p53DNA-阿霉素—聚氰基丙烯酸正丁酯纳米粒的制备和药物含量的研究
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摘要
目的：制备阿霉素-聚氰基丙烯酸正丁酯纳米粒；研究影响其阿霉素药物含量的因素水平；以粒径、粒形，药物含量为指标，优化制备工艺；制备药物含量较高，同时包封阿霉素(DOX)和质粒p53的纳米粒，为靶向治疗和基因治疗联合化学治疗奠定基础。
方法：应用一步法制备纳米粒，初选影响阿霉素-聚氰基丙烯酸正丁酯纳米粒的药物含量的因素，在此基础上采用均匀设计，多因素分析各因素水平的相互作用，优化阿霉素-聚氰基丙烯酸正丁酯纳米粒制备工艺。通过改变体系的Zeta电位和加入离子对试剂，进一步提高纳米粒的DOX药物含量，同时采用二步法制备p53DNA-阿霉素-聚氰基丙烯酸正丁酯纳米粒。分别以紫外分光光度法和荧光分光光度法测定阿霉素和p53DNA的包封率，计算载药量。
结果：
1．影响阿霉素-聚氰基丙烯酸正丁酯纳米粒药物含量的因素有：阿霉素用量、BCA单体用量、体系PH值、表面活性剂。
2．建立了具有良好预报作用的多元线性回归方程Y=19.456+0.113X_1-1.66X_2-6.121X_3-0.839X_4，F=0.888，R=0.686，R~2=0.57，P=0.544。优化工艺后制备了粒径粒形好，阿霉素药物含量高的聚氰基丙烯酸正丁酯纳米粒。优化的处方为DOX 10.0 mg，BCA 0.25ml，
PH二2 .5，1 .5%Dextran7。。
3.加入电解质改变体系的Ze七a电位，硫酸钠组与对照组相比包
封率和载药量增加(P<0.05)，而NaCI，NaZCO。的影响不大(P>.05)。
加入NaZSO。的4.0，8.0，12.Omg/ml三个剂量水平组包封率和载药量
均较未加NaSO性组增高(P<0.05)，Na25O，浓度为12.Omg/ml时，阿霉
素药物含量进一步提高到ER=84.26十产。.27，LD= 3.57+/一0.01
(P<0.05)。但当NaZSO;达16.Omg/ml时药物含量反而较12mg/m1组
降低(P<0.05)。对应的Zeta电位亦有相同改变(P<0.05)。Zeta电
位的改变与阿霉素一聚氰基丙烯酸正丁酚纳米粒的包封率和载药量呈
负相关(r护0.9671，坑。刁.9125，P<。.05)。加入离子对试剂后阿霉素
药物含量亦进一步提高(P<0.05)。
4.制备出了P53邓可霉素一聚氰基丙烯酸正丁酷纳米粒。加入季胺
盐CTAB后，p53DNA的包封率明显提高(P<0.05)，而阿霉素的包封
率变化不大(P>?05)，分别为Dox:82.17+/一0.22，p53
49.67+/一1.26。
结论:阿霉素用量、Q一氰基丙烯酸正丁‘酉旨单体用量、介质的PH
值、表面活性剂的种类和量是影响聚氰基丙烯酸正丁醋纳米粒药物含
量的重要因素，搅拌速度没有影响。经优化工艺制备的阿霉素一聚氰
基丙烯酸正丁醋纳米粒粒径、粒形好，药物含量高。优化的阿霉素-
聚氰基丙烯酸正丁酷纳米粒制备工艺为其进一步研究奠定了基础。包
封p53DNA和阿霉素于同一载体是可以行的。改变纳米粒的Ze七a电位
和加入离子对试剂均可以有效提高阿霉素一聚氰基丙烯酸正丁酷纳米
粒的药物含量。电解质NaSO4对阿霉素一聚氰基丙烯酸正丁酒旨纳米粒
的药物含量的提高与纳米粒的Zeta电位改变密切相关。p53DNA一阿霉
素一聚氰基丙烯酸正丁酷纳米粒的制备成功为靶向治疗和基因治疗与
化学治疗的联合应用提供了一条新思路。
Objective: To investigation the determinate factors and levels on procedures of Doxorubicin-polybutylcyanoacrylate nanoparticles (DOX-PBCA-NP) and to optimize preparation procedures and then product the Dox-PBCA-NP entrapped Doxorubicin and plasmid p53,in order to reveal its' possibility in combining gene therapy with targeting therapy.
