉素纳米化在体外模型中的分布情况,探讨柔红霉素纳米粒透过血脑屏障的体外模型通过率。
结果:①在优化条件下制备的DAM-PBCA-NP为乳红色胶体溶液,毫微粒外形圆整光滑、分布均匀、不粘连,平均粒径(82±3)nm,包封率为(92.65±1.87)%,载药量为(68.52±1.56)%.②用我们的分离培养方法获得的脑微血管内皮细胞在倒置显微镜下细胞呈多角形或“铺路石”形,单层贴壁生长。培养的细胞Ⅷ因子相关抗原免疫组化试验阳性,细胞纯度为90%.③柔红霉素纳米粒透过血脑屏障的体外模型通过率为(70.15±1.23)%。
讨论:①通过优选工艺所制得的柔红霉素纳米粒可以满足本实验研究的需要,该优化条件可作为DAM-PBCA-NP的最佳制备工艺。②我们实验采用的两步滤过获得脑微血管段,胶原酶消化,低分子量右旋糖昔密度离心可分离和培养大鼠脑微血管内皮细胞。这一方法较其他方法程序简单,获得细胞纯度高,适合用于体外BBB模型的建立。③柔红霉素纳米粒透过血脑屏障的体外模型通过率为(70.15±1.23)%,表明对进一步的体内试验及临床试验提供了实验依据。
Objective: To prepare nanoparticle drug carriers for daunorbic, and to simulate a simple in-vitro cell model of blood brain barrier, as well as to provide evidence on clinical application of daunorbic nanoparticles.
Methods:①Polybutylcyano acrylate was selected to be drug carrier, and daunorubicin polybutylcyano acrylate nanoparticles were prepared using emulsifi-polymerization. DAM-PBCA-NP, examined with single factor experiment and equality test, was analyzed and evaluated on multi factors, in terms of drug load of daunorubicin, coating rate, and average particle diameter, for optimizing preparation of daunorubicin nanoparticle.②SD rats were selected as the experimental material. Micro-blood vessels were attained using two-step filtration. Assimilated by collagen enzyme and centrifuged with low-molecular dextran, micro-blood vessel endothelium cells were gained. Cell culture base was changed 4 hours after inoculation. With inversion microscope, cellular purification was immunohistochemically determined byⅧF-Ag, and cellular activation was examined using MTT method. The second generation micro-blood vessel endothelium cells were inoculated inner Transwell and cultured for 1 week. Rate of permeation through blood brain barrier was determined by DAM-PBCA-NP distribution in the in-vitro model.
Results:①DAM-PBCA-NP solution presented red with optimized preparation. Particle shape was round and smooth, well distributed, with an average diameter of (82±3) nm. Its coating rate reached (92.65±1.87)%, and drug load was (68.52±1.56)%.②Micro-blood vessel endothelium cells gained with separated culture approach had multi-angle shape with monolayer growth. Immunohistochemical test showed positive, and cellular purification reached 90%.③Rate of permeation through blood brain barrier was (70.15±1.23) %.
Discussion:①The optimized preparation of DAM-PBCA-NP satis fied the needs in the present study, which could be considered the best preparation.②The two-step method for attaining rat micro-blood vess el endothelium cells is easy to approach, and the high cellular purification was suitable for BBB model.③Rate of permeation through blood brain barrier was (70.15±1.23) %, which has provided clinical evidence for further in-vivo experiments.
引文
[1]Gerald AG,Joan AN,Damir J.Understanding the physiology of the blood-brain barrier:In vitro models.New Physiol sci.1998;13:287-293
[2]邓孔昭、黄如训编著.血脑屏障.北京.人民卫生出版社.1994.20-21
[3]Sobue K,Yamamoto N,Yoneda K,et al.Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.Neurosci Res.1999,35:155-164.
[4]Janigro D,Leaman SM,Stanness KA.Dynamic in vitro modeling of the blood-brain barrier:A novel tool for studies of drug delivery to the brain.Pharm Sci Technol Today.1999,2:7-12.
[5]Muruganandam A,Herx LM,Monete R,et al.Development of immortalized cerebromicrovascular endothelial cell line as an in vitro model of the human blood brain barrier.FASEB J.1997,11:1187-1197.
[6]Gaillard PJ,Voorwinden LH,Nielsen JL,et al.Establishment and functional characterization of an in vitro model of the blood-brain barrier,comprising a co-culture of brain capillary endothelial cells and astrocytes.Eur J Pharm Sci.2001,12:215-222.
[7]Kido Y,Tamai I,Okamoto M,Suzuki F,et al.Functional clarification of MCTI-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies.Pharm Res.2000,17:55-62.
[8]Duport S,Robert E Muller D,et al.An in vitro blood-brain barrier model:cocultures between endothelial cells and organotypic brain slice culture.Proc Natl Acad Sci.1998;95:1840-1855.
[9]Rubin L,Hall DE,Porter S,et al.A cell culture model of the blood-brain barrier.
[10]J Cell Biology.1991;115(6):1725-1735.
