基于阳离子聚合物/DNA纳米复合物微针给药系统的研究
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摘要
DNA疫苗是将含有编码抗原基因的真核表达质粒直接接种体内,在体内表达相应抗原,以刺激机体产生针对该抗原的免疫应答,产生保护性免疫。它是在基因治疗和转基因技术基础上产生的第三代疫苗,是疫苗的一次革命。但目前DNA疫苗的给药系统比较单一,主要是质粒DNA冻干粉针,肌注给药后,DNA进入组织细胞,特别是到达树状突细胞(DCs)等抗原提呈细胞(antigen-presenting cells,APCs)的转运效率较低,因而导致抗原表达水平低,免疫效果不好。目前关键是在于缺乏高效低毒的DNA疫苗的传递系统。皮肤特别是在活性表皮层中存在着大量的树状突细胞(DCs)—郎格汉氏细胞(Langerhans cells,500-1000 cells mm~(-2)),这是一种高效的抗原提呈细胞(APCs),可产生很强的免疫效应,因此活性表皮层被看作疫苗接种的最佳部位。然而DNA疫苗这样的亲水性大分子药物是无法穿透皮肤角质层的屏障,并且皮肤内的酶系会对DNA疫苗产生降解作用,而使抗原提呈细胞无法捕获DNA疫苗。为此我们设计了阳离子聚合物/DNA疫苗复合物微针给药系统。微针(Microneedles)是指通过微电子机械工艺技术(MEMS)制作的,尺寸在微米级,长度100μm以上呈针状的复杂结构,材料可以为硅、聚合物、金属等。微针在生物医学领域有广泛的应用。微针用于药物传输系统具有精确、无痛、高效、便利的优势。利用微针给药系统可主动靶向于皮肤特定深度,即活性表皮层的郎格汉氏细胞。为了提高DNA疫苗在体内的稳定性,合成了基于聚乙烯亚胺(PEI)的基因载体,选用两亲性的嵌段共聚物Pluronic(聚氧乙烯-聚环氧丙烷-聚氧乙烯嵌端共聚物)和Solutol(聚乙二醇-12羟基硬脂酸酯)修饰支链高分子量PEI,不但可以增加PEI/DNA复合物的稳定性,屏蔽复合物表面的正电荷,减小其对细胞的毒性作用,还因为Pluronic和Solutol分子中含有疏水基团,对细胞具有一定的亲和力,不会显著降低DNA疫苗体内外转染效率。以乙肝DNA疫苗为模型药物,考察了阳离子聚合物/DNA疫苗复合物微针透皮给药后的小鼠体内免疫效果。
第一部分使用琥珀酰亚胺酯法活化非离子表面活性剂Pluronic或Solutol的末端羟基,得到活化率高,反应活度大的产物。使用活化后的聚合物按不同比例对PEI进行修饰。经过葡聚糖凝胶纯化,超滤脱盐,冷冻干燥,成功的合成了一系列不同修饰度(每个PEI分子上分别接枝1个、2个、5个、10个和20个Pluronic或Solutol)的Graft-PEI聚合物。~1H-NMR结果显示,本方法合成的阳离子聚合物接枝率高,控制投药量能定量地对PEI进行修饰,其接枝率与反应时投药量的摩尔比呈良好的线性关系,说明该合成方法比较稳定、可靠,产率高,具有良好的可控性。~1H-NMR图谱中未见其它杂质峰,证明这些阳离子聚合物的纯度较高。
第二部分重点考察了阳离子接枝聚合物/DNA复合物的相关性质。凝胶电泳阻滞分析结果显示,完全阻滞DNA所需的N/P值随着PEI修饰度的增加而增大,随着修饰物分子量的减小而减小。本实验所制备的PEI/DNA、graft-PEI/DNA复合物粒径在60-400 nm范围内。随着复合物N/P值的增加,粒径有减小的趋势。高分子量的修饰物,在高修饰度时复合物的粒径明显增大。复合物的zeta电位随N/P值的增加而增加;相同N/P值的复合物,其zeta电位随表面活性剂接枝数的增加而减少;复合物的zeta电位在相同修饰度且相同N/P值时,随表面活性剂分子量的减小而略有增加。MTT法测定细胞毒性的结果表明,使用表面活性剂修饰PEI可以不同程度地降低其细胞毒性,在低浓度时每个PEI接枝一个表面活性剂的分子即可显著降低细胞毒性。在高浓度时每个PEI接枝≥5个表面活性剂分子则可显著降低细胞毒性。
第三部分评价了阳离子接枝聚合物/DNA复合物体内外转染效果。选用了两种分别编码绿色荧光蛋白和虫荧光素酶的质粒。为了获得虫荧光素酶在体内较高的表达,将CMV启动子克隆到虫荧光素酶表达质粒pGL3-basic Luciferase Reporter vector中,构建成重组的pGL3-CMV Luciferase Reporter vector。阳离子接枝聚合物/DNA复合物在Hela细胞上的转染效果表明,低分子量的L61和Solutol修饰的PEI在低修饰度时不会显著地降低转染效果,低P123修饰度的PEI的最佳转染效果与未修饰的PEI相似。随着表面活性剂分子量增大,接枝率增加,其最佳转染效率随之下降。ICR小鼠尾静脉注射复合物,测定各个脏器中虫荧光素酶的表达,除了肺部外,低修饰度的PEI最佳转染效率显著优于未修饰的PEI。
第四部分使用微电机技术制备了SU-8聚合物微针,考察了微针的机械强度,结果表明该微针阵列可以容易地穿破铝箔和离体皮肤,并具有一定的坚韧度,不易折断。考察了微针对不同药物的体外透皮促进作用。首先考察了四种不同油水分配系数的小分子药物(吲哚美辛、酮洛芬、美索巴莫、更昔洛韦),结果表明吲哚美辛的皮肤渗透速率增加4.05倍,酮洛芬的皮肤渗透速率增加4.77倍,美索巴莫的皮肤渗透速率增加6.06倍,更昔洛韦的皮肤渗透速率增加12.49倍,说明logP值是影响药物经微针透皮传递速率的重要因素,微针对于水溶性药物的透皮促进作用较脂溶性药物更加有利。又考察了四种不同分子量的生物大分子药物(胰岛素、溶菌酶、卵清白蛋白、人血清白蛋白),结果表明与无法透过完整的皮肤相比,生物大分子药物可以透过微针阵列处理过的皮肤,经皮渗透速率可以达到小分子药物的水平。生物大分子药物分子量与经皮渗透速率成反比。
第五部分主要考察了DNA疫苗的体内免疫效果。通过细胞吞噬实验证明了修饰后的PEI其细胞吞噬过程未发生改变。抗DNAaseⅠ的保护实验表明复合物可以有效地保护DNA不被降解。微针阵列可刺入小鼠活体皮肤,并在皮肤表皮层产生几十微米的孔洞,提供了复合物穿透角质层的通道。以乙肝疫苗为模型药物,考察体内免疫效果,分别测定血清中的抗体滴度,脾细胞分泌的细胞因子(γ-INF,IL-4),及脾细胞的增殖。与乙肝DNA疫苗复合物肌注和DNA疫苗水溶液肌注相比,阳离子聚合物/DNA疫苗复合物微针经皮给药在第4周后可以显著提高血清中的抗体滴度,同时显著提高了细胞免疫水平,刺激机体产生了很强的免疫应答。
第六部分主要考察了阳离子聚合物/DNA疫苗复合物微针给药系统的组织刺激性。DNA疫苗复合物微针或肌注给药后都表现出了一定的组织刺激反应,微针给药组的炎症反应明显低于肌注给药组,说明阳离子聚合物/DNA疫苗复合物微针给药系统具有较好的生理适应性。
DNA vaccination is a technique to inoculate directly eukaryotic expression plasmid containing encoding antigenic gene into body and then the plasmid express the related antigen to produce an immunological response for protecting an organism against disease. It is vaccine of third generation on the basis of gene therapy and transgenic technology, which is a revolution for vaccine. But the DNA vaccine drug delivery system is single relatively. The major is plasmid freeze-dried powder injection which was given by intramuscular injection to delivery DNA into rhagiocrine cell, especially antigen-presenting cells (APCs) including dendritic cells(DCs).Because of the low transfection efficiency, the degree of antigen presentation was low and the immunity effect is not satified. There are a lot of dendritic cells(DCs)(Langerhans cells, 500-1000 cells mm~(-2) ) , a high-performance antigen presenting cell(APCs), which have strong immunological effect, so activity cuticular layer refers to the best part to inoculate vaccine. But hydrophilia macromolecule drug like DNA vaccine can not penetrate the keratoderma and enzyme system in skin will degrade DNA vaccine so that antigen presenting cell can not catch DNA vaccine. According that, we designed.