Methods: The influential factors were determined preliminarily by one-step methods. On the base of this attempt, uniform design allowed to find the interaction among all possible factors and its' levels. The Zeta potential of suspension systems was altered and ion-pair reagents were added so as to elevate drug content of nanoparticles. By two-step method, p53DNA-Doxorubcin-polybutylcyanoacrylate nanoparticles was prepared.
Results:
1. The influential factors of drug content of PBCA-NP included: quantity of DOX in initial system; added volume of alpha-BCA monomer; PH value of media; class and volume of surfacant.
2. Multiple factors linear regression equation was established: Y =
19.456+0.113X1-1.66X2-6.121X3-0.839X4, F=0.888, R=0.686, R2=0.57, P=0.544, and high content of Doxorubicin of PBCA-NP with fine particle size and shape were producted successfully followed the optimized procedure.The optimized recipe for Doxorubicin-polybutylcyanoacrylate nanoparticles is as follow: DOX lO.Omg, BCA 0.25ml, PH=2.5, 1.5%Dextran70.
3. The drug content of PBCA-NP was enhanced by addition of iron-pare reagents and increasing Zeta potential of colloid systems. Compared with controlled groups, enhancement of entrapment rate and drug loading capacity were obtained in group with additional Na2SO4 (P<0.05 ) ,but the similar results couldn't be found in NaCl and Na2CO3 groups (P>0.05 ) .Entrapment rate and drug loading capacity of Doxorubicin in 4.0, 8.0, 12.0mg/ml Na2SO4 groups were higher than 0 mg/ml one (P<0.05) ,the biggest of which was ER=84.26+/- 0.27 ,LD =3.57+7-0.01 when system concentration of Na2SO4 was 12.0mg/ml. while system concentration of Na2SO4 increased to 16.0mg/ml, the drug content declined compaired with 12.0mg/ml group (P<0.05 ) . In the same time ,Zeta potential of colloid systems followed this trend (P<0.05) and its' strength are negatively related to the scale of drug content of DOX-PBCA-NP (rER=0.9671, rLD=0.9125, P<0.05) . The drug content of PBCA-NP was intensified by additional ion-pair reagents too (P<0.05). 4.High drug content nanoparticles, p53DNA-DOX-PBCA-NP was
prepared successfully. ER(%) of p53DNA rised markedly ( ER-49.67+7-1.26%, P<0.05) . but there was no difference for Doxorubicin (ER= 82.17+/ -0.22%,P>0.05 ) .
Conclusion: The quantity of Doxorubicin and butylcyanoacrylate polymer, PH value in medium, the varies and volume of the surfactants were key factors on drug content of PBCA-NP. The influence of stirring speed is negligible.Taken the optimized recipe, DOX-PBCA-Np with fine particle size and morphology is available and it's drug content is intensified too. The optimized technique of preparation of nanoparticles entrapped Doxorubicin will facilitate the further study. Both change of Zeta potential and employment of ion-pair reagents can elevate the drug content of NPs. the higher drug content of DOX-PBCA-NP resulted from electrolyte Na2SO4 takes on negative correlation with it's alternation of Zeta potential. The presence of p53- DOX-PBCA-NP provides a novel method for studying combination of gene therapy with chemical therapy.