[11]Reinhardt CA,Gloor SM.Co-culture blood-brain barrier models and their use for pharmatoxicological screening[J].Toxicol In Vitro,1997,11(5):513-517.
[12]王建民,施永德,步燕芳,等.大鼠脑血管内皮细胞的分离培养与形态学观察[J].解剖学杂志,1998,21(6):495-499.
[13]Brendel K,Meezan E,and Carlson EC.Isolated brain microvessels:a purified,metabolically active preparation from bovine cerebral cortex[J].Science,1974,185(4155):953-935.
[14]Duan CG,Xiu RJ.Cultivation of endothdial cells from rat brain microvessels[J].Chinese Med J,1988,101(2):649-652.
[15]张兆旺 主编.中药药剂学(川.北京:中国中医药出版社,2002,507-511
[16]贺石林 主编.中医科研设计与统计方法.长沙:湖南科学技术出版社,1989 328-332
[17]刘明芝主编.中医药统计学[M].长沙:湖南科学技术出版社,1999,231-243,287-312
[18]Zobel HP.Cationic polyhexyleyanoacrlate nanoparticles as carders for antisense oligucl otides[J].Antisense Nucleic Acid Drug Dev,1997,7(1):483-489
[19]Fattal E,Vauthier C,Aynie I,et al.Biodegradable polyalkycyanoaerylate nanopartieles for the delivery of oligonucleotides[J].Journal of Controlled Release,1998,53(2):137-143.
[20]Godard G..Antisense effects of cholesteroioligodeoxynucleotide conjugates associated with poly(alkyleyanoaerylate)nanopartieles[J].Eur J Biochem,1995,232(2):404-410.
[21]Lambert G,Fattal E,Pinto-Alphandary H,et al.Polyisobutyleyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides[J].Int J Pharm,2001,214(1-2):13-16.
[22]Zimmer A.Antisense oligonucleotide delivery with polyhexylcyanoarylate nanop articles as carriers[J].Methods,1999,18(2):286-295.
[23]Calvo P,Gouritin B,Villarroya H,et al.Quantification and Locolization of PEGylated polyalkyleyanoacrylate nanoparticles in brain and spinal cord during experimental allergic encephalomyelitis in the rat[J].Eur J Neurosci,2002,15(8):1317-1326.
[24]张志荣.聚氰基丙烯酸酯毫微球的研究进展[J].中国药学杂志,1998,29(6):323-326.
[25]Regina Z.Comparative in vitro biocompatibility testing of polycyanoacrylates and poly(D,L-laetide-eo-glycolide)using different mouse fibroblast(L929)biocompatibility test models[J].Eur J Pharm Biopharm,1997,44(1):149-157.
[26]Karate B,Couvreur P,Dubois-Krack,et al.Toxicity of polyalkylcyanoacrylate nanoparticles:free nanoparticles[J].J Pharm Sic,1982,71(7):786-790.
[27]Gipps EM,Groscurth P,Kreuter J,et al.Distribution of polyhexylcyanoacrylate nanoparticles in nude mice over extended times and after repeated injection[J].J Pharm Sci,1988,77(3):208-209.
[28]陆彬主编,药物新剂型新技术[M].人民卫生出版社,1998,p211-213.
[29]廖工铁主编,药物靶向制剂[M].四川科学技术出版社,1997,p192-423.
[30] Sommerfeld P, Schroeder U, Sabel B. Long-term stability of PBCA nanoparticles suspensions suggests clinical usefulness[J]. International Journal of Pharmaceutics, 1997,155(2):201-207.
[31] Kreuter J, Alyautdin RN, Kharkevich DA, et al. Passage of peptides through theblood-brain barrier with colloidal polymer particles (nanoparticles) [J]. Brain Res, 1995,647 (1):171-174.
[32] Ramge P, Unger RE, Oltrogge JB, et al. Polysorbate 80-coating enhances uptake of polybutyleyanoacrylate(PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells[J].Eur J Neurosci,2000,12(6): 1931 -1940.
[33] Gulyaev AE, Gelperina SE, Skind IN, et al. Significant transport of doxorubicin into the brain with polysorbate-80-coated nanoparticles[J]. Pharm Res,1999,16(10):1564-1569.
[34] Kreuter J, Petroy VE, Kharkevich DA,et al. Influence of the type of surfactant on the analgesic effects induced by the peptide dalarin after its delivery across the blood-brain barrier using surfactant-coated nanoparticles[J]. J Controlled Release, 1997,49(1):81-87.
[35] Sun W, Me CS, Wang HF, et al. Specific role polysorbate 80 coating on the targeting of nanoparticles to the brain[J]. Biomaterials,2004,25(6):3065-3071.
[36] Pilar C, Bruno G, Helene C et al. Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery[J]. Pharm Res,2001,18(8): 1157-1166.
[37] Yang SC, Lu LF, Cai Y, et al. Body distribution in mice of intravenously injected camptot hecin solid lipid nanoparticles and targeting effect on brain[J]. J Control Release, 1999,59 (3):299-307.