Microneedles refers to a needle-shaped microstructure the needles on which have the length of more than 100μm and micrometer size. It fabricated by micro-electro-mechanical systems (MEMS) technology using different material, including silicon, polymer or metal. There has a broad use in the range of biomedicine for microneedle wjich as the drug delivery system have the advantage of exact dosage, high performance, painless and convenient use. The microneedle drug delivery system can be used to activitily target to the definite depth of skin, the Langerhans cell in active cuticular layer. The genetic carrier is synthesized on the base of PEI to enhance the stability DNA in vivo. The Pluronic and Solutol with amphipathic properties are chosed to modify the chain of PEI with high molecular weight in order to enhance the stability of PEI/DNA compounds and shield the positive charge on the surface of compound to diminish the toxicity. Besides, because the hydrophobic group of Pluronic and Solutol have a certain affinity to the cell, the transfection efficiency of DNA vaccine won't be lowered in vivo and vitro. Using the hepatitis DNA vaccine as model drug, to investigate the immunity effect of mouse in vivo after giving the compound of cationic polymer and DNA vaccine by transdermal application of microneedle.
In the first part, succinimide ester was used to activation the terminal hydroxyl of Pluronic or solutol, nonionic wetting agent,to get a product with high activation efficiency and activity series. The activation compound was modified with PEI in the different proportion. After the purification through polydextran gel, the product was ultrafiltrated to get rid of salt and then cryodesiccated. According to the above process, we successfully got Graft-PEI compound with a series of different degree of modification per one PEI macromolecule at 1,2,5,10 and 20 Pluronic or Solutol. The result of ~1H-NMR showed the cationic polymerization synthesized by this method had a high graft ratio and PEI can be modified quantitatively by controlling administration dosage molar ratio of which had a satisfactory linear relationship with graft ratio. All that demonstrated that that synthetic method was stability, safety, controllability and had a high yield. There is no other impunity peak, so the purity of these cationic polymerization was high.
In the second part, the related property of catiomc grafte polymer was investigated. By analysis blockage of electrophoresis, the N/P ratios need to completely block DNA were increased with the increment of modified degree of PEI, and decreased with the diminished molecular of reactor. The diameter of PEI/DNA and graft-PEI/DNA compound prepared were between 60-400nm. As the N/P ratios increased, the diameter of compound was decreased. The diameter of compound was obviously increased if it modified by macromolecular PEI at high modified degree. The zeta potential of compound with the same N/P ratios decreased with the increase of grafting amount of surfactant. If the modified degree and N/P ratio are same, zeta potential of compound increased a litter with the decrease of molecular of surfactant. According to the result of MTT method to test cytotoxicity, using surfactant to modify PEI can decrease the cytotoxicity in some degree. In the low concentration, the cytotoxicity can be decreased obviously if every PEI was grafted a surfactant, while in the high concentration, the cytotoxity won't decrease unless every PEI was grafted more than five surfactant.
In the third part, cationic graft polymer to DNA compound was evaluated for transfection in vivo and vitro. Enhanced green fluorescent protein(GFP) plasmid and Luciferase plasmid was selected as the report genes, respectively. In order to enhance the expression of Luciferase, CMV promoter was cloned into thepGL3-basic Luciferase Reporter vector to construct recombinant plasmid pGL3-CMV Luciferase Reporter vector. In vitro transfection experiment of cationic graft polymer/DNA compound on Hela cell showed the PEI modified by low moleculae L61 and solutol did not significantly decreas the transfection efficiency, and the best transfection effect of PEI modified by low P123 is similar with that of PEI unmodified. As the increasing molecular of surfactant, grafting ratio rise and the decrease of the best transfection efficiency was followed. Expect bellows, investigate the express of Luciferase in all organ of ICR mouse who were injected compound through vena caudalis. The best transfection efficiency of low modified degree is better than that of PEL.
In the forth part, the mechanical strength of SU-8 polymer microneedle prepared by MEMS was investigated to indicated that the microneedle array can tresis the aluminium foil and ex vivo skin easily and is difficult to break into two with certain toughness and hardness. This part was also to investigate the promotion of transdermal permeation of different kinds of drag. At first, three micromolecule drag, ketoprofen, methocarbamol and Ganciclovir were selected because of their different octanol-water partition coefficient. The transdermal permeation rate of ketoprofen increase by 4.77 times, methocarbamol 6.06 and Ganciclovir 12.49, which demonstrated that logP is an important fact which can influence the transdermal permeation rate of drag and the promotion of transdermal permeation of water-solubility drag is better than liposolubility drag, with the help of microneedle. Then four macromolecule drag having different molecular weight, insuline, lysozyme, human serum albumin and ovi albumin albumin, were investigated to get the result that macromolecule drag can permeate the skin treated with microneedle array on the lever of the transdermal permeation rate of micromolecule drag while it can not permeate the intact skin and the molecular weight of biomacromolecule drag is inversely proportional to transdermal permeation rate.
