载基因酸度敏感聚合物微球的研究
|
摘要
酸度敏感的药物载体可以在人体内的酸性环境释放药物,实现特异性的胃肠道、癌症、炎症等部位的给药。不仅如此,酸度敏感药物载体还可以实现内涵体逃逸,从而实现细胞内的药物传递。本论文旨在利用含缩醛基团的酸度敏感聚合物作为基因载体制备微球制剂,微球制剂在靶向基团的作用下到达细胞被有效摄取,进入细胞后在酸度敏感基团的作用下,破坏细胞内涵体膜进入细胞浆中,实现基因的细胞内释放,提高基因的转染效率,这为基因疫苗的发展提供了新的思路。
本文利用缩醛修饰聚乙二醇作为酸度敏感链段,与丙交酯共聚得到酸度敏感聚合物(PBELA),进一步通过Click反应接枝半乳糖基团,得到带半乳糖残基的酸度敏感聚合物(PGBELA),并以聚乳酸-聚乙二醇共聚物(PELA)为对照。本文以模型质粒(pDNA)与聚乙烯亚胺复合粒子为对象,利用复乳法制备了聚合物微球,通过优化微球制备工艺参数,成功制备了粒径在2-3μm、包裹量在0.4-0.6‰的PELA、PBELA和PGBELA微球。电泳检测结果表明微球制备过程中可较好保护pDNA的超螺旋结构,酶保护试验中结果表明,三种聚合物微球对包裹pDNA结构有较好的保护作用。
在pH为7.4、6.0和5.0的缓冲溶液中,系统研究了三种不同聚合物微球对红细胞膜破坏程度、载pDNA复合粒子微球中pDNA释放和基质聚合物的降解行为。结果表明PELA微球基质聚合物的降解和复合粒子的释放在中性和酸性条件下的行为相近,复合粒子释放较为缓慢。但PBELA和PGBELA微球在酸性条件下的降解和pDNA复合粒子的释放明显加快,表现出明显的酸度敏感性。
使用巨噬细胞RAW264.7考察载pDNA复合粒子聚合物微球的细胞毒性和转染效率。结果表明将复合粒子包裹到微球中降低了复合粒子的细胞毒性。转染实验结果表明,酸度敏感微球PBELA和PGBELA的转染效果显著高于PELA微球,中等pDNA复合粒子携载量的微球在具有较高目标蛋白表达量的同时细胞保持良好的增殖率。PGBELA微球表面半乳糖残基促进了细胞对微球的吞噬作用,提高了转染效率,显示出微球的靶向性特征。
Drug delivery systems with pH-sensitive polymer carriers can targetingly transfer drugs to acidic environments, such as gastrointestinal tract, tumor, and inflammatory site. Besides this, acid-labile carriers can lead endosomal escape and deliver drugs into cytoplasm, because of its rapid degradation in the acidic environment. In present study, plasmid DNA (pDNA) encapsuated microspheres from matrix polymers containing acetal groups were prepared by double emulsion method. Target groups of matrix polymers lead an effective cell uptake, while acid-labile groups lead endosomal rupture, which should enhance the cell transfection. This study could provide a new method for the delivery of DNA vaccines.
The acetal group modified PEG was used as pH-sensitive segments, which was copolymerized with lactide to obtain pH-sensitive polymers of PBELA. Galactose residues were conjugated onto the backbone of PBELA through click reaction to obtain PGBELA. Polyethyleneimine (PEI) condensed pDNA (pDNA-PEI) were wrapped within acid-labile polymers of PBELA and PGBELA by double emulsion method, using polylactide-poly(ethylene glycol) (PELA) microspheres as controlled. All the pDNA-PEI encapsulated microspheres possessed the size of 2-3μm and the loading amount of 0.4-0.6%o. The analyses of agarose gel electrophoresis and enzyme protection tests exhibited that the microsphere formation processes did not significantly influence the structure integrity of pDNA, and the microspheres could protect pDNA from enzymolysis.
The hemolytic effect, pDNA-PEI release profile and matrix polymer degradation were investigated on PELA, PBELA, and PGBELA microspheres in media with pH values of 7.4, 6.0, and 5.0. The results indicated that the release rate of pDNA-PEI from PELA microspheres was slow, and there were no remarkable difference after incubation in neutral and acidic media. However, the pDNA-PEI release and matrix polymer degradation of PBELA and PGBELA microspheres were significantly accelerated in acidic enivronment, which confirmed the pH sensitivity of matrix polymers.
The cytotoxicity and cell transfection efficiency of pDNA-PEI encapsulated microspheres with different matrix polymer were investigated by incubating with RAW264.7 cells. The results showed that the cytotoxicity of pDNA-PEI was decreased after encapsulation into microsphere, and the cell transfection efficiency of acid-labile microspheres was significantly higher than that of PELA microspheres. As the high transfection efficiency always led high cytotoxicity, a middle loading level of pDNA-PEI led a balance of transfection efficiency and cell viability. Furthermore, the galactose residues on the surface of PGBELA microsphere ehanced the cell uptake and promoted the cell transfection efficiency, which confirmed the targeting capabilities.