[38] Kreuter J. Nanoparticulate systems for brain delivery of drugs[J]. Adv Drug Deliv Rev, 2001,47 (1):65-81.
[39] Calvo P, Gouritin B, Villarroya H, et al. Quantification and localization of PEGylated polycyanoacrylate nanoparticles in brain and spinal cord during experimental allergic encephalomyelitis in the rat[J]. Eur J Neurosci,2002,15(2): 1317-1326.
[40] Renad N, Reichel A, Ramge P, et al. Interaction of Poly(butylcyanoacrylate) nanoparticles with the Blood-Brain barrier in vivo and vitro[J]. Journal of Drug Targeting,2001,9(3):209- 221.
[41] Qaddoumi, MG, Ueda H, Yang J, et al. Uptake of biodegradable nanoparticles in pigmented rabbit conjunctival epithelial cells[J]. Invest Ophthalmol Vis Sci 2000,41(2):765-781.
[1]王建民,施永德,步燕芳,等.大鼠脑微血管内皮细胞的分离培养与形态学观察.[J].解剖学杂志.1998;21(6):495-499.
[2]Franke H,Galla H-J,Beuckmann CT.An improved low permeability in vitro model of the blood-brain barrier:Transport studies on retinods,sucrose,haloperidol,cafeine and mannitol.Brain Res.1999,818:65-71.
[3]Fenke H,Galla H J,Beuckmann CT.Primary cultures of brain micovessel endothelial cells:a valid and flexible model to study drug transport through the blood-brain barrier in vitro[J].Brain Res Prot,2000,5(2):248-256.
[4]罗玉敏.内皮细胞与脑缺血[J].国外医学脑血管疾病分册,1998,6(5):271-273.
[5]Hess DC,Howard E,Cheng C,et al.Hypertonic mannitol loading of NF-κB transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1[J].Stroke,2000,31(5):1179-1186.
[6]Nobuhiro I,Kohiji N,Hidenari H,et al.Isolation and primary culture of rat cerebral for studying drug transport in vitro[J].JPM,1996,36(3):45-56.
[7]Fern J,Barkalow k,Michael J.Rapid communication culture murine cerebral microvaseular endothelial cells contain yon willebrand factor-positive weibel-palade bodies and support rapid eytokine-indueed neutrophil adhesion[J].Microcireulation,1996,3(1):19-30.
[8]Pham YT,Regina A,Farinoti R,et al.Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalized rat brain capillary endothelial cell line,GPNT[J].Biochim Biophys Acta,2000,1524(2-3):212-219.
[9]Kido Y,Tamai I,Okamoto M,et al.Functional clarification of MCTI-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies.Pharm Res,2000,17(1):55-62.
[10]Reinhardt CA,Gloor SM.Co-culture blood-brain barrier models and their use for pharmatoxicological screening[J].Toxicol In Vitro,1997,11(5):513-517.
[11]王建民,施永德,步燕芳,等.大鼠脑血管内皮细胞的分离培养与形态学观察[J].解剖学杂志,1998,21(6):495-499.
[1] Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov, 2002,1 (2): 131-139
[2] BEGL EY DJ . The blood-brain barrier : principles for targeting peptides and drugs to the central nervous system[J]. J Pharm Pharmacol, 1996,48 (2) :136-146.
[3] Miller G. Drug targeting. Breaking down barriers. Science , 2002 , 297 : 1116— 1118.
[4] Kreuter J . Nanoparticulate systems for brain delivery of drugs [J] .Adv Drug Deliv Rev , 2001,47 (1) :65-81.
[5] Kumaresh S , Soppimath , et al . Biodegradable polymeric nanoparticles as drug delivery devices[J]. J Control Rel ,2000,70,120
[6] M.L. Hans A. M. Lowman .Biodegradable nanoparticles for drug delivery and targeting[J]. Current Opinion in Solid State and Materials Science, 2002,6 :319-327.
[7] Plank C , Tang MX, Wolfe AR, et al. Branched cationic peptides for gene therapy : role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum Gene Ther, 1999,10 :319-332.
[8] Shi N, Pardridge WM. Non2invasive gene targeting to the brain. Proc Natl Acad Sci USA , 2000, 97 :7567-7572.
[9] Zhang Y, Pardridge WM. Global nonviral gene transfer to the primate brain following intravenous administration. Mol Ther, 2003 ,7(1) :11—18.
[10] Lockman RJ , Mumper MA , Khan , et al. Nanoparticle technology for drug delivery across the blood2brain barrier. Drug Dev Ind Pharm, 2002, 28 (1): 1—13.
[11] Alyaudtin RN , Gother D, Petrov V. Analgesic activity of the hexapeptide dalargin absorbed on the surface of polysorbate-80 coated polybutylcyanoacrylate nanoparticles. Euro J Pharm Biopharm, 1995 ,41 :44-48.
[12] Fenart L , Casanova A , Dehouck B , et al. Evaluation of effect of charge and lipid coating on ability of 602nm nanoparticles to cross an in vitro model of the blood-brain barrier. J Pharmacol Exp Ther, 1999,291 :1017-1022.