方法：应用一步法制备纳米粒，初选影响阿霉素-聚氰基丙烯酸正丁酯纳米粒的药物含量的因素，在此基础上采用均匀设计，多因素分析各因素水平的相互作用，优化阿霉素-聚氰基丙烯酸正丁酯纳米粒制备工艺。通过改变体系的Zeta电位和加入离子对试剂，进一步提高纳米粒的DOX药物含量，同时采用二步法制备p53DNA-阿霉素-聚氰基丙烯酸正丁酯纳米粒。分别以紫外分光光度法和荧光分光光度法测定阿霉素和p53DNA的包封率，计算载药量。
结果：
1．影响阿霉素-聚氰基丙烯酸正丁酯纳米粒药物含量的因素有：阿霉素用量、BCA单体用量、体系PH值、表面活性剂。
2．建立了具有良好预报作用的多元线性回归方程Y=19.456+0.113X_1-1.66X_2-6.121X_3-0.839X_4，F=0.888，R=0.686，R~2=0.57，P=0.544。优化工艺后制备了粒径粒形好，阿霉素药物含量高的聚氰基丙烯酸正丁酯纳米粒。优化的处方为DOX 10.0 mg，BCA 0.25ml，
PH二2 .5，1 .5%Dextran7。。
3.加入电解质改变体系的Ze七a电位，硫酸钠组与对照组相比包
封率和载药量增加(P<0.05)，而NaCI，NaZCO。的影响不大(P>.05)。
加入NaZSO。的4.0，8.0，12.Omg/ml三个剂量水平组包封率和载药量
均较未加NaSO性组增高(P<0.05)，Na25O，浓度为12.Omg/ml时，阿霉
素药物含量进一步提高到ER=84.26十产。.27，LD= 3.57+/一0.01
(P<0.05)。但当NaZSO;达16.Omg/ml时药物含量反而较12mg/m1组
降低(P<0.05)。对应的Zeta电位亦有相同改变(P<0.05)。Zeta电
位的改变与阿霉素一聚氰基丙烯酸正丁酚纳米粒的包封率和载药量呈
负相关(r护0.9671，坑。刁.9125，P<。.05)。加入离子对试剂后阿霉素
药物含量亦进一步提高(P<0.05)。
4.制备出了P53邓可霉素一聚氰基丙烯酸正丁酷纳米粒。加入季胺
盐CTAB后，p53DNA的包封率明显提高(P<0.05)，而阿霉素的包封
率变化不大(P>?05)，分别为Dox:82.17+/一0.22，p53
49.67+/一1.26。
结论:阿霉素用量、Q一氰基丙烯酸正丁‘酉旨单体用量、介质的PH
值、表面活性剂的种类和量是影响聚氰基丙烯酸正丁醋纳米粒药物含
量的重要因素，搅拌速度没有影响。经优化工艺制备的阿霉素一聚氰
基丙烯酸正丁醋纳米粒粒径、粒形好，药物含量高。优化的阿霉素-
聚氰基丙烯酸正丁酷纳米粒制备工艺为其进一步研究奠定了基础。包
封p53DNA和阿霉素于同一载体是可以行的。改变纳米粒的Ze七a电位
和加入离子对试剂均可以有效提高阿霉素一聚氰基丙烯酸正丁酷纳米
粒的药物含量。电解质NaSO4对阿霉素一聚氰基丙烯酸正丁酒旨纳米粒
的药物含量的提高与纳米粒的Zeta电位改变密切相关。p53DNA一阿霉
素一聚氰基丙烯酸正丁酷纳米粒的制备成功为靶向治疗和基因治疗与
化学治疗的联合应用提供了一条新思路。
Objective: To investigation the determinate factors and levels on procedures of Doxorubicin-polybutylcyanoacrylate nanoparticles (DOX-PBCA-NP) and to optimize preparation procedures and then product the Dox-PBCA-NP entrapped Doxorubicin and plasmid p53,in order to reveal its' possibility in combining gene therapy with targeting therapy.
Methods: The influential factors were determined preliminarily by one-step methods. On the base of this attempt, uniform design allowed to find the interaction among all possible factors and its' levels. The Zeta potential of suspension systems was altered and ion-pair reagents were added so as to elevate drug content of nanoparticles. By two-step method, p53DNA-Doxorubcin-polybutylcyanoacrylate nanoparticles was prepared.