本文利用缩醛修饰聚乙二醇作为酸度敏感链段,与丙交酯共聚得到酸度敏感聚合物(PBELA),进一步通过Click反应接枝半乳糖基团,得到带半乳糖残基的酸度敏感聚合物(PGBELA),并以聚乳酸-聚乙二醇共聚物(PELA)为对照。本文以模型质粒(pDNA)与聚乙烯亚胺复合粒子为对象,利用复乳法制备了聚合物微球,通过优化微球制备工艺参数,成功制备了粒径在2-3μm、包裹量在0.4-0.6‰的PELA、PBELA和PGBELA微球。电泳检测结果表明微球制备过程中可较好保护pDNA的超螺旋结构,酶保护试验中结果表明,三种聚合物微球对包裹pDNA结构有较好的保护作用。
在pH为7.4、6.0和5.0的缓冲溶液中,系统研究了三种不同聚合物微球对红细胞膜破坏程度、载pDNA复合粒子微球中pDNA释放和基质聚合物的降解行为。结果表明PELA微球基质聚合物的降解和复合粒子的释放在中性和酸性条件下的行为相近,复合粒子释放较为缓慢。但PBELA和PGBELA微球在酸性条件下的降解和pDNA复合粒子的释放明显加快,表现出明显的酸度敏感性。
使用巨噬细胞RAW264.7考察载pDNA复合粒子聚合物微球的细胞毒性和转染效率。结果表明将复合粒子包裹到微球中降低了复合粒子的细胞毒性。转染实验结果表明,酸度敏感微球PBELA和PGBELA的转染效果显著高于PELA微球,中等pDNA复合粒子携载量的微球在具有较高目标蛋白表达量的同时细胞保持良好的增殖率。PGBELA微球表面半乳糖残基促进了细胞对微球的吞噬作用,提高了转染效率,显示出微球的靶向性特征。
Drug delivery systems with pH-sensitive polymer carriers can targetingly transfer drugs to acidic environments, such as gastrointestinal tract, tumor, and inflammatory site. Besides this, acid-labile carriers can lead endosomal escape and deliver drugs into cytoplasm, because of its rapid degradation in the acidic environment. In present study, plasmid DNA (pDNA) encapsuated microspheres from matrix polymers containing acetal groups were prepared by double emulsion method. Target groups of matrix polymers lead an effective cell uptake, while acid-labile groups lead endosomal rupture, which should enhance the cell transfection. This study could provide a new method for the delivery of DNA vaccines.
The acetal group modified PEG was used as pH-sensitive segments, which was copolymerized with lactide to obtain pH-sensitive polymers of PBELA. Galactose residues were conjugated onto the backbone of PBELA through click reaction to obtain PGBELA. Polyethyleneimine (PEI) condensed pDNA (pDNA-PEI) were wrapped within acid-labile polymers of PBELA and PGBELA by double emulsion method, using polylactide-poly(ethylene glycol) (PELA) microspheres as controlled. All the pDNA-PEI encapsulated microspheres possessed the size of 2-3μm and the loading amount of 0.4-0.6%o. The analyses of agarose gel electrophoresis and enzyme protection tests exhibited that the microsphere formation processes did not significantly influence the structure integrity of pDNA, and the microspheres could protect pDNA from enzymolysis.
The hemolytic effect, pDNA-PEI release profile and matrix polymer degradation were investigated on PELA, PBELA, and PGBELA microspheres in media with pH values of 7.4, 6.0, and 5.0. The results indicated that the release rate of pDNA-PEI from PELA microspheres was slow, and there were no remarkable difference after incubation in neutral and acidic media. However, the pDNA-PEI release and matrix polymer degradation of PBELA and PGBELA microspheres were significantly accelerated in acidic enivronment, which confirmed the pH sensitivity of matrix polymers.
The cytotoxicity and cell transfection efficiency of pDNA-PEI encapsulated microspheres with different matrix polymer were investigated by incubating with RAW264.7 cells. The results showed that the cytotoxicity of pDNA-PEI was decreased after encapsulation into microsphere, and the cell transfection efficiency of acid-labile microspheres was significantly higher than that of PELA microspheres. As the high transfection efficiency always led high cytotoxicity, a middle loading level of pDNA-PEI led a balance of transfection efficiency and cell viability. Furthermore, the galactose residues on the surface of PGBELA microsphere ehanced the cell uptake and promoted the cell transfection efficiency, which confirmed the targeting capabilities.
引文
[1]成军,陈菊梅.基因疫苗临床研究进展.国外医学流行病学传染病学册.2000,27(6)：247-250.
[2]聂青和.基因疫苗的基础研究及应用现状.世界华人消化杂志.2003,11(2)：125-129.
[3]Cai H, Hu X, Yu D, LI S, Tian X, Zhu Y. Combined DNA vaccine encapsulated in microspheres enhanced protection efficacy against Mycobacterium tuberculosis infection of mice. Vaccine.2005,23:4167-4174.
[4]Kobayashi H, Nagato T, O ikawa K. Recognition of p rostate and breast tumor cells by helper T lymphocytes specific for a p rostate and breast tumor associated antigen, Tarp. Clin Cancer Res.2005,11 (10):3869-3878.
[5]王连艳,马国辉.疫苗控释微球研究进展.国外医学预防诊断治疗用生物制品分册.2004,27(2)：79-83.