[13] Schroeder U , Sabel BA , Schroeder H. Diffusion enhancement of drugs by loaded nanoparticles in vitro. Prog Neuropsychopharmacol Biol Psychiatry, 1999, 23 : 941 - 949.
[14] Schroeder U , Sommerfeld P, Ulrich S , et al. Nanoparticle technology for delivery of drugs across the blood2brain barrier. J Pharm Sci, 1998 , 87 :1305 - 1307.
[15] Alyautdin RN , Tezikov EB , Ramge P , et al . Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 802coated polybutylcyanoacrylate nanoparticles : an in situ brain perfusion study. J Microencapsul, 1998 ,15 : 67 - 74.
[16]Sun W,Xie C,Wang H,et al.Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain.Biomaterials,2004,25:3065-3071.
[17]Kreuter J,Shamenkov D,Petrov V,et al.Apolipoprotein-mediated transport of nanoparticle-bound drags across the blood-brain barrier.J Drug Target,2002,10:317-325.
[18]KREUTER J,AL YAUTDIN RN,KHARKEVICH DA,et al.Passage of peptides through the blood2brain barrier with colloidal polymer particles(nanoparticles)[J].Brain Res,1995,647(1):171-174.
[19]BORCHARD G,AUDUS KL,SHI FL,et al.Uptake of surfactant-coated poly(methyl methacrylate)nanoparticles by bovine brain microvessel endothelial monolayers[J].Int J Pharm,1994,110(1):29-35.
[20]AL YAUTDIN RN,RRICHEL A,LOBENBERG R,et al.Interaction of poly(butylcya noacrylate)nanoparticles with the blood-brain barrier i n vivo and i n vitro[J].J Drug Target,2001,9(3):209-221.
[21]CALVO P,GOURITIN B,BRIGGER I,et al.PEGylated poly-cyanoacrylate nanoparticles as vector for drug delivery in prion diseases[J].J Neurosci Methods,2001,111(2):151-155.
[22]Schroeder U,Sommeffeld P,Ulrich S,et al.Nanoparticle technology for delivery of drugs across the blood-brain barrier.J Pharm Sci,1998,87:1305 - 1307.
[23]GESSNER A,OLBRICH C,SCHRODER W,et al.The role of plasma proteins in brain targeting:species dependent protein adsorption patterns on brain-specific lipid drug conjugate(LDC)nanoparticles[J].Int J Pharm,2001,214(122):87-91.
[24]DEHOUCK B,FENART L,DEHOUCK MP,et al.A new function for the LDL receptor:transcytosis of LDL across the blood-brain barrier[J].J Cell Biol,1997,138(4):877-889.
[25]NERURKAR MM,BURTO PS,BORCHARD RT.The use of surfactants to enhance the permeability of peptides through Caco2-cells by inhibition of an apically polarized efflux system[J].Pharm Res,1996,13(4):528-534.
[26]Lockman PR,Mumper RJ,Khan MA,et al.Nanoparticle technology for drug delivery across the blood2brain barrier.Drug Dev Ind Pharm,2002,28:1-13.
[27]王杰,张强.长循环纳米粒[J].国外医药·药学分册,1999,26(12):350-354.
[28]Yang SC,Lu L F,Cai Y,et al.Body distribution in mice of intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain[J].J Cont rol Rel,1999,59(3):299 -307.
[29] Calvo P , Gouritin B , Villarroya H , et al . Quantification and localization of PEGylated polycyanoacrylate nanoparticles in brain and spinal cord during experimental allergic encephalomyelitis in the rat. Eur J Neurosci, 2002,15:1317-1326.
[30] YANG SC , LU L F , CAI Y, et al . Body distribution in mice of intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain[J]. J Control Release , 1999, 59(3):299-307.
[31] YANG SC , ZHU JB , LU Y, et al . Body distribution of camptothecin solid lipid nanoparticles after oral administration [J] .Pharm Res, 1999,16 (5) :751-757.
[32] Calvo P, Gouritin B , Brigger I, et al. PEGylated polycyanoacrylate nanoparticles as vector for drug delivery in prion diseases. J Neurosci Methods, 2001, 111 :151 -155.
[33] Gelperina SE , Smirnova ZS , Khalanskiy AS , et al. Chemotherapy of brain tumours using doxorubiein bound to polysorbate 802coated nanoparticles. Proceedings of the 3rd World Meeting APV/ APGI, Berlin, 2000.441 - 442.
[34] Wang JX, Sun X, Zhang ZR. Enhanced brain targeting by synthesis of 3',5'-dioctanoyl-5-flu ro-2'-deoxyuridine and incorporation into solid lipid nanoparticles. Eur J Pharm Biopharm, 2002 , 54 :285 - 290.