The fifth part of this paper was to investigate the immune effect of DNA vaccine in vivo. The cello-phagocytosis test verificated that the phagocytosis of PEI modified remained. The anti-DNAase protection test showed the compound can protect the cell from degradation. Microneedle array can prick mouse' skin so that there were apertures about fifth micrometers in mouse' epidermis and these apertures provided passages through which compound can penetrate cuticle. Using hepatitis DNA vaccine as a model drag ,we investigated immunity effect in vivo by determining antibody titer in blood serum, cytokine excreted by spleen cell (γ-INF, IL-4) and proliferation of spleen cell. Compared with injecting cationic graft polymer to DNA compound intramuscularly and injecting hepatitis DNA vaccine aqueous solution intramuscularly, cationic polymerization/DNA vaccine compound was given by transdermal application of microneedle,after four weeks, antibody titre in blood serum and immunity effect were significantly enhanced ,strong immune response was emerged.
The sixth part of this paper was to investigate constitution irritation of cationic polymerization/DNA vaccine compound. Constitution irritation was discovered after cationic graft polymer to DNA compound was given by intramuscular injection and transdermal application of microneedle. Inflammatory reaction of intramuscular injection group was more severe than microneedle group, which illustrated cationic polymerization/DNA vaccine compound had well physiological adaption.
第一部分使用琥珀酰亚胺酯法活化非离子表面活性剂Pluronic或Solutol的末端羟基,得到活化率高,反应活度大的产物。使用活化后的聚合物按不同比例对PEI进行修饰。经过葡聚糖凝胶纯化,超滤脱盐,冷冻干燥,成功的合成了一系列不同修饰度(每个PEI分子上分别接枝1个、2个、5个、10个和20个Pluronic或Solutol)的Graft-PEI聚合物。~1H-NMR结果显示,本方法合成的阳离子聚合物接枝率高,控制投药量能定量地对PEI进行修饰,其接枝率与反应时投药量的摩尔比呈良好的线性关系,说明该合成方法比较稳定、可靠,产率高,具有良好的可控性。~1H-NMR图谱中未见其它杂质峰,证明这些阳离子聚合物的纯度较高。
第二部分重点考察了阳离子接枝聚合物/DNA复合物的相关性质。凝胶电泳阻滞分析结果显示,完全阻滞DNA所需的N/P值随着PEI修饰度的增加而增大,随着修饰物分子量的减小而减小。本实验所制备的PEI/DNA、graft-PEI/DNA复合物粒径在60-400 nm范围内。随着复合物N/P值的增加,粒径有减小的趋势。高分子量的修饰物,在高修饰度时复合物的粒径明显增大。复合物的zeta电位随N/P值的增加而增加;相同N/P值的复合物,其zeta电位随表面活性剂接枝数的增加而减少;复合物的zeta电位在相同修饰度且相同N/P值时,随表面活性剂分子量的减小而略有增加。MTT法测定细胞毒性的结果表明,使用表面活性剂修饰PEI可以不同程度地降低其细胞毒性,在低浓度时每个PEI接枝一个表面活性剂的分子即可显著降低细胞毒性。在高浓度时每个PEI接枝≥5个表面活性剂分子则可显著降低细胞毒性。
第三部分评价了阳离子接枝聚合物/DNA复合物体内外转染效果。选用了两种分别编码绿色荧光蛋白和虫荧光素酶的质粒。为了获得虫荧光素酶在体内较高的表达,将CMV启动子克隆到虫荧光素酶表达质粒pGL3-basic Luciferase Reporter vector中,构建成重组的pGL3-CMV Luciferase Reporter vector。阳离子接枝聚合物/DNA复合物在Hela细胞上的转染效果表明,低分子量的L61和Solutol修饰的PEI在低修饰度时不会显著地降低转染效果,低P123修饰度的PEI的最佳转染效果与未修饰的PEI相似。随着表面活性剂分子量增大,接枝率增加,其最佳转染效率随之下降。ICR小鼠尾静脉注射复合物,测定各个脏器中虫荧光素酶的表达,除了肺部外,低修饰度的PEI最佳转染效率显著优于未修饰的PEI。
第四部分使用微电机技术制备了SU-8聚合物微针,考察了微针的机械强度,结果表明该微针阵列可以容易地穿破铝箔和离体皮肤,并具有一定的坚韧度,不易折断。考察了微针对不同药物的体外透皮促进作用。首先考察了四种不同油水分配系数的小分子药物(吲哚美辛、酮洛芬、美索巴莫、更昔洛韦),结果表明吲哚美辛的皮肤渗透速率增加4.05倍,酮洛芬的皮肤渗透速率增加4.77倍,美索巴莫的皮肤渗透速率增加6.06倍,更昔洛韦的皮肤渗透速率增加12.49倍,说明logP值是影响药物经微针透皮传递速率的重要因素,微针对于水溶性药物的透皮促进作用较脂溶性药物更加有利。又考察了四种不同分子量的生物大分子药物(胰岛素、溶菌酶、卵清白蛋白、人血清白蛋白),结果表明与无法透过完整的皮肤相比,生物大分子药物可以透过微针阵列处理过的皮肤,经皮渗透速率可以达到小分子药物的水平。生物大分子药物分子量与经皮渗透速率成反比。
第五部分主要考察了DNA疫苗的体内免疫效果。通过细胞吞噬实验证明了修饰后的PEI其细胞吞噬过程未发生改变。抗DNAaseⅠ的保护实验表明复合物可以有效地保护DNA不被降解。微针阵列可刺入小鼠活体皮肤,并在皮肤表皮层产生几十微米的孔洞,提供了复合物穿透角质层的通道。以乙肝疫苗为模型药物,考察体内免疫效果,分别测定血清中的抗体滴度,脾细胞分泌的细胞因子(γ-INF,IL-4),及脾细胞的增殖。与乙肝DNA疫苗复合物肌注和DNA疫苗水溶液肌注相比,阳离子聚合物/DNA疫苗复合物微针经皮给药在第4周后可以显著提高血清中的抗体滴度,同时显著提高了细胞免疫水平,刺激机体产生了很强的免疫应答。
第六部分主要考察了阳离子聚合物/DNA疫苗复合物微针给药系统的组织刺激性。DNA疫苗复合物微针或肌注给药后都表现出了一定的组织刺激反应,微针给药组的炎症反应明显低于肌注给药组,说明阳离子聚合物/DNA疫苗复合物微针给药系统具有较好的生理适应性。
DNA vaccination is a technique to inoculate directly eukaryotic expression plasmid containing encoding antigenic gene into body and then the plasmid express the related antigen to produce an immunological response for protecting an organism against disease. It is vaccine of third generation on the basis of gene therapy and transgenic technology, which is a revolution for vaccine. But the DNA vaccine drug delivery system is single relatively. The major is plasmid freeze-dried powder injection which was given by intramuscular injection to delivery DNA into rhagiocrine cell, especially antigen-presenting cells (APCs) including dendritic cells(DCs).Because of the low transfection efficiency, the degree of antigen presentation was low and the immunity effect is not satified. There are a lot of dendritic cells(DCs)(Langerhans cells, 500-1000 cells mm~(-2) ) , a high-performance antigen presenting cell(APCs), which have strong immunological effect, so activity cuticular layer refers to the best part to inoculate vaccine. But hydrophilia macromolecule drug like DNA vaccine can not penetrate the keratoderma and enzyme system in skin will degrade DNA vaccine so that antigen presenting cell can not catch DNA vaccine. According that, we designed.