[6]Jang JH, Shea LD. Intramuscular delivery of DNA releasing microspheres:Microsphere propertiesand transgene expression. J Control Release.2006,112:120-128.
[7]Zhou XF, Liu B, Yu X, Zha X, Zhang XZ, Chen Y, Wang XY, Jin YH, Wu YG, Chen Y, Shan Y, Chen Y, Liu JQ, Kong W, Shen J. Controlled Release of PEI/DNA complexes from mannose-bearing chitosanmicrospheres as a potent delivery system to enhance immune response to HBV DNA vaccine. J Control Release.2007,121:200-207.
[8]朱敏艳,袁喜英,宗莉.基因疫苗微粒系统黏膜免疫的研究进展.药物生物技术.2008,15(3)：223-226.
[9]Wanga D, Xua J, Fenga Y, Liua Y, Mchengaa S.S.S, Shana FP,Ji S, Lua CL. Liposomal oral DNA vaccine (mycobacterium DNA) elicits immune response. Vaccine. 2010,28:3134-3142.
[10]Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Delivery Rev. 2005,57:637-651.
[11]Xiong XB, Huang Y, Lu WL, Xuan Zhang, Zhang H, Nagai T, Zhang Q, Enhanced intracellular delivery and improved antitumor efficacy of doxo rubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic. J Control Release.2005, 107:262-275.
[12]Bareford LM, Swaan PW. Endocytic mechanisms for targeted drug delivery. Adv Drug Delivery Rev.2007,59:748-758.
[13]彭洁,管涛,高洪.线粒体超微病变与线粒体病.中国畜牧兽医.2006,33(8)：50-52.
[14]Dittoa AJ, Shahb PN., Lopinab ST., Yun YH. Nanospheres formulated from ltyrosine polyphosphate as a potential intra-cellular delivery device. Int J Pharm.2009,368: 199-206.
[15]Dietrich G, Gentschev I, Hess J. Delivery of DNA vaccines by attenuated intracellular bacteria. Immunol Today.1999,20:251-253.
[16]Saar K, Lindgren Mari, Hansen M, Eiriksdottir E, Jiang Yang, Aizman KR, Sassian M, Lu angel. Cell-penetrating peptides:A comparative membrane toxicity study. Anal Biochem.2005,345:55-65
[17]Dietz G.P., Bahr M.. Delivery of bioactive molecules into the cell:the Trojan horse approach. Mol. Cell. Neurosci.2004,27:85-131.
[18]Schwarm SR, Dowdy SF. In vivo protein transduction:Intracellular delivery of biologieally active proteins compounds and DNA. Trends Pharmacol Sci.2000,21: 45-48.
[19]Lloyd JB. Lysosome membrane permeability:implications for drug delivery. Adv Drug Delivery Rev.2000,41:189-200.
[20]Bergstrand N, Arfvidsson MC, Kim JM, Thompson DH, Edwards K. Interactions between pH-sensitive liposomes and model membranes. Biophys Chem.2003,104: 361-379.
[21]Obata Y, Tajima S, Takeoka S. Evaluation of pH-responsive liposomes containing amino acid-based zwitterionic lipids for improving intracellular drug delivery in vitro and in vivo. J Control Release.2010,142:267-276.
[22]Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery:The need for precision in reporting particle size parameters. Eur J Pharm Biopharm.2008,69:1-9.
[23]Chan Y, Bulmus V, Zareie MH, Byrne FL, Barner L, Kavallaris M. Acid-cleavable polymeric core-shell particles for delivery of hydrophobic drugs. J Control Release.
2006,115:197-207.
[24]Kyriakides TR, Cheung CY, Murthy N, Bornstein P, Stayton PS, Hoffman AS. pH-Sensitive polymers that enhance intracellular drug delivery in vivo. J Control Release.2002,78:295-303.
[25]Couvreur P, Patile, Malvy C.pH-sensitive liposomes:all intelligent system for delivery of antisense oligonucleotides. J Lipos Res.1997,7(1):1-18.
[26]Oliveira MC, Rosilioa V, Lesieur P, Bourgaux C, Couvreura P, Ollivona M, Duberneta C. pH-Sensitive liposomes as a carrier for oligonucleotides:a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in excess water. Biophys Chem.2000,87:127-137.
[27]Momekovaa D, Rangelovb S, Yanevc S, Nikolovad E, Konstantinove S, Rombergf B, Stormf G, Lambov N. Long-circulating, pH-sensitive liposomes sterically stabilized by copolymers bearing short blocks of lipid-mimetic units. Eur J Pharm Sci.2007,32: 308-317.
[28]Shi GF, Guo WJ, Stephenson SM, Lee RJ. Effecient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid. J Control Release.2002,80:309-319.
[29]裴勇,陈捷.环境敏感水凝胶的研究进展.现代化工.2004,24：76-79
[30]余灯广,杨祥良,徐辉碧.pH敏感型水凝胶在多肽和蛋白质药物给药系统研究中的应用.中国医院药学杂志.2005,25(8)：762-763.
[31]Yue YM, Sheng X, Wang PX. Fabrication and characterization of microstructured and pH sensitive interpenetrating networks hydrogel films and application in drug. Eur Polym J.2009,45:309-315.