[35] A. E. Gulyaev , S. E. Gelperina , I. N. Skidan , A. S. Antropov , G. Y. Kivama ,J . Kreuter. Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticle [J]. Pharm Res ,1999,16 :1564-1569
[36] S. E. Gelperina, Z. S. Smirnova ,A. S. Khalanskiy, I. N. Skidan ,A. I. Bobruskin ,J . Kreuter. Chemotherapy of brain tumours using doxorubicin bound to polysorbate 802coated nanopartiles. Proceedings of the 3rd World Meeting APV/ APGI .Berlin ,2000 ,3/ 6th April 2000 ,441-442.
[37] L I YP , PEI YY, ZHANG XY, et al. PEGylated PL GA nanoparticles as protein carriers : synthesis , preparation and biodistribution in rats[J] . J Controlled Release , 2001 ,71 (2):203-211.
[38] SCHRODER U , SOMMERFELD P , SABEL BA. Efficacy of oral dalargin-loaded nanoparticle delivery across the blood-brain barrier[J]. Peptides, 1998,19 (4) :777-780.
[39] KREUTER J , PETROV VE, KHARKEVICH DA , et al. Influence of the type of surfactant on the analgesic effects induced by the peptide dalargin after its delivery across the blood2br ain barrier using surfactant2coated nanoparticles [J] . J Controlled Release , 1997 ,49 (1) :81-87.
[40]CALVO P,GOURITIN B,BRIGGER I,et al.PEGylated polycyanoaerylate nanoparticles as vector for drug delivery in prion diseases[J].J Neurosci Methods,2001,111(2):151-155.
[41]Muller RH,Mader K,Gohla S.Solid lipid nanopartieles(SLN)for controlled drug delivery -a review of the state of the art.Eur J Pharm Biopharm,2000,50:161-177.
[2]邓孔昭、黄如训编著.血脑屏障.北京.人民卫生出版社.1994.20-21
[3]Sobue K,Yamamoto N,Yoneda K,et al.Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.Neurosci Res.1999,35:155-164.
[4]Janigro D,Leaman SM,Stanness KA.Dynamic in vitro modeling of the blood-brain barrier:A novel tool for studies of drug delivery to the brain.Pharm Sci Technol Today.1999,2:7-12.
[5]Muruganandam A,Herx LM,Monete R,et al.Development of immortalized cerebromicrovascular endothelial cell line as an in vitro model of the human blood brain barrier.FASEB J.1997,11:1187-1197.
[6]Gaillard PJ,Voorwinden LH,Nielsen JL,et al.Establishment and functional characterization of an in vitro model of the blood-brain barrier,comprising a co-culture of brain capillary endothelial cells and astrocytes.Eur J Pharm Sci.2001,12:215-222.
[7]Kido Y,Tamai I,Okamoto M,Suzuki F,et al.Functional clarification of MCTI-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies.Pharm Res.2000,17:55-62.
[8]Duport S,Robert E Muller D,et al.An in vitro blood-brain barrier model:cocultures between endothelial cells and organotypic brain slice culture.Proc Natl Acad Sci.1998;95:1840-1855.
[9]Rubin L,Hall DE,Porter S,et al.A cell culture model of the blood-brain barrier.
[10]J Cell Biology.1991;115(6):1725-1735.
[11]Reinhardt CA,Gloor SM.Co-culture blood-brain barrier models and their use for pharmatoxicological screening[J].Toxicol In Vitro,1997,11(5):513-517.
[12]王建民,施永德,步燕芳,等.大鼠脑血管内皮细胞的分离培养与形态学观察[J].解剖学杂志,1998,21(6):495-499.
[13]Brendel K,Meezan E,and Carlson EC.Isolated brain microvessels:a purified,metabolically active preparation from bovine cerebral cortex[J].Science,1974,185(4155):953-935.
[14]Duan CG,Xiu RJ.Cultivation of endothdial cells from rat brain microvessels[J].Chinese Med J,1988,101(2):649-652.
[15]张兆旺 主编.中药药剂学(川.北京:中国中医药出版社,2002,507-511
[16]贺石林 主编.中医科研设计与统计方法.长沙:湖南科学技术出版社,1989 328-332
[17]刘明芝主编.中医药统计学[M].长沙:湖南科学技术出版社,1999,231-243,287-312
[18]Zobel HP.Cationic polyhexyleyanoacrlate nanoparticles as carders for antisense oligucl otides[J].Antisense Nucleic Acid Drug Dev,1997,7(1):483-489
[19]Fattal E,Vauthier C,Aynie I,et al.Biodegradable polyalkycyanoaerylate nanopartieles for the delivery of oligonucleotides[J].Journal of Controlled Release,1998,53(2):137-143.
[20]Godard G..Antisense effects of cholesteroioligodeoxynucleotide conjugates associated with poly(alkyleyanoaerylate)nanopartieles[J].Eur J Biochem,1995,232(2):404-410.
[21]Lambert G,Fattal E,Pinto-Alphandary H,et al.Polyisobutyleyanoacrylate nanocapsules containing an aqueous core for the delivery of oligonucleotides[J].Int J Pharm,2001,214(1-2):13-16.
[22]Zimmer A.Antisense oligonucleotide delivery with polyhexylcyanoarylate nanop articles as carriers[J].Methods,1999,18(2):286-295.