Results:
1. The influential factors of drug content of PBCA-NP included: quantity of DOX in initial system; added volume of alpha-BCA monomer; PH value of media; class and volume of surfacant.
2. Multiple factors linear regression equation was established: Y =
19.456+0.113X1-1.66X2-6.121X3-0.839X4, F=0.888, R=0.686, R2=0.57, P=0.544, and high content of Doxorubicin of PBCA-NP with fine particle size and shape were producted successfully followed the optimized procedure.The optimized recipe for Doxorubicin-polybutylcyanoacrylate nanoparticles is as follow: DOX lO.Omg, BCA 0.25ml, PH=2.5, 1.5%Dextran70.
3. The drug content of PBCA-NP was enhanced by addition of iron-pare reagents and increasing Zeta potential of colloid systems. Compared with controlled groups, enhancement of entrapment rate and drug loading capacity were obtained in group with additional Na2SO4 (P<0.05 ) ,but the similar results couldn't be found in NaCl and Na2CO3 groups (P>0.05 ) .Entrapment rate and drug loading capacity of Doxorubicin in 4.0, 8.0, 12.0mg/ml Na2SO4 groups were higher than 0 mg/ml one (P<0.05) ,the biggest of which was ER=84.26+/- 0.27 ,LD =3.57+7-0.01 when system concentration of Na2SO4 was 12.0mg/ml. while system concentration of Na2SO4 increased to 16.0mg/ml, the drug content declined compaired with 12.0mg/ml group (P<0.05 ) . In the same time ,Zeta potential of colloid systems followed this trend (P<0.05) and its' strength are negatively related to the scale of drug content of DOX-PBCA-NP (rER=0.9671, rLD=0.9125, P<0.05) . The drug content of PBCA-NP was intensified by additional ion-pair reagents too (P<0.05). 4.High drug content nanoparticles, p53DNA-DOX-PBCA-NP was
prepared successfully. ER(%) of p53DNA rised markedly ( ER-49.67+7-1.26%, P<0.05) . but there was no difference for Doxorubicin (ER= 82.17+/ -0.22%,P>0.05 ) .
Conclusion: The quantity of Doxorubicin and butylcyanoacrylate polymer, PH value in medium, the varies and volume of the surfactants were key factors on drug content of PBCA-NP. The influence of stirring speed is negligible.Taken the optimized recipe, DOX-PBCA-Np with fine particle size and morphology is available and it's drug content is intensified too. The optimized technique of preparation of nanoparticles entrapped Doxorubicin will facilitate the further study. Both change of Zeta potential and employment of ion-pair reagents can elevate the drug content of NPs. the higher drug content of DOX-PBCA-NP resulted from electrolyte Na2SO4 takes on negative correlation with it's alternation of Zeta potential. The presence of p53- DOX-PBCA-NP provides a novel method for studying combination of gene therapy with chemical therapy.