[32]Kadlubowski S, Henke A, Ulanski P, Rosiak JM, Bromberg L, Hatton TA. Hydrogels ofpolyvinylpyrrolidone (PVP) and poly(acrylic acid) (PAA) synthesized by photoinduced crosslinking of homopolymers. Polymer.2007,48:4974-4981.
[33]Shao YY, Zhu BQ, Li J, Liu XR, Tan X, Yang XL. Novel chitosan microsphere-templatedmicrocapsules suitable for spontaneous loading of heparin. Mater Sci Eng C.2009,29:936-941.
[34]Guliyeva U, Oner F, Ozsoy S, Haziroglu R. Chitosan microparticles containing plasmid DNA as potential oral gene delivery system. Eur J Pharm Biopharm.2006,62:17-25.
[35]lemma F, Spizzirri UG, Puoci R, Muzzalupo R, Trombino S, Cassano R, Leta S, Picci N. pH-Sensitive hydrogels based on bovine serum albumin for oral drug delivery. Int J Pharm.2006,312:151-157.
[36]Ma LW, Liu MZ, Liu HL, Chen J, Cui DP. In vitro cytotoxicity and drug release properties of pH-and temperature-sensitive core-shell hydrogel microspheres. Int J Pharm.2010,385:86-91.
[37]Wu J, Wei W, Wang LY, Su ZG, Ma GH. Preparation of uniform-sized pH-sensitive quaternized chitosan. Colloid Surf B.2008,63:164-175.
[38]Colinet I, Dulong V, Mocanu G, Picton L, Cerf DL. New amphiphilic and pH-sensitive hydrogel for controlledled release of a model poorly water-soluble drug. Eur J Pharm Biopharm.2009,73:345-350.
[39]Abdellaoui KS, Heller J, Gurny R. Hydrolysis and erosion studies of autocatalyzed poly(ortho esters) containing lactoyl-lactyl acid dimers. Macromolecule.1999,32: 301-307.
[40]Nguyen DN, Raghavan SS, Tashima LM, Lin EC, Fredette SJ, Langer RS, Wang C. Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine). Biomaterals.2008,29:2783-2793.
[41]Qi M, Li X, Yang Y, Zhou SB. Electrospun fibers of acid-labile biodegradable polymers containing ortho ester groups for controlledled release of paracetamol. Eur J Pharm Biopharm.2008,70:445-452.
[42]Prabaharan M, Grailer JJ, Pilla S, Steeber DA, Gong SQ. Amphiphilic multi-arm-block copolymer conjugated. with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery. Biomaterials.2008,29:2783-2793.
[43]Hruby M, Konak C, Ulbrich K. Polymeric micellar pH-sensitive drug delivery system for doxorubicin. J Control Release.2005,103:137-148.
[44]Chen W, Meng FH, Cheng R, Zhong ZY. pH-sensitive degradable polymersomes for triggered release of anticancer drugs:A comparative study with micelles. J Control Release,2010,142:40-46.
[45]傅晓源,张浩伟,谢灏,丁诚,彭锴,张阳德.聚乙烯亚胺载基因纳米颗粒的制备及其相关特性的研究.中国现代医学杂志.2008,18(24)：3570-3576.
[46]赵梦丹,杨峰亮,陈坚,虞和永.多聚乙烯亚胺介导基因转染的体外研究.中国药学杂志.2008,43(16)：1211-1214.
[47]Biebery T, Elsasser HP. Preparation of a low molecular weight polyethylenimine for efficient cell transfection. Biotechniques,2001,30(1):74-81.
[48]王星,姚静,周建平.低相对分子质量聚乙烯亚胺及其衍生物作为基因载体的应用.中国药学杂志.2008,43(8)：561-564
[49]Kunath K, Harpe AV, Fische D, Petersen H, Bickel U, Voigt k, Kissel T. Low molecularweight polyethylenimine as a non viral vector for DNA delivery:comparison of physicochemical properties, transfection efficiencyand in vivo distribution with high molecular weight polyethylenimine. J Control Release.2003,89:113-125.
[50]Ahn C H, Chae S Y, Bae Y H, Kim S W. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J Control Release.2002,80:273-282.
[51]陈磊,田华雨,陈学思,朴泰宽,丸山厚,景遐斌.交联型聚乙烯亚胺智能基因载体的制备及PEG化影响.高分子学报.2009,(6)：499-505.
[52]Ogris M, Walker G, Blessing T, Kircheis R, Wolschek M, Wagner E. Tumor-targeted gene therapy:strategies for the preparation of ligand-polyethylene glycol polyethylenimine/DNA complexes. J Control Release.2003,91:173-181.
[53]Wolschek MF, Thallinger C, Kursa M, Rossler V, Allen M, Lichtenberger C, Kircheis R, Lucas T, Willheim M, Reinisch W, Gangl A, Wagner E, Jansen B. Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. Hepatology.2002,36(5):1106-1114
[54]Ogris M, Brunner S, Schuller S. PEGylated DNA/transferrin-PEI complexes:reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther.1999,6(4):595-605.