[23]Calvo P,Gouritin B,Villarroya H,et al.Quantification and Locolization of PEGylated polyalkyleyanoacrylate nanoparticles in brain and spinal cord during experimental allergic encephalomyelitis in the rat[J].Eur J Neurosci,2002,15(8):1317-1326.
[24]张志荣.聚氰基丙烯酸酯毫微球的研究进展[J].中国药学杂志,1998,29(6):323-326.
[25]Regina Z.Comparative in vitro biocompatibility testing of polycyanoacrylates and poly(D,L-laetide-eo-glycolide)using different mouse fibroblast(L929)biocompatibility test models[J].Eur J Pharm Biopharm,1997,44(1):149-157.
[26]Karate B,Couvreur P,Dubois-Krack,et al.Toxicity of polyalkylcyanoacrylate nanoparticles:free nanoparticles[J].J Pharm Sic,1982,71(7):786-790.
[27]Gipps EM,Groscurth P,Kreuter J,et al.Distribution of polyhexylcyanoacrylate nanoparticles in nude mice over extended times and after repeated injection[J].J Pharm Sci,1988,77(3):208-209.
[28]陆彬主编,药物新剂型新技术[M].人民卫生出版社,1998,p211-213.
[29]廖工铁主编,药物靶向制剂[M].四川科学技术出版社,1997,p192-423.
[30] Sommerfeld P, Schroeder U, Sabel B. Long-term stability of PBCA nanoparticles suspensions suggests clinical usefulness[J]. International Journal of Pharmaceutics, 1997,155(2):201-207.
[31] Kreuter J, Alyautdin RN, Kharkevich DA, et al. Passage of peptides through theblood-brain barrier with colloidal polymer particles (nanoparticles) [J]. Brain Res, 1995,647 (1):171-174.
[32] Ramge P, Unger RE, Oltrogge JB, et al. Polysorbate 80-coating enhances uptake of polybutyleyanoacrylate(PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells[J].Eur J Neurosci,2000,12(6): 1931 -1940.
[33] Gulyaev AE, Gelperina SE, Skind IN, et al. Significant transport of doxorubicin into the brain with polysorbate-80-coated nanoparticles[J]. Pharm Res,1999,16(10):1564-1569.
[34] Kreuter J, Petroy VE, Kharkevich DA,et al. Influence of the type of surfactant on the analgesic effects induced by the peptide dalarin after its delivery across the blood-brain barrier using surfactant-coated nanoparticles[J]. J Controlled Release, 1997,49(1):81-87.
[35] Sun W, Me CS, Wang HF, et al. Specific role polysorbate 80 coating on the targeting of nanoparticles to the brain[J]. Biomaterials,2004,25(6):3065-3071.
[36] Pilar C, Bruno G, Helene C et al. Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery[J]. Pharm Res,2001,18(8): 1157-1166.
[37] Yang SC, Lu LF, Cai Y, et al. Body distribution in mice of intravenously injected camptot hecin solid lipid nanoparticles and targeting effect on brain[J]. J Control Release, 1999,59 (3):299-307.
[38] Kreuter J. Nanoparticulate systems for brain delivery of drugs[J]. Adv Drug Deliv Rev, 2001,47 (1):65-81.
[39] Calvo P, Gouritin B, Villarroya H, et al. Quantification and localization of PEGylated polycyanoacrylate nanoparticles in brain and spinal cord during experimental allergic encephalomyelitis in the rat[J]. Eur J Neurosci,2002,15(2): 1317-1326.
[40] Renad N, Reichel A, Ramge P, et al. Interaction of Poly(butylcyanoacrylate) nanoparticles with the Blood-Brain barrier in vivo and vitro[J]. Journal of Drug Targeting,2001,9(3):209- 221.
[41] Qaddoumi, MG, Ueda H, Yang J, et al. Uptake of biodegradable nanoparticles in pigmented rabbit conjunctival epithelial cells[J]. Invest Ophthalmol Vis Sci 2000,41(2):765-781.
[1]王建民,施永德,步燕芳,等.大鼠脑微血管内皮细胞的分离培养与形态学观察.[J].解剖学杂志.1998;21(6):495-499.
[2]Franke H,Galla H-J,Beuckmann CT.An improved low permeability in vitro model of the blood-brain barrier:Transport studies on retinods,sucrose,haloperidol,cafeine and mannitol.Brain Res.1999,818:65-71.
[3]Fenke H,Galla H J,Beuckmann CT.Primary cultures of brain micovessel endothelial cells:a valid and flexible model to study drug transport through the blood-brain barrier in vitro[J].Brain Res Prot,2000,5(2):248-256.
[4]罗玉敏.内皮细胞与脑缺血[J].国外医学脑血管疾病分册,1998,6(5):271-273.
[5]Hess DC,Howard E,Cheng C,et al.Hypertonic mannitol loading of NF-κB transcription factor decoys in human brain microvascular endothelial cells blocks upregulation of ICAM-1[J].Stroke,2000,31(5):1179-1186.