Microneedles refers to a needle-shaped microstructure the needles on which have the length of more than 100μm and micrometer size. It fabricated by micro-electro-mechanical systems (MEMS) technology using different material, including silicon, polymer or metal. There has a broad use in the range of biomedicine for microneedle wjich as the drug delivery system have the advantage of exact dosage, high performance, painless and convenient use. The microneedle drug delivery system can be used to activitily target to the definite depth of skin, the Langerhans cell in active cuticular layer. The genetic carrier is synthesized on the base of PEI to enhance the stability DNA in vivo. The Pluronic and Solutol with amphipathic properties are chosed to modify the chain of PEI with high molecular weight in order to enhance the stability of PEI/DNA compounds and shield the positive charge on the surface of compound to diminish the toxicity. Besides, because the hydrophobic group of Pluronic and Solutol have a certain affinity to the cell, the transfection efficiency of DNA vaccine won't be lowered in vivo and vitro. Using the hepatitis DNA vaccine as model drug, to investigate the immunity effect of mouse in vivo after giving the compound of cationic polymer and DNA vaccine by transdermal application of microneedle.
In the first part, succinimide ester was used to activation the terminal hydroxyl of Pluronic or solutol, nonionic wetting agent,to get a product with high activation efficiency and activity series. The activation compound was modified with PEI in the different proportion. After the purification through polydextran gel, the product was ultrafiltrated to get rid of salt and then cryodesiccated. According to the above process, we successfully got Graft-PEI compound with a series of different degree of modification per one PEI macromolecule at 1,2,5,10 and 20 Pluronic or Solutol. The result of ~1H-NMR showed the cationic polymerization synthesized by this method had a high graft ratio and PEI can be modified quantitatively by controlling administration dosage molar ratio of which had a satisfactory linear relationship with graft ratio. All that demonstrated that that synthetic method was stability, safety, controllability and had a high yield. There is no other impunity peak, so the purity of these cationic polymerization was high.
In the second part, the related property of catiomc grafte polymer was investigated. By analysis blockage of electrophoresis, the N/P ratios need to completely block DNA were increased with the increment of modified degree of PEI, and decreased with the diminished molecular of reactor. The diameter of PEI/DNA and graft-PEI/DNA compound prepared were between 60-400nm. As the N/P ratios increased, the diameter of compound was decreased. The diameter of compound was obviously increased if it modified by macromolecular PEI at high modified degree. The zeta potential of compound with the same N/P ratios decreased with the increase of grafting amount of surfactant. If the modified degree and N/P ratio are same, zeta potential of compound increased a litter with the decrease of molecular of surfactant. According to the result of MTT method to test cytotoxicity, using surfactant to modify PEI can decrease the cytotoxicity in some degree. In the low concentration, the cytotoxicity can be decreased obviously if every PEI was grafted a surfactant, while in the high concentration, the cytotoxity won't decrease unless every PEI was grafted more than five surfactant.
In the third part, cationic graft polymer to DNA compound was evaluated for transfection in vivo and vitro. Enhanced green fluorescent protein(GFP) plasmid and Luciferase plasmid was selected as the report genes, respectively. In order to enhance the expression of Luciferase, CMV promoter was cloned into thepGL3-basic Luciferase Reporter vector to construct recombinant plasmid pGL3-CMV Luciferase Reporter vector. In vitro transfection experiment of cationic graft polymer/DNA compound on Hela cell showed the PEI modified by low moleculae L61 and solutol did not significantly decreas the transfection efficiency, and the best transfection effect of PEI modified by low P123 is similar with that of PEI unmodified. As the increasing molecular of surfactant, grafting ratio rise and the decrease of the best transfection efficiency was followed. Expect bellows, investigate the express of Luciferase in all organ of ICR mouse who were injected compound through vena caudalis. The best transfection efficiency of low modified degree is better than that of PEL.
In the forth part, the mechanical strength of SU-8 polymer microneedle prepared by MEMS was investigated to indicated that the microneedle array can tresis the aluminium foil and ex vivo skin easily and is difficult to break into two with certain toughness and hardness. This part was also to investigate the promotion of transdermal permeation of different kinds of drag. At first, three micromolecule drag, ketoprofen, methocarbamol and Ganciclovir were selected because of their different octanol-water partition coefficient. The transdermal permeation rate of ketoprofen increase by 4.77 times, methocarbamol 6.06 and Ganciclovir 12.49, which demonstrated that logP is an important fact which can influence the transdermal permeation rate of drag and the promotion of transdermal permeation of water-solubility drag is better than liposolubility drag, with the help of microneedle. Then four macromolecule drag having different molecular weight, insuline, lysozyme, human serum albumin and ovi albumin albumin, were investigated to get the result that macromolecule drag can permeate the skin treated with microneedle array on the lever of the transdermal permeation rate of micromolecule drag while it can not permeate the intact skin and the molecular weight of biomacromolecule drag is inversely proportional to transdermal permeation rate.
The fifth part of this paper was to investigate the immune effect of DNA vaccine in vivo. The cello-phagocytosis test verificated that the phagocytosis of PEI modified remained. The anti-DNAase protection test showed the compound can protect the cell from degradation. Microneedle array can prick mouse' skin so that there were apertures about fifth micrometers in mouse' epidermis and these apertures provided passages through which compound can penetrate cuticle. Using hepatitis DNA vaccine as a model drag ,we investigated immunity effect in vivo by determining antibody titer in blood serum, cytokine excreted by spleen cell (γ-INF, IL-4) and proliferation of spleen cell. Compared with injecting cationic graft polymer to DNA compound intramuscularly and injecting hepatitis DNA vaccine aqueous solution intramuscularly, cationic polymerization/DNA vaccine compound was given by transdermal application of microneedle,after four weeks, antibody titre in blood serum and immunity effect were significantly enhanced ,strong immune response was emerged.
The sixth part of this paper was to investigate constitution irritation of cationic polymerization/DNA vaccine compound. Constitution irritation was discovered after cationic graft polymer to DNA compound was given by intramuscular injection and transdermal application of microneedle. Inflammatory reaction of intramuscular injection group was more severe than microneedle group, which illustrated cationic polymerization/DNA vaccine compound had well physiological adaption.