[55]Kichler A, Chillon M, Leborgne C, Danos O, Frisch B. Intranasal gene delivery with a polyethylenimine-PEG conjugate. J Control Release.2002,81:379-388.
[56]Hu-Lin Jiang, Kim YK, Arote R, Nah JW, Cho MH, Choi YJ, Akaike T, Cho CS. Chitosan-graft-polyethylenimine as a gene carrier. J Control Release.2007,117: 273-280.
[57]Jiang HL, Kima YK, Arotea R, Jerea D, Quana JS, Yua JH, Choia YJ, Nahd JW, Choe MH, Choa CS. Mannosylated chitosan-graft-polyethylenimine as a gene carrier for Raw 264.7 cell targeting. Int J Pharm.2009,375:133-39.
[58]Rorkel S, Keeney M, Pandit A. Non-viral polyplexes:scaffold mediated delivery for gene therapy. Prog polym Sci.2010,35:441-458.
[59]Holladay C, Keeney M, Greiser U, Murphy M, Brien TO, Pandit A. A matrix reservoir for improved controlled of non-viral gene delivery. J Control Release.2009,136: 220-225.
[60]Jang JH, Bengali Z, Houchin TL, Shea LD. Surface adsorption of DNA to tissue engineering scaffoldsfor efficient gene delivery. J Biomed Mater Res A.2006,77(1): 50-58.
[61]Rosa G, Quaglia F, Bochot A, Ungaro F, Fattal E. Long-term release and improved intracellular penetration of oligonucleotide polyethylenimine complexes entrapped in biodegradable microspheres. Biomacromolecules.2003,4:529-536.
[62]McKeever U, Barman S, Hao T, Chambers P, Song, L. Lunsford SL,Hsu YY, Roy K, Hedley ML. Protective immune responses elicited in mice by immunization with for mulations of poly(lactide-co-glycolide) microparticles. Vaccine.2002,20:1524-1531.
[63]Godbey WT, Barry MA,Saggau P, Wu KK, Mikos AG Poly(ethylenimine)-mediated transfection:a new paradigm for gene delivery. J Biomed Mater Res.2000,51(3): 321-328.
[64]Walter E, Dreher D, Kok M, Thiele L, Kiama S, Gehr P, Merkle H. Hydrophilic poly(DL-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells. J Control Release.2001,76:149-168.
[65]程龙.静电纺聚合物超细纤维作为基因控释体系的研究.西南交通大学硕士毕业论文.2009：16-19
[66]Cui WG, Qi MB, Li XH,Huang SZ, Zhou SB, Weng J. Electrospun fibers of acid-labile biodegradable polymers with acetal groups as potential drug carriers. Int J Pharm.2008, 361:47-55.
[67]吴冠男.半乳糖修饰酸度敏感聚合物及其载药微球的制备与表征,西南交通大学硕士毕业论文.2009：22-29.
[68]Sipos P, Csom I, Srcic K. Influence of preparation conditions on the properties of Eudragit microspheres produced by a double emulsion method. Drug Dev Res.2005,64: 41-44.
[69]汪自然,徐启勇,叶燕青.复乳法制备聚乳酸及其共聚物微球参数的研究进展.国际生物医学工程杂志.2006,29(4)：238-242.
[70]Intra J, Salem AK. Fabrication, characterization and in vitro evaluation of poly(D, L-lactide-co-glycolide) microparticles loaded with polyamidoamine-plasmid DNA dendriplexes fo applications in nonviral gene delivery. J Pharm Sci.2010,99:368-364.
[71]Ding CX, Gu J X, Qu XZ, Yang ZZ. Preparation of multifunctional drug carrier for tumor-specific uptake and enhanced intracellular delivery through the conjugation of weak acid labile linker. Bioconjugate Chem.2009,20:1163-1170.
[72]Yogesh P, Jayanth P. Polymeric nanoparticles for siRNA delivery and gene silencing. Int J Pharm.2009,367:195-203.
[73]Li XH, Zhang YH, Yan RH, Deng XM, and Huang ZT. Body distribution of poly-DL-lactide-poly(ethylene glycol) microspheres containing Leptospira Interrogans antigens after intravenous and oral administration. J. Pharm. Pharmcol.,2000,52(7): 763-770.
[74]Li ZH, Huang L. Sustained delivery and expression of plasmid DNA based on biodegradable polyester, poly (D, L-lactide-co-4-hydroxy-L-proline). J Control Release. 2004,98:437-446.
[75]Nie H, Wang CH. Fabrication and characterization of PLGA/HAp composite scaffolds for delivery of BMP-2 plasmid DNA. J Control Release.2007,120:111-121.
[2]聂青和.基因疫苗的基础研究及应用现状.世界华人消化杂志.2003,11(2)：125-129.
[3]Cai H, Hu X, Yu D, LI S, Tian X, Zhu Y. Combined DNA vaccine encapsulated in microspheres enhanced protection efficacy against Mycobacterium tuberculosis infection of mice. Vaccine.2005,23:4167-4174.
[4]Kobayashi H, Nagato T, O ikawa K. Recognition of p rostate and breast tumor cells by helper T lymphocytes specific for a p rostate and breast tumor associated antigen, Tarp. Clin Cancer Res.2005,11 (10):3869-3878.