引文
[1] 吴孟超。原发性肝癌的诊断和治疗进展。中华外科杂志，1998：36(9)：515
[2] Somo CE, Dubemet C ,Bentolila D et al .Revertion of multidrug resistence by co-encapsuition of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles[J]. Biomaterals, 1999;20(14): 1269-1275
[3] Schwab G, Chanvany C, Duroux I, et al.Antisense oligonuleotides absorbed to polyalkylcyanoacrylate nanoparticles specificially inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice[J]. Pro Natl Acad Sci USA, 1994;91 (22): 10460-10464
[4] Fattle E,Vauthier C,Aynie I,et al.Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides[J]. J Control Release , 1998;53(1-3): 137-143
[5] Zimmer A.Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers[J].Methods,1999;18(3):286-295
[6] 张阳德，龚连生。磁性阿霉素白蛋白纳米粒的研制。中国现代医学杂志，2001；11(3)：1-3
[7] 张阳德，龚连生。阿霉素白蛋白磁性纳米粒在正常肝脏的靶向性。中国现代医学杂志，2001；11(3)：4-7
[8] 张阳德，毛先海，龚连生等。给药途径对磁性白蛋白纳米粒靶向分布的影响。中国现代医学杂志，2001；11(3)：11-13
[9] Lambert G,.Fattal E,Couvreur P, et al.nanoparticulate systems for the delivery of Antisense oligonucleotides[J].Adv Drag Deliv Rev,2001;47(1):99-112
[10] Cohen H, Levy RJ, Gao J, et al.Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles [J]. Gene Therapy,2000;7:1896-1905
[11] Labhasetwar V, et al.A DNA controlled-release coating for gene transfer:transfection in skeletal and cardiac mulscle[J]. J Pharm Sci, 1998;87:1347-1350
[12] Leong KW, et al.DNA-polycation nanospheres as non-vires gene delivery vehicles[J]. J Control Release, 1998;53:183-193
[13] De Verdiere AC, Dubemet C, Nemati F, et al. Revertion of muitidrug resistance with polyalkylcyanoacrylate nanoparticles:towards a mechanism of action [J]. British J Cancer, 1997;76(2):198-205.
[14] Gonzalez-Martin G, Isabel Merino M, Rodriguez-Cabezas N, et al. Characterization and trypanocidal activity of Nifurtimox-containing and empty nanoparticles of polyethylcyanoacrylate [J].J Pharm Pharmacol, 1998;50(1):29-35
[15] Li YP, Pei YY, Zhou ZH,et al. PEGylated nanoparticles as tumours necrosis factor-Alpha carriers [J]. J Control Release, 2001;71(3):287-296
[16] 方开泰．均匀设计-数论方法在试验设计的应用．应用数学学报，1980：3(4)：363-372．
[17] 卢圣栋主编．现代分子生物学实验技术(第二版)．北京：中国协和医科大学出版社，1999；394．
[18] F 奥斯伯，R布伦特，R E金斯等著．严子颖等译．精编分子生物学实验指南．北京：科学出版社，1998．
[19] 何勤，张志荣，刘戟等．荧光法测定载基因纳米粒的包封率．华西药学杂志，2002：17(1)：49-50
[20] Jia K Li,Nuo Wang,Xue S, et al.A novel biodegradable system based on gelatin nanoparticles and poly(lactide-co-glycolic acid)microspheres for protein and peptide drug[J]. J Pharm Sci, 1997;86(8):891-895
[21] Lin W, Garnett MC, Davis S, et al. Preparation and characterization of rose bengal-loaded surface modified albumin nanoparticles [J]. J Control Release, 2001;71(1):117-126
[22] Govender T, Stolnik S, Garnett MC,etal. PLGA nanoparticles prepared by nanoprecipitation drug loading and release studies of a water soluble drug [J]. J Control release, 1999;57:171-185
[23] Gasper MM, Blanco D, Cruz ME, et al. Formulation of L-asparaginase-loaded poly(lactide-co-glycolide) nanoparticles:influence of polymer properties on enzyme loading,activity and in vitro release [J]. J Control Release, 1998;51(1-2):53-62
[24] Ahlin P, Kristl J, Kristl A, et al. Investigation of polymeric nanoparticles as carriers of enalaprilat for oral administration [J]. Int J Pharm, 2002; 239(1-2):113-120