引文
1.孙树汉.核酸疫苗.上海,第二军医大学出版社,2000:1-7.
2.Belakova J,Horynova M,Krupka M,et al.DNA vaccines:are they still just a powerful tool for the future[J]? Arch Immunol Ther Exp,2007,55(6):387-398
3.Lu S,Wang S,Grimes-Serrano JM.Current progress of DNA vaccine studies in humans[J].Expert Rev Vaccines,2008,7(2):175-191.
4.Nchinda G,Kuroiwa J,Oks M,et al.The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells[J].J Clin Invest,2008,118(4):1427-1436.
5.Foged C,Sundblad A,Hovgaard L.Targeting vaccines to dendritic cells[J].Pharm Res,2002,19(3):229-238.
6.Manam S,Ledmith BJ,Barnum AB,et al.Plasmid DNA vaccines:tissue distribution and effect of DNA sequence,adjuvants and delivery method on integration into host DNA[J].Intervirolgy,2000,43(4-6):273-281.
7.Robertson JS,Griffiths E.Assuring the quality,safety,and efficacy of DNA vaccines[J].Mol Biotechnol,2001,17(2):143-149.
8.Gebhart CL,Kabanov AV.Evaluation of polyplexes as gene transfer agents[J].J Control Release,2001,73(2-3):401-416.
9.Boussif O,Lezoualc'h F,Zanta MA,et al.A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine[J].Proc Natl Acad Sci USA,1995,92(16):7297-7301.
10.Godbey WT,Wu KK,Midos AG,et al.Tracking the intracelular path of poly(ethylenimine) DNA complexes for gene delivery[J].Proc Natl Acad Sci USA,1999,96(9):5177-5181.
11.Godbey WT,Wu KK,Mikos AG.Poly(ethylenimine) mediated gene delivery affects endothelial cell function and viability[J].Biomaterials,2001,22(5):471-480.
12.Dailey LA,Kleemann E,Merdan T,et al.Modified polyethylenimines as non viral gene delivery systems for aerosol therapy:effects of nebulization on cellular uptake and transfection efficiency[J].J of Control Release,2004,100(3):425-436.
13.Kabanov AV,Lemieux P,Vinogradov S,et al.Pluronic block copolymers:novel functional molecules for gene therapy[J].Adv Drug Deliv Rev,2002,54(2):223-233.
14.许宝建,金庆辉,赵建龙.基于MEMS微针技术的研究现状与展望.微纳电子技术,2005,42(4):150-1566.
15.Foged C,Sundblad A,Hovgaard L.Targeting vaccines to dendritic cells[J].Pharm Res,2002,19(3):229-238.
16. Omid Akbari, Naveed Panjwani, Sylvie Garcia, et al. DNA vaccination: transfection and activation of dedritic cells as key events for immunity[J]. J Exp Med, 1999,189(1):169-177.
17. Mikszta JA, Alarcon JB, Brittingham JM, et al. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery[J]. Nature, 2002, 8(4):415-419.
18. Matriano JA, Cormier M, Johnson J, et al. Macroflux microprojection array patch technology: a new and efficient approach for intracutaneous immunization[J]. Pharm Res, 2002, 19(1):63-70.
19. Li S, Huang L. Nonviral gene therapy: promises and challenges[J]. Gene Ther, 2000, 7(1):31-34.
20. Thomas M, Klibanov AM. Non-viral gene therapy: polycation-mediated DNA delivery[J]. Appl Microbiol Biotechnol, 2003, 62(1):27-34.
21. Fischer D, Li Y, Ahlemeyer B, et al. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis[J]. Biomaterials, 2003,24(7):1121-1131.
22. Fischer D, Bieber T, Li YX, et al. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity[J]. Pharm Res, 16(8):1273-1279.
23. Hong JW, Park JH, Huh KM, et al. PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system[J]. J Control Release, 99(1): 167-176.
24. Gebhart CL, Sriadibhatla S, Vinogradov S, et al. Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer[J]. Bioconjug Chem, 2002, 13(5):937-944.
25. Shmuel ZE. Active carbonates of polyalkylene oxides for modification of polypeptides [P]. US, A,US5122614,1992-06-16.
26. Miron T, Wilchek M. A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters[J]. Anal Biochem, 1982, 126(2):433-435
27. Snyder SL, Sobocinski PZ. An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines[J]. Anal Biochem, 1975(1), 64:284-8.
28. Cho KC, Choi SH, Park TG. Low molecular weight pei conjugated pluronic copolymer: useful additive for enhancing gene transfection efficiency[J]. Macromol Res, 2006, 14(3): 348-353.
29. Kim TH, Cook SE, Arote RB, et al. A degradable hyperbranched poly(ester amine) based on poloxamer diacrylate and polyethylenimine as a gene carrier[J]. Macromol Biosci, 2007, 7(5):611-619.
30．成俊然,文佳,邰瑞链．双(三氯甲基)碳酸酯的制备和应用[J]．化学通报,1999,4()：20-25．
31. Gebhart CL, Sriadibhatla S, Vinogradov S, et al. Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer[J]. Bioconjug Chem, 2002, 13(5):937-944.
32. Nguyen HK, Lemieux P, Vinogradov SV, et al. Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents[J]. Gene Ther, 2000, 7(2): 126-38.
33. Jeon E, Kim HD, Kim JS. Pluronic-grafted poly-(L)-lysine as a new synthetic gene carrier[J]. J Biomed Mater Res A, 2003, 66(4):854-859.
34. Ogris M, Steinlein P, Kursa M, et al. The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells[J]. Gene Ther, 1998,1425-1433.
35. Tang GP, Zeng JM, Gao SJ, et al. Polyethylene glycol modified polyethylenimine for improved CNS gene transfer: effects of PEGylation extent[J]..Biomaterials, 2003,24(13):2351-2362.
36. Choosakoonkriang S, Lobo BA, Koe GS. Biophysical characterization of PEI/DNA complexes[J]. J Pharm Sci, 2003,92(8): 1710-1719.
37. Ogris M, Wallker G, Blessing T. Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes[J]. J Control Release, 2003, 91(1-2):173-181.
38. Erbacher P, Bettinger T, Brion E. Genuine DNA/polyethylenimine(PEI) complexes improve transfection properties and cell survival[J]. J Drug Target, 2004, 12(4):223-236.
39. Ongpipattnakul B, Burnette RR, Potts RO, et al. Evidencethat oleic acid exists in a separate phase within stratum corneum lipids[J]. Pharm Res, 1991, 8(3):350-354
40. McAllister DV, Wang PM, Davis SP, et al. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies[J]. Proc Nat Acad Sci, 2003,100(24): 13755-13760.