[5]王连艳,马国辉.疫苗控释微球研究进展.国外医学预防诊断治疗用生物制品分册.2004,27(2)：79-83.
[6]Jang JH, Shea LD. Intramuscular delivery of DNA releasing microspheres:Microsphere propertiesand transgene expression. J Control Release.2006,112:120-128.
[7]Zhou XF, Liu B, Yu X, Zha X, Zhang XZ, Chen Y, Wang XY, Jin YH, Wu YG, Chen Y, Shan Y, Chen Y, Liu JQ, Kong W, Shen J. Controlled Release of PEI/DNA complexes from mannose-bearing chitosanmicrospheres as a potent delivery system to enhance immune response to HBV DNA vaccine. J Control Release.2007,121:200-207.
[8]朱敏艳,袁喜英,宗莉.基因疫苗微粒系统黏膜免疫的研究进展.药物生物技术.2008,15(3)：223-226.
[9]Wanga D, Xua J, Fenga Y, Liua Y, Mchengaa S.S.S, Shana FP,Ji S, Lua CL. Liposomal oral DNA vaccine (mycobacterium DNA) elicits immune response. Vaccine. 2010,28:3134-3142.
[10]Gupta B, Levchenko TS, Torchilin VP. Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. Adv Drug Delivery Rev. 2005,57:637-651.
[11]Xiong XB, Huang Y, Lu WL, Xuan Zhang, Zhang H, Nagai T, Zhang Q, Enhanced intracellular delivery and improved antitumor efficacy of doxo rubicin by sterically stabilized liposomes modified with a synthetic RGD mimetic. J Control Release.2005, 107:262-275.
[12]Bareford LM, Swaan PW. Endocytic mechanisms for targeted drug delivery. Adv Drug Delivery Rev.2007,59:748-758.
[13]彭洁,管涛,高洪.线粒体超微病变与线粒体病.中国畜牧兽医.2006,33(8)：50-52.
[14]Dittoa AJ, Shahb PN., Lopinab ST., Yun YH. Nanospheres formulated from ltyrosine polyphosphate as a potential intra-cellular delivery device. Int J Pharm.2009,368: 199-206.
[15]Dietrich G, Gentschev I, Hess J. Delivery of DNA vaccines by attenuated intracellular bacteria. Immunol Today.1999,20:251-253.
[16]Saar K, Lindgren Mari, Hansen M, Eiriksdottir E, Jiang Yang, Aizman KR, Sassian M, Lu angel. Cell-penetrating peptides:A comparative membrane toxicity study. Anal Biochem.2005,345:55-65
[17]Dietz G.P., Bahr M.. Delivery of bioactive molecules into the cell:the Trojan horse approach. Mol. Cell. Neurosci.2004,27:85-131.
[18]Schwarm SR, Dowdy SF. In vivo protein transduction:Intracellular delivery of biologieally active proteins compounds and DNA. Trends Pharmacol Sci.2000,21: 45-48.
[19]Lloyd JB. Lysosome membrane permeability:implications for drug delivery. Adv Drug Delivery Rev.2000,41:189-200.
[20]Bergstrand N, Arfvidsson MC, Kim JM, Thompson DH, Edwards K. Interactions between pH-sensitive liposomes and model membranes. Biophys Chem.2003,104: 361-379.
[21]Obata Y, Tajima S, Takeoka S. Evaluation of pH-responsive liposomes containing amino acid-based zwitterionic lipids for improving intracellular drug delivery in vitro and in vivo. J Control Release.2010,142:267-276.
[22]Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery:The need for precision in reporting particle size parameters. Eur J Pharm Biopharm.2008,69:1-9.
[23]Chan Y, Bulmus V, Zareie MH, Byrne FL, Barner L, Kavallaris M. Acid-cleavable polymeric core-shell particles for delivery of hydrophobic drugs. J Control Release.
2006,115:197-207.
[24]Kyriakides TR, Cheung CY, Murthy N, Bornstein P, Stayton PS, Hoffman AS. pH-Sensitive polymers that enhance intracellular drug delivery in vivo. J Control Release.2002,78:295-303.
[25]Couvreur P, Patile, Malvy C.pH-sensitive liposomes:all intelligent system for delivery of antisense oligonucleotides. J Lipos Res.1997,7(1):1-18.
[26]Oliveira MC, Rosilioa V, Lesieur P, Bourgaux C, Couvreura P, Ollivona M, Duberneta C. pH-Sensitive liposomes as a carrier for oligonucleotides:a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in excess water. Biophys Chem.2000,87:127-137.
[27]Momekovaa D, Rangelovb S, Yanevc S, Nikolovad E, Konstantinove S, Rombergf B, Stormf G, Lambov N. Long-circulating, pH-sensitive liposomes sterically stabilized by copolymers bearing short blocks of lipid-mimetic units. Eur J Pharm Sci.2007,32: 308-317.
[28]Shi GF, Guo WJ, Stephenson SM, Lee RJ. Effecient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid. J Control Release.2002,80:309-319.
[29]裴勇,陈捷.环境敏感水凝胶的研究进展.现代化工.2004,24：76-79
[30]余灯广,杨祥良,徐辉碧.pH敏感型水凝胶在多肽和蛋白质药物给药系统研究中的应用.中国医院药学杂志.2005,25(8)：762-763.