[25] Oyewumi MO, Mumper RJ.Gadolinlum-loaded nanoparticles engneered from microemulsion templates [J]. Drug Del Ind Pharm, 2002,28(3):317-328
[26] Elizabelth M Gipps, Oroscurth P, Keuter J, etal. Distribution of polyhexylcyanoacrylate nanoparticles in nude mice extended times and after repeated injection [J]. J Pharm Sci, 1998;77(3): 1208-1209
[27] Chavany C, Ledoan T, Couveur P, etal. Polyalkylcyanoacrylate nanoparticles as
polymeric carrers for antisense oligonuleotides [J]. Pharm Res, 1992;9:441-449
[28] Cavalli R,Caputo O, Gasco MR, et al. Solid lipospheres of doxorubicin and idarubicin[J]. Int J Pharm, 1993,89:R9-R12
[29] Pepin X, Attali L, Domrault C, etal. On the use of ion-pair chromatography to elucidate doxrubincin release mechanism from Polyalkylcyanoacrylate nanoparticles at celluar level [J]. J Chromatography B, 1997;702:181-191
[30] Pertkins WR, Ahmad I, Li X, et al. Novel therapeutic nanoparticles (lipocores): trapping poorly water soluble compounds[J]. Int J Pharm,2000;200:27-39
[31] Calvo P, Remunan-Lopez C, Vila-Jato JL, et al. Chitosan and Chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines [J]. Pharm Res, 1997; 14(10):1431-1436
[32] Jia K Li, Nuo Wang, Xue S, et al.A novel biodegradable system based on gelatin nanoparticles and poly(lactide-co-glycolic acid) microspheres for protein and peptide drug [J]. J Pharm Sci, 1997;86(8):891-895
[33] Berton M, Allemann E, Stein Cy A, etal.Highly loaded nanoparticle carrer using an hydrophobic antisense oligonuleotide complex [J].Euro J Pharm Sci, 1999,9:163-170
[34] Deprez-De Campeneere.Comparative study in mice of toxicity, pharmacology and therapeutic activity of daunorubicin-DNA and doxorubicin-DNA complexes [J]. Cancer Chemother Pharmacol, 1979; 1:25
[2] Somo CE, Dubemet C ,Bentolila D et al .Revertion of multidrug resistence by co-encapsuition of doxorubicin and cyclosporin A in polyalkylcyanoacrylate nanoparticles[J]. Biomaterals, 1999;20(14): 1269-1275
[3] Schwab G, Chanvany C, Duroux I, et al.Antisense oligonuleotides absorbed to polyalkylcyanoacrylate nanoparticles specificially inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice[J]. Pro Natl Acad Sci USA, 1994;91 (22): 10460-10464
[4] Fattle E,Vauthier C,Aynie I,et al.Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides[J]. J Control Release , 1998;53(1-3): 137-143
[5] Zimmer A.Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers[J].Methods,1999;18(3):286-295
[6] 张阳德，龚连生。磁性阿霉素白蛋白纳米粒的研制。中国现代医学杂志，2001；11(3)：1-3
[7] 张阳德，龚连生。阿霉素白蛋白磁性纳米粒在正常肝脏的靶向性。中国现代医学杂志，2001；11(3)：4-7
[8] 张阳德，毛先海，龚连生等。给药途径对磁性白蛋白纳米粒靶向分布的影响。中国现代医学杂志，2001；11(3)：11-13
[9] Lambert G,.Fattal E,Couvreur P, et al.nanoparticulate systems for the delivery of Antisense oligonucleotides[J].Adv Drag Deliv Rev,2001;47(1):99-112
[10] Cohen H, Levy RJ, Gao J, et al.Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles [J]. Gene Therapy,2000;7:1896-1905
[11] Labhasetwar V, et al.A DNA controlled-release coating for gene transfer:transfection in skeletal and cardiac mulscle[J]. J Pharm Sci, 1998;87:1347-1350
[12] Leong KW, et al.DNA-polycation nanospheres as non-vires gene delivery vehicles[J]. J Control Release, 1998;53:183-193
[13] De Verdiere AC, Dubemet C, Nemati F, et al. Revertion of muitidrug resistance with polyalkylcyanoacrylate nanoparticles:towards a mechanism of action [J]. British J Cancer, 1997;76(2):198-205.