[6]Nobuhiro I,Kohiji N,Hidenari H,et al.Isolation and primary culture of rat cerebral for studying drug transport in vitro[J].JPM,1996,36(3):45-56.
[7]Fern J,Barkalow k,Michael J.Rapid communication culture murine cerebral microvaseular endothelial cells contain yon willebrand factor-positive weibel-palade bodies and support rapid eytokine-indueed neutrophil adhesion[J].Microcireulation,1996,3(1):19-30.
[8]Pham YT,Regina A,Farinoti R,et al.Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalized rat brain capillary endothelial cell line,GPNT[J].Biochim Biophys Acta,2000,1524(2-3):212-219.
[9]Kido Y,Tamai I,Okamoto M,et al.Functional clarification of MCTI-mediated transport of monocarboxylic acids at the blood-brain barrier using in vitro cultured cells and in vivo BUI studies.Pharm Res,2000,17(1):55-62.
[10]Reinhardt CA,Gloor SM.Co-culture blood-brain barrier models and their use for pharmatoxicological screening[J].Toxicol In Vitro,1997,11(5):513-517.
[11]王建民,施永德,步燕芳,等.大鼠脑血管内皮细胞的分离培养与形态学观察[J].解剖学杂志,1998,21(6):495-499.
[1] Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses. Nat Rev Drug Discov, 2002,1 (2): 131-139
[2] BEGL EY DJ . The blood-brain barrier : principles for targeting peptides and drugs to the central nervous system[J]. J Pharm Pharmacol, 1996,48 (2) :136-146.
[3] Miller G. Drug targeting. Breaking down barriers. Science , 2002 , 297 : 1116— 1118.
[4] Kreuter J . Nanoparticulate systems for brain delivery of drugs [J] .Adv Drug Deliv Rev , 2001,47 (1) :65-81.
[5] Kumaresh S , Soppimath , et al . Biodegradable polymeric nanoparticles as drug delivery devices[J]. J Control Rel ,2000,70,120
[6] M.L. Hans A. M. Lowman .Biodegradable nanoparticles for drug delivery and targeting[J]. Current Opinion in Solid State and Materials Science, 2002,6 :319-327.
[7] Plank C , Tang MX, Wolfe AR, et al. Branched cationic peptides for gene therapy : role of type and number of cationic residues in formation and in vitro activity of DNA polyplexes. Hum Gene Ther, 1999,10 :319-332.
[8] Shi N, Pardridge WM. Non2invasive gene targeting to the brain. Proc Natl Acad Sci USA , 2000, 97 :7567-7572.
[9] Zhang Y, Pardridge WM. Global nonviral gene transfer to the primate brain following intravenous administration. Mol Ther, 2003 ,7(1) :11—18.
[10] Lockman RJ , Mumper MA , Khan , et al. Nanoparticle technology for drug delivery across the blood2brain barrier. Drug Dev Ind Pharm, 2002, 28 (1): 1—13.
[11] Alyaudtin RN , Gother D, Petrov V. Analgesic activity of the hexapeptide dalargin absorbed on the surface of polysorbate-80 coated polybutylcyanoacrylate nanoparticles. Euro J Pharm Biopharm, 1995 ,41 :44-48.
[12] Fenart L , Casanova A , Dehouck B , et al. Evaluation of effect of charge and lipid coating on ability of 602nm nanoparticles to cross an in vitro model of the blood-brain barrier. J Pharmacol Exp Ther, 1999,291 :1017-1022.
[13] Schroeder U , Sabel BA , Schroeder H. Diffusion enhancement of drugs by loaded nanoparticles in vitro. Prog Neuropsychopharmacol Biol Psychiatry, 1999, 23 : 941 - 949.
[14] Schroeder U , Sommerfeld P, Ulrich S , et al. Nanoparticle technology for delivery of drugs across the blood2brain barrier. J Pharm Sci, 1998 , 87 :1305 - 1307.
[15] Alyautdin RN , Tezikov EB , Ramge P , et al . Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 802coated polybutylcyanoacrylate nanoparticles : an in situ brain perfusion study. J Microencapsul, 1998 ,15 : 67 - 74.
[16]Sun W,Xie C,Wang H,et al.Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain.Biomaterials,2004,25:3065-3071.
[17]Kreuter J,Shamenkov D,Petrov V,et al.Apolipoprotein-mediated transport of nanoparticle-bound drags across the blood-brain barrier.J Drug Target,2002,10:317-325.
[18]KREUTER J,AL YAUTDIN RN,KHARKEVICH DA,et al.Passage of peptides through the blood2brain barrier with colloidal polymer particles(nanoparticles)[J].Brain Res,1995,647(1):171-174.
[19]BORCHARD G,AUDUS KL,SHI FL,et al.Uptake of surfactant-coated poly(methyl methacrylate)nanoparticles by bovine brain microvessel endothelial monolayers[J].Int J Pharm,1994,110(1):29-35.