41. Moon SJ, Lee SS. Fabrication of microneedle array using inclined LIGA process. 12-(th) Int. Conf. S-State Sensors, Actuators and Microsystem S.Boston, June 8-12,2003.
42. Finin BC, Morgan TM. Transdermal penetration enhancers: applications, limitations, and potential[J]. J Pharm Sci, 1999, 88(10):955-958.
43. Costello CT, Jeske A H. Iontophoresis: applications in transdermal medication delivery[J]. Phys Ther, 1995,75(6):554-563.
44. Davis SP, Martanto W, Allen MG, et al. Hollow metal microneedles for insulin delivery to diabetic rats[J]. IEEE Trans Biomed Eng, 2005, 52(5):909-915.
45. Martanto W, Davis SP, Holiday NR, et al. Transdermal delivery of insulin using microneedles in vivo[J]. Pharm Res, 2004,21(6):947-952
46. Kim S, Shetty S, Price D, Bhansali S. Skin penetration of silicon dioxide microneedle arrays[J]. Conf Proc IEEE Eng Med Biol Soc, 2006,1:4088-4091.
47. Teo MA, Shearwood C, Ng KC, Lu J, et al. In vitro and in vivo characterization of MEMS microneedles[J]. Biomed Microdevices, 2005,7(1):47-52.
48. Park JH, Yoon YK, Choi SO, et al. Tapered conical polymer microneedles fabricated using an integrated lens technique for transdermal drug delivery[J]. IEEE Trans Biomed Eng, 2007, 54(5):903-913.
49. Park JH, Allen MG, Prausnitz MR. Polymer microneedles for controlled-release drug delivery[J]. Pharm Res. 2006, 23(5): 1008-1019.
50. Wu XM, Todo H, Sugibayashi K. Effects of pretreatment of needle puncture and sandpaper abrasion on the in vitro skin permeation of fluorescein isothiocyanate (FITC)-dextran[J]. Int J Pharm, 2006, 316(1-2): 102-108.
51. Pearton M, Allender C, Brain K, et al. Gene Delivery to the Epidermal Cells of Human Skin Explants Using Microfabricated Microneedles and Hydrogel Formμlations[J]. Pharm Res, 2007, 25(2): 407-416.
52. Wang J, Hu JH, Li FQ, Liu GZ, et al. Strong cellular and humoral immune responses induced by transcutaneous immunization with HBsAg DNA-cationic deformable liposome complex[J]. Exp Dermatol, 2007, 16(9):724-729.
53. Mahor S, Rawat A, Dubey PK, et al. Cationic transfersomes based topical genetic vaccine against hepatitis B[J]. Int J Pharm, 2007, 340(1-2): 13-19.
54. McCloskey PS, Salo RJ. Flow cytometric analysis of group B streptococci phagocytosis and oxidative burst in human neutrophils and monocytes[J]. FEMS Immunol Med Microbiol, 2000, 27(1):59-65.
55. DeYulia GJ Jr, Carcamo JM, B6rquez-Ojeda O, et al. Hydrogen peroxide generated extracellularly by receptor-ligand interaction facilitates cell signaling[J]. Proc Natl Acad Sci U S A, 2005, 102(14):5044-5049
56. Pachuk CJ, Mccallus DE, Weiner DB, et al. DNA vaccines-challenges in delivery[J]. Curr Opin Mol Ther, 2000, 2(2):188-198.
57. Dubensky TW Jr, Liu MA, Mlmer JB. Delivery systems for gene-based vaccines[J]. Mol Med, 2000,6(9):723-732.
58. Coulman SA, Barrow D, Anstey A, et al. Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles[J]. Curr Drug Deliv, 2006, 3(1):65-75.
59. Belakova J, Horynova M, Krupka M, et al. DNA vaccines: are they still just a powerful tool for the future[J]? Arch Immunol Ther Exp, 2007, 55(6):387-398
60. Lu S, Wang S, Grimes-Serrano JM. Current progress of DNA vaccine studies in humans[J]. Expert Rev Vaccines, 2008, 7(2): 175-191.
61. Nchinda G, Kuroiwa J, Oks M, et al. The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells[J]. J Clin Invest, 2008,118(4): 1427-1436.
62. Foged C, Sundblad A, Hovgaard L. Targeting vaccines to dendritic cells[J]. Pharm Res, 2002, 19(3):229-238.
63. Debenedictis C, Joubeh S, Zhang G, et al. Immune functions of the skin[J]. Clin Dermatol, 2001, 19(5):573-585.
64. Berger CL, Edelson R. The life cycle of cutaneous T cell lymphoma reveals opportunities for targeted drug therapy[J]. Curr Cancer Drug Targets, 2004,4(7):609-619.
65. Li L, Hoffman RM. The feasibility of targeted selective gene therapy of the hair follicle[J]. Nat Med, 1995, 1:705-706.
66. Fan H, Lin Q, Morrissey GM, et al. Immunization via hair follicles by topical application of naked DNA to normal skin[J]. Nat Biotechnol, 1999,17:870-872.
67. Shi Z, Curiel DT, Tang DC. DNA-based non-invasive vaccination onto the skin[J]. Vaccine, 1999, 17(17):2136-2141.
68. Watabe S, Xin KQ, Ihata A, et al. Protection against influenza virus challenge by topical application of influenza DNA vaccine[J]. Vaccine, 2001,19(31):4434-4444.
69. Mahor S, Rawat A, Dubey PK, et al. Cationic transfersomes based topical genetic vaccine against hepatitis B[J]. Int J Pharm, 2007,340(1-2):13-19.
70. Wang J, Hu JH, Li FQ, et al. Strong cellular and humoral immune responses induced by transcutaneous immunization with HBsAg DNA-cationic deformable liposome complex[J]. Exp Dermatol, 2007, 16(9):724-729.
71. Nasseri B. Efect of cholesterol and temperature on the elastic properties of niosomal membranes[J]. Int J Pharm, 2005, 300(1-2): 95-101
72. Vyas SP, Singh RP, Jain S, et al. Non-ionic surfactant based vesicles (niosomes) for non-invasive topical genetic immunization against hepatitis B[J]. Int J Pharm, 2005,296(1-2):80-6.
73. Wu H, Ramachandran C, Bielinska AU, et al. Topical transfection using plasmid DNA in a water-in-oil nanoemulsion[J]. Int J Pharm, 2001, 221(1-2):23-34.
74. Cui Z, Fountain W, Clark M, et al. Novel ethanol-in-fluorocarbon microemulsions for topical genetic immunization[J]. Pharm Res, 2003, 20(1): 16-23.
75. Cui Z, Mumper RJ. Chitosan-based nanoparticles for topical genetic immunization[J]. J Control Release, 2001,75(3):409-419.