[14] Gonzalez-Martin G, Isabel Merino M, Rodriguez-Cabezas N, et al. Characterization and trypanocidal activity of Nifurtimox-containing and empty nanoparticles of polyethylcyanoacrylate [J].J Pharm Pharmacol, 1998;50(1):29-35
[15] Li YP, Pei YY, Zhou ZH,et al. PEGylated nanoparticles as tumours necrosis factor-Alpha carriers [J]. J Control Release, 2001;71(3):287-296
[16] 方开泰．均匀设计-数论方法在试验设计的应用．应用数学学报，1980：3(4)：363-372．
[17] 卢圣栋主编．现代分子生物学实验技术(第二版)．北京：中国协和医科大学出版社，1999；394．
[18] F 奥斯伯，R布伦特，R E金斯等著．严子颖等译．精编分子生物学实验指南．北京：科学出版社，1998．
[19] 何勤，张志荣，刘戟等．荧光法测定载基因纳米粒的包封率．华西药学杂志，2002：17(1)：49-50
[20] Jia K Li,Nuo Wang,Xue S, et al.A novel biodegradable system based on gelatin nanoparticles and poly(lactide-co-glycolic acid)microspheres for protein and peptide drug[J]. J Pharm Sci, 1997;86(8):891-895
[21] Lin W, Garnett MC, Davis S, et al. Preparation and characterization of rose bengal-loaded surface modified albumin nanoparticles [J]. J Control Release, 2001;71(1):117-126
[22] Govender T, Stolnik S, Garnett MC,etal. PLGA nanoparticles prepared by nanoprecipitation drug loading and release studies of a water soluble drug [J]. J Control release, 1999;57:171-185
[23] Gasper MM, Blanco D, Cruz ME, et al. Formulation of L-asparaginase-loaded poly(lactide-co-glycolide) nanoparticles:influence of polymer properties on enzyme loading,activity and in vitro release [J]. J Control Release, 1998;51(1-2):53-62
[24] Ahlin P, Kristl J, Kristl A, et al. Investigation of polymeric nanoparticles as carriers of enalaprilat for oral administration [J]. Int J Pharm, 2002; 239(1-2):113-120
[25] Oyewumi MO, Mumper RJ.Gadolinlum-loaded nanoparticles engneered from microemulsion templates [J]. Drug Del Ind Pharm, 2002,28(3):317-328
[26] Elizabelth M Gipps, Oroscurth P, Keuter J, etal. Distribution of polyhexylcyanoacrylate nanoparticles in nude mice extended times and after repeated injection [J]. J Pharm Sci, 1998;77(3): 1208-1209
[27] Chavany C, Ledoan T, Couveur P, etal. Polyalkylcyanoacrylate nanoparticles as
polymeric carrers for antisense oligonuleotides [J]. Pharm Res, 1992;9:441-449
[28] Cavalli R,Caputo O, Gasco MR, et al. Solid lipospheres of doxorubicin and idarubicin[J]. Int J Pharm, 1993,89:R9-R12
[29] Pepin X, Attali L, Domrault C, etal. On the use of ion-pair chromatography to elucidate doxrubincin release mechanism from Polyalkylcyanoacrylate nanoparticles at celluar level [J]. J Chromatography B, 1997;702:181-191
[30] Pertkins WR, Ahmad I, Li X, et al. Novel therapeutic nanoparticles (lipocores): trapping poorly water soluble compounds[J]. Int J Pharm,2000;200:27-39
[31] Calvo P, Remunan-Lopez C, Vila-Jato JL, et al. Chitosan and Chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines [J]. Pharm Res, 1997; 14(10):1431-1436
[32] Jia K Li, Nuo Wang, Xue S, et al.A novel biodegradable system based on gelatin nanoparticles and poly(lactide-co-glycolic acid) microspheres for protein and peptide drug [J]. J Pharm Sci, 1997;86(8):891-895
[33] Berton M, Allemann E, Stein Cy A, etal.Highly loaded nanoparticle carrer using an hydrophobic antisense oligonuleotide complex [J].Euro J Pharm Sci, 1999,9:163-170
[34] Deprez-De Campeneere.Comparative study in mice of toxicity, pharmacology and therapeutic activity of daunorubicin-DNA and doxorubicin-DNA complexes [J]. Cancer Chemother Pharmacol, 1979; 1:25