[20]AL YAUTDIN RN,RRICHEL A,LOBENBERG R,et al.Interaction of poly(butylcya noacrylate)nanoparticles with the blood-brain barrier i n vivo and i n vitro[J].J Drug Target,2001,9(3):209-221.
[21]CALVO P,GOURITIN B,BRIGGER I,et al.PEGylated poly-cyanoacrylate nanoparticles as vector for drug delivery in prion diseases[J].J Neurosci Methods,2001,111(2):151-155.
[22]Schroeder U,Sommeffeld P,Ulrich S,et al.Nanoparticle technology for delivery of drugs across the blood-brain barrier.J Pharm Sci,1998,87:1305 - 1307.
[23]GESSNER A,OLBRICH C,SCHRODER W,et al.The role of plasma proteins in brain targeting:species dependent protein adsorption patterns on brain-specific lipid drug conjugate(LDC)nanoparticles[J].Int J Pharm,2001,214(122):87-91.
[24]DEHOUCK B,FENART L,DEHOUCK MP,et al.A new function for the LDL receptor:transcytosis of LDL across the blood-brain barrier[J].J Cell Biol,1997,138(4):877-889.
[25]NERURKAR MM,BURTO PS,BORCHARD RT.The use of surfactants to enhance the permeability of peptides through Caco2-cells by inhibition of an apically polarized efflux system[J].Pharm Res,1996,13(4):528-534.
[26]Lockman PR,Mumper RJ,Khan MA,et al.Nanoparticle technology for drug delivery across the blood2brain barrier.Drug Dev Ind Pharm,2002,28:1-13.
[27]王杰,张强.长循环纳米粒[J].国外医药·药学分册,1999,26(12):350-354.
[28]Yang SC,Lu L F,Cai Y,et al.Body distribution in mice of intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain[J].J Cont rol Rel,1999,59(3):299 -307.
[29] Calvo P , Gouritin B , Villarroya H , et al . Quantification and localization of PEGylated polycyanoacrylate nanoparticles in brain and spinal cord during experimental allergic encephalomyelitis in the rat. Eur J Neurosci, 2002,15:1317-1326.
[30] YANG SC , LU L F , CAI Y, et al . Body distribution in mice of intravenously injected camptothecin solid lipid nanoparticles and targeting effect on brain[J]. J Control Release , 1999, 59(3):299-307.
[31] YANG SC , ZHU JB , LU Y, et al . Body distribution of camptothecin solid lipid nanoparticles after oral administration [J] .Pharm Res, 1999,16 (5) :751-757.
[32] Calvo P, Gouritin B , Brigger I, et al. PEGylated polycyanoacrylate nanoparticles as vector for drug delivery in prion diseases. J Neurosci Methods, 2001, 111 :151 -155.
[33] Gelperina SE , Smirnova ZS , Khalanskiy AS , et al. Chemotherapy of brain tumours using doxorubiein bound to polysorbate 802coated nanoparticles. Proceedings of the 3rd World Meeting APV/ APGI, Berlin, 2000.441 - 442.
[34] Wang JX, Sun X, Zhang ZR. Enhanced brain targeting by synthesis of 3',5'-dioctanoyl-5-flu ro-2'-deoxyuridine and incorporation into solid lipid nanoparticles. Eur J Pharm Biopharm, 2002 , 54 :285 - 290.
[35] A. E. Gulyaev , S. E. Gelperina , I. N. Skidan , A. S. Antropov , G. Y. Kivama ,J . Kreuter. Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticle [J]. Pharm Res ,1999,16 :1564-1569
[36] S. E. Gelperina, Z. S. Smirnova ,A. S. Khalanskiy, I. N. Skidan ,A. I. Bobruskin ,J . Kreuter. Chemotherapy of brain tumours using doxorubicin bound to polysorbate 802coated nanopartiles. Proceedings of the 3rd World Meeting APV/ APGI .Berlin ,2000 ,3/ 6th April 2000 ,441-442.
[37] L I YP , PEI YY, ZHANG XY, et al. PEGylated PL GA nanoparticles as protein carriers : synthesis , preparation and biodistribution in rats[J] . J Controlled Release , 2001 ,71 (2):203-211.
[38] SCHRODER U , SOMMERFELD P , SABEL BA. Efficacy of oral dalargin-loaded nanoparticle delivery across the blood-brain barrier[J]. Peptides, 1998,19 (4) :777-780.
[39] KREUTER J , PETROV VE, KHARKEVICH DA , et al. Influence of the type of surfactant on the analgesic effects induced by the peptide dalargin after its delivery across the blood2br ain barrier using surfactant2coated nanoparticles [J] . J Controlled Release , 1997 ,49 (1) :81-87.
[40]CALVO P,GOURITIN B,BRIGGER I,et al.PEGylated polycyanoaerylate nanoparticles as vector for drug delivery in prion diseases[J].J Neurosci Methods,2001,111(2):151-155.
[41]Muller RH,Mader K,Gohla S.Solid lipid nanopartieles(SLN)for controlled drug delivery -a review of the state of the art.Eur J Pharm Biopharm,2000,50:161-177.