76. Cui Z, Mumper RJ. Topical immunization using nanoengineered genetic vaccines[J]. J Control Release, 2002, 81(1-2):173-84.
77. Heckert RA, Elankumaran S, Oshop GL, et al. A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization[J]. Vet Immunol Immunopathol, 2002, 89(1-2):67-81.
78. Lori F, Trocio J, Bakare N, et al. DermaVir, a novel HIV immunisation technology[J]. Vaccine, 2005, 23(17-18):2030-2034.
79. Calarota SA, Weiner DB, Lori F, et al. Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine[J]. Vaccine, 2007,25(16):3070-3074.
80. Drabick JJ, Glasspool-Malone J, King A, et al. Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization[J]. Mol Ther, 2001, 3(2):249-255.
81. Luxembourg A, Hannaman D, Ellefsen B, et al. Enhancement of immune responses to an HBV DNA vaccine by electroporation[J]. Vaccine, 2006,24(21):4490-4493
82. Birchall J, Coulman S, Anstey A, et al. Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation[J]. Int J Pharm, 2006, 312(1-2):15-23.
83. Fang JY, Lee WR, Shen SC, et al. Transdermal delivery of macromolecules by erbium:YAG laser[J]. J Control Release, 2004, 100(1):75-85.
84. Lee WR, Shen SC, Liu CR, et al. Erbium: YAG laser-mediated oligonucleotide and DNA delivery via the skin: an animal study[J]. J Control Release, 2006, 115(3):344-353.
85. Liu LJ, Watabe S, Yang J, et al. Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses[J]. Vaccine, 2001,20(1-2):42-8.
86. Gregersen JP. DNA vaccines[J]. Naturwissenschaften, 2001, 88(12):504-513.
87. Steele KE, Stabler K, VanderZanden L. Cutaneous DNA vaccination against Ebola virus by particle bombardment: histopathology and alteration of CD3-positive dendritic epidermal cells[J]. Vet Pathol,2001,38(2):203-15.
88. Raju PA, McSloy N, Truong NK, et al. Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles[J]. Vaccine, 2006 , 24(21):4644-7.
89. Braun RP, Babiuk LA, Loehr BI, et al. Particle-mediated DNA immunization of cattle confers long-lasting immunity against bovine herpesvirus-1. Virology, 1999,265(1):46-56
90. Gaffal E, Schweichel D, Tormo D, et al. Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells[J]. Eur J Cell Biol, 2007, 86(11-12):817-26.
91. Stoecklinger A, Grieshuber I, Scheiblhofer S, et al. Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization[J]. J Immunol, 2007, 179(2):886-93
92. Walther W, Stein U, Fichtner I, et al. Intratumoral low-volume jet-injection for efficient nonviral gene transfer[J]. Mol Biotechnol, 2002, 21(2):105-l 15.
93. Cui Z, Baizer L, Mumper RJ. Intradermal immunization with novel plasmid DNA-coated nanoparticles via a needle-free injection device. J Biotechnol, 2003, 102(2): 105-115
94. Henry S, McAllister DV, Allen MG, et al. Microfabricated microneedles: A novel approach to transdermal drug delivery[J]. J Pharm Sci, 1999, 88(9):948.
95. McAllister DV, Wang PM, Davis SP, et al. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies[J]. Proc Nat Acad Sci, 2003,100(24): 13755-13760
96. Cormier M, Johnson B, Ameri M, et al. Transdermal delivery of desmopressin using a coated microneedle array patch system[J]. J Control Release, 2004, 97 (3): 503-511.
97. Coulman SA, Barrow D, Anstey A, et al. Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles[J]. Curr Drug Deliv, 2006, 3(1):65-75
98. Chabri F, Bouris K, Jones T, et al. Microfabricated silicon microneedles for nonviral cutaneous gene delivery[J]. Br J Dermatol, 2004, 150(5):869-877.
99. Mikszta JA, Alarcon JB, Brittingham JM, et al. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery[J]. Nature, 2002,8(4):415-419.
100. Alarcon JB, Hartley AW, Harvey NG, et al. Preclinical evaluation of microneedle technology for intradermal delivery of influenza vaccines. Clin Vaccine Immunol, 2007, 14(4):375-381.
101. Gill HS, Prausnitz MR. Coated microneedles for transdermal delivery[J]. J Control Release, 2007, 117(2):227-237.
102. Shirkhanzadeh M. Microneedles coated with porous calcium phosphate ceramics: effective vehicles for transdermal delivery of solid trehalose[J]. J Mater Sci Mater Med, 2005, 16(1):37-45.
103. Matriano JA, Cormier M, Johnson J, et al. Macroflux microprojection array patch technology: a new and efficient approach for intracutaneous immunization[J]. Pharm Res, 2002, 19(1):63-70.
104. Lin W, Cormier M, Samiee A, et al. Transdermal delivery of antisense oligonucleotides with microprojection patch (Macroflux)technology[J], Pharm Res, 2001,18 : 1789-1793.
105. Hooper JW, Golden JW, Ferro AM, et al. Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge[J]. Vaccine, 2007, 25(10):1814-1823.
106. Pearton M, Allender C, Brain K, et al. Gene delivery to the epidermal cells of human skin explants using microfabricated microneedles and hydrogel formulations[J]. Pharm Res, 2008, 25(2):407-16.
2.Belakova J,Horynova M,Krupka M,et al.DNA vaccines:are they still just a powerful tool for the future[J]? Arch Immunol Ther Exp,2007,55(6):387-398
3.Lu S,Wang S,Grimes-Serrano JM.Current progress of DNA vaccine studies in humans[J].Expert Rev Vaccines,2008,7(2):175-191.
4.Nchinda G,Kuroiwa J,Oks M,et al.The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells[J].J Clin Invest,2008,118(4):1427-1436.
5.Foged C,Sundblad A,Hovgaard L.Targeting vaccines to dendritic cells[J].Pharm Res,2002,19(3):229-238.
6.Manam S,Ledmith BJ,Barnum AB,et al.Plasmid DNA vaccines:tissue distribution and effect of DNA sequence,adjuvants and delivery method on integration into host DNA[J].Intervirolgy,2000,43(4-6):273-281.
7.Robertson JS,Griffiths E.Assuring the quality,safety,and efficacy of DNA vaccines[J].Mol Biotechnol,2001,17(2):143-149.
8.Gebhart CL,Kabanov AV.Evaluation of polyplexes as gene transfer agents[J].J Control Release,2001,73(2-3):401-416.
9.Boussif O,Lezoualc'h F,Zanta MA,et al.A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:polyethylenimine[J].Proc Natl Acad Sci USA,1995,92(16):7297-7301.
10.Godbey WT,Wu KK,Midos AG,et al.Tracking the intracelular path of poly(ethylenimine) DNA complexes for gene delivery[J].Proc Natl Acad Sci USA,1999,96(9):5177-5181.