[31]Yue YM, Sheng X, Wang PX. Fabrication and characterization of microstructured and pH sensitive interpenetrating networks hydrogel films and application in drug. Eur Polym J.2009,45:309-315.
[32]Kadlubowski S, Henke A, Ulanski P, Rosiak JM, Bromberg L, Hatton TA. Hydrogels ofpolyvinylpyrrolidone (PVP) and poly(acrylic acid) (PAA) synthesized by photoinduced crosslinking of homopolymers. Polymer.2007,48:4974-4981.
[33]Shao YY, Zhu BQ, Li J, Liu XR, Tan X, Yang XL. Novel chitosan microsphere-templatedmicrocapsules suitable for spontaneous loading of heparin. Mater Sci Eng C.2009,29:936-941.
[34]Guliyeva U, Oner F, Ozsoy S, Haziroglu R. Chitosan microparticles containing plasmid DNA as potential oral gene delivery system. Eur J Pharm Biopharm.2006,62:17-25.
[35]lemma F, Spizzirri UG, Puoci R, Muzzalupo R, Trombino S, Cassano R, Leta S, Picci N. pH-Sensitive hydrogels based on bovine serum albumin for oral drug delivery. Int J Pharm.2006,312:151-157.
[36]Ma LW, Liu MZ, Liu HL, Chen J, Cui DP. In vitro cytotoxicity and drug release properties of pH-and temperature-sensitive core-shell hydrogel microspheres. Int J Pharm.2010,385:86-91.
[37]Wu J, Wei W, Wang LY, Su ZG, Ma GH. Preparation of uniform-sized pH-sensitive quaternized chitosan. Colloid Surf B.2008,63:164-175.
[38]Colinet I, Dulong V, Mocanu G, Picton L, Cerf DL. New amphiphilic and pH-sensitive hydrogel for controlledled release of a model poorly water-soluble drug. Eur J Pharm Biopharm.2009,73:345-350.
[39]Abdellaoui KS, Heller J, Gurny R. Hydrolysis and erosion studies of autocatalyzed poly(ortho esters) containing lactoyl-lactyl acid dimers. Macromolecule.1999,32: 301-307.
[40]Nguyen DN, Raghavan SS, Tashima LM, Lin EC, Fredette SJ, Langer RS, Wang C. Enhancement of poly(orthoester) microspheres for DNA vaccine delivery by blending with poly(ethylenimine). Biomaterals.2008,29:2783-2793.
[41]Qi M, Li X, Yang Y, Zhou SB. Electrospun fibers of acid-labile biodegradable polymers containing ortho ester groups for controlledled release of paracetamol. Eur J Pharm Biopharm.2008,70:445-452.
[42]Prabaharan M, Grailer JJ, Pilla S, Steeber DA, Gong SQ. Amphiphilic multi-arm-block copolymer conjugated. with doxorubicin via pH-sensitive hydrazone bond for tumor-targeted drug delivery. Biomaterials.2008,29:2783-2793.
[43]Hruby M, Konak C, Ulbrich K. Polymeric micellar pH-sensitive drug delivery system for doxorubicin. J Control Release.2005,103:137-148.
[44]Chen W, Meng FH, Cheng R, Zhong ZY. pH-sensitive degradable polymersomes for triggered release of anticancer drugs:A comparative study with micelles. J Control Release,2010,142:40-46.
[45]傅晓源,张浩伟,谢灏,丁诚,彭锴,张阳德.聚乙烯亚胺载基因纳米颗粒的制备及其相关特性的研究.中国现代医学杂志.2008,18(24)：3570-3576.
[46]赵梦丹,杨峰亮,陈坚,虞和永.多聚乙烯亚胺介导基因转染的体外研究.中国药学杂志.2008,43(16)：1211-1214.
[47]Biebery T, Elsasser HP. Preparation of a low molecular weight polyethylenimine for efficient cell transfection. Biotechniques,2001,30(1):74-81.
[48]王星,姚静,周建平.低相对分子质量聚乙烯亚胺及其衍生物作为基因载体的应用.中国药学杂志.2008,43(8)：561-564
[49]Kunath K, Harpe AV, Fische D, Petersen H, Bickel U, Voigt k, Kissel T. Low molecularweight polyethylenimine as a non viral vector for DNA delivery:comparison of physicochemical properties, transfection efficiencyand in vivo distribution with high molecular weight polyethylenimine. J Control Release.2003,89:113-125.
[50]Ahn C H, Chae S Y, Bae Y H, Kim S W. Biodegradable poly(ethylenimine) for plasmid DNA delivery. J Control Release.2002,80:273-282.
[51]陈磊,田华雨,陈学思,朴泰宽,丸山厚,景遐斌.交联型聚乙烯亚胺智能基因载体的制备及PEG化影响.高分子学报.2009,(6)：499-505.
[52]Ogris M, Walker G, Blessing T, Kircheis R, Wolschek M, Wagner E. Tumor-targeted gene therapy:strategies for the preparation of ligand-polyethylene glycol polyethylenimine/DNA complexes. J Control Release.2003,91:173-181.
[53]Wolschek MF, Thallinger C, Kursa M, Rossler V, Allen M, Lichtenberger C, Kircheis R, Lucas T, Willheim M, Reinisch W, Gangl A, Wagner E, Jansen B. Specific systemic nonviral gene delivery to human hepatocellular carcinoma xenografts in SCID mice. Hepatology.2002,36(5):1106-1114
[54]Ogris M, Brunner S, Schuller S. PEGylated DNA/transferrin-PEI complexes:reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther.1999,6(4):595-605.
[55]Kichler A, Chillon M, Leborgne C, Danos O, Frisch B. Intranasal gene delivery with a polyethylenimine-PEG conjugate. J Control Release.2002,81:379-388.
[56]Hu-Lin Jiang, Kim YK, Arote R, Nah JW, Cho MH, Choi YJ, Akaike T, Cho CS. Chitosan-graft-polyethylenimine as a gene carrier. J Control Release.2007,117: 273-280.
[57]Jiang HL, Kima YK, Arotea R, Jerea D, Quana JS, Yua JH, Choia YJ, Nahd JW, Choe MH, Choa CS. Mannosylated chitosan-graft-polyethylenimine as a gene carrier for Raw 264.7 cell targeting. Int J Pharm.2009,375:133-39.
[58]Rorkel S, Keeney M, Pandit A. Non-viral polyplexes:scaffold mediated delivery for gene therapy. Prog polym Sci.2010,35:441-458.
[59]Holladay C, Keeney M, Greiser U, Murphy M, Brien TO, Pandit A. A matrix reservoir for improved controlled of non-viral gene delivery. J Control Release.2009,136: 220-225.
[60]Jang JH, Bengali Z, Houchin TL, Shea LD. Surface adsorption of DNA to tissue engineering scaffoldsfor efficient gene delivery. J Biomed Mater Res A.2006,77(1): 50-58.
[61]Rosa G, Quaglia F, Bochot A, Ungaro F, Fattal E. Long-term release and improved intracellular penetration of oligonucleotide polyethylenimine complexes entrapped in biodegradable microspheres. Biomacromolecules.2003,4:529-536.
[62]McKeever U, Barman S, Hao T, Chambers P, Song, L. Lunsford SL,Hsu YY, Roy K, Hedley ML. Protective immune responses elicited in mice by immunization with for mulations of poly(lactide-co-glycolide) microparticles. Vaccine.2002,20:1524-1531.
[63]Godbey WT, Barry MA,Saggau P, Wu KK, Mikos AG Poly(ethylenimine)-mediated transfection:a new paradigm for gene delivery. J Biomed Mater Res.2000,51(3): 321-328.
[64]Walter E, Dreher D, Kok M, Thiele L, Kiama S, Gehr P, Merkle H. Hydrophilic poly(DL-lactide-co-glycolide) microspheres for the delivery of DNA to human-derived macrophages and dendritic cells. J Control Release.2001,76:149-168.
[65]程龙.静电纺聚合物超细纤维作为基因控释体系的研究.西南交通大学硕士毕业论文.2009：16-19
[66]Cui WG, Qi MB, Li XH,Huang SZ, Zhou SB, Weng J. Electrospun fibers of acid-labile biodegradable polymers with acetal groups as potential drug carriers. Int J Pharm.2008, 361:47-55.
[67]吴冠男.半乳糖修饰酸度敏感聚合物及其载药微球的制备与表征,西南交通大学硕士毕业论文.2009：22-29.
[68]Sipos P, Csom I, Srcic K. Influence of preparation conditions on the properties of Eudragit microspheres produced by a double emulsion method. Drug Dev Res.2005,64: 41-44.
[69]汪自然,徐启勇,叶燕青.复乳法制备聚乳酸及其共聚物微球参数的研究进展.国际生物医学工程杂志.2006,29(4)：238-242.
[70]Intra J, Salem AK. Fabrication, characterization and in vitro evaluation of poly(D, L-lactide-co-glycolide) microparticles loaded with polyamidoamine-plasmid DNA dendriplexes fo applications in nonviral gene delivery. J Pharm Sci.2010,99:368-364.
[71]Ding CX, Gu J X, Qu XZ, Yang ZZ. Preparation of multifunctional drug carrier for tumor-specific uptake and enhanced intracellular delivery through the conjugation of weak acid labile linker. Bioconjugate Chem.2009,20:1163-1170.
[72]Yogesh P, Jayanth P. Polymeric nanoparticles for siRNA delivery and gene silencing. Int J Pharm.2009,367:195-203.
[73]Li XH, Zhang YH, Yan RH, Deng XM, and Huang ZT. Body distribution of poly-DL-lactide-poly(ethylene glycol) microspheres containing Leptospira Interrogans antigens after intravenous and oral administration. J. Pharm. Pharmcol.,2000,52(7): 763-770.
[74]Li ZH, Huang L. Sustained delivery and expression of plasmid DNA based on biodegradable polyester, poly (D, L-lactide-co-4-hydroxy-L-proline). J Control Release. 2004,98:437-446.
[75]Nie H, Wang CH. Fabrication and characterization of PLGA/HAp composite scaffolds for delivery of BMP-2 plasmid DNA. J Control Release.2007,120:111-121.