11.Godbey WT,Wu KK,Mikos AG.Poly(ethylenimine) mediated gene delivery affects endothelial cell function and viability[J].Biomaterials,2001,22(5):471-480.
12.Dailey LA,Kleemann E,Merdan T,et al.Modified polyethylenimines as non viral gene delivery systems for aerosol therapy:effects of nebulization on cellular uptake and transfection efficiency[J].J of Control Release,2004,100(3):425-436.
13.Kabanov AV,Lemieux P,Vinogradov S,et al.Pluronic block copolymers:novel functional molecules for gene therapy[J].Adv Drug Deliv Rev,2002,54(2):223-233.
14.许宝建,金庆辉,赵建龙.基于MEMS微针技术的研究现状与展望.微纳电子技术,2005,42(4):150-1566.
15.Foged C,Sundblad A,Hovgaard L.Targeting vaccines to dendritic cells[J].Pharm Res,2002,19(3):229-238.
16. Omid Akbari, Naveed Panjwani, Sylvie Garcia, et al. DNA vaccination: transfection and activation of dedritic cells as key events for immunity[J]. J Exp Med, 1999,189(1):169-177.
17. Mikszta JA, Alarcon JB, Brittingham JM, et al. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery[J]. Nature, 2002, 8(4):415-419.
18. Matriano JA, Cormier M, Johnson J, et al. Macroflux microprojection array patch technology: a new and efficient approach for intracutaneous immunization[J]. Pharm Res, 2002, 19(1):63-70.
19. Li S, Huang L. Nonviral gene therapy: promises and challenges[J]. Gene Ther, 2000, 7(1):31-34.
20. Thomas M, Klibanov AM. Non-viral gene therapy: polycation-mediated DNA delivery[J]. Appl Microbiol Biotechnol, 2003, 62(1):27-34.
21. Fischer D, Li Y, Ahlemeyer B, et al. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis[J]. Biomaterials, 2003,24(7):1121-1131.
22. Fischer D, Bieber T, Li YX, et al. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: effect of molecular weight on transfection efficiency and cytotoxicity[J]. Pharm Res, 16(8):1273-1279.
23. Hong JW, Park JH, Huh KM, et al. PEGylated polyethylenimine for in vivo local gene delivery based on lipiodolized emulsion system[J]. J Control Release, 99(1): 167-176.
24. Gebhart CL, Sriadibhatla S, Vinogradov S, et al. Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer[J]. Bioconjug Chem, 2002, 13(5):937-944.
25. Shmuel ZE. Active carbonates of polyalkylene oxides for modification of polypeptides [P]. US, A,US5122614,1992-06-16.
26. Miron T, Wilchek M. A spectrophotometric assay for soluble and immobilized N-hydroxysuccinimide esters[J]. Anal Biochem, 1982, 126(2):433-435
27. Snyder SL, Sobocinski PZ. An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines[J]. Anal Biochem, 1975(1), 64:284-8.
28. Cho KC, Choi SH, Park TG. Low molecular weight pei conjugated pluronic copolymer: useful additive for enhancing gene transfection efficiency[J]. Macromol Res, 2006, 14(3): 348-353.
29. Kim TH, Cook SE, Arote RB, et al. A degradable hyperbranched poly(ester amine) based on poloxamer diacrylate and polyethylenimine as a gene carrier[J]. Macromol Biosci, 2007, 7(5):611-619.
30．成俊然,文佳,邰瑞链．双(三氯甲基)碳酸酯的制备和应用[J]．化学通报,1999,4()：20-25．
31. Gebhart CL, Sriadibhatla S, Vinogradov S, et al. Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer[J]. Bioconjug Chem, 2002, 13(5):937-944.
32. Nguyen HK, Lemieux P, Vinogradov SV, et al. Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents[J]. Gene Ther, 2000, 7(2): 126-38.
33. Jeon E, Kim HD, Kim JS. Pluronic-grafted poly-(L)-lysine as a new synthetic gene carrier[J]. J Biomed Mater Res A, 2003, 66(4):854-859.
34. Ogris M, Steinlein P, Kursa M, et al. The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells[J]. Gene Ther, 1998,1425-1433.
35. Tang GP, Zeng JM, Gao SJ, et al. Polyethylene glycol modified polyethylenimine for improved CNS gene transfer: effects of PEGylation extent[J]..Biomaterials, 2003,24(13):2351-2362.
36. Choosakoonkriang S, Lobo BA, Koe GS. Biophysical characterization of PEI/DNA complexes[J]. J Pharm Sci, 2003,92(8): 1710-1719.
37. Ogris M, Wallker G, Blessing T. Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes[J]. J Control Release, 2003, 91(1-2):173-181.
38. Erbacher P, Bettinger T, Brion E. Genuine DNA/polyethylenimine(PEI) complexes improve transfection properties and cell survival[J]. J Drug Target, 2004, 12(4):223-236.
39. Ongpipattnakul B, Burnette RR, Potts RO, et al. Evidencethat oleic acid exists in a separate phase within stratum corneum lipids[J]. Pharm Res, 1991, 8(3):350-354
40. McAllister DV, Wang PM, Davis SP, et al. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies[J]. Proc Nat Acad Sci, 2003,100(24): 13755-13760.
41. Moon SJ, Lee SS. Fabrication of microneedle array using inclined LIGA process. 12-(th) Int. Conf. S-State Sensors, Actuators and Microsystem S.Boston, June 8-12,2003.
42. Finin BC, Morgan TM. Transdermal penetration enhancers: applications, limitations, and potential[J]. J Pharm Sci, 1999, 88(10):955-958.
43. Costello CT, Jeske A H. Iontophoresis: applications in transdermal medication delivery[J]. Phys Ther, 1995,75(6):554-563.
44. Davis SP, Martanto W, Allen MG, et al. Hollow metal microneedles for insulin delivery to diabetic rats[J]. IEEE Trans Biomed Eng, 2005, 52(5):909-915.
45. Martanto W, Davis SP, Holiday NR, et al. Transdermal delivery of insulin using microneedles in vivo[J]. Pharm Res, 2004,21(6):947-952
46. Kim S, Shetty S, Price D, Bhansali S. Skin penetration of silicon dioxide microneedle arrays[J]. Conf Proc IEEE Eng Med Biol Soc, 2006,1:4088-4091.
47. Teo MA, Shearwood C, Ng KC, Lu J, et al. In vitro and in vivo characterization of MEMS microneedles[J]. Biomed Microdevices, 2005,7(1):47-52.
48. Park JH, Yoon YK, Choi SO, et al. Tapered conical polymer microneedles fabricated using an integrated lens technique for transdermal drug delivery[J]. IEEE Trans Biomed Eng, 2007, 54(5):903-913.
49. Park JH, Allen MG, Prausnitz MR. Polymer microneedles for controlled-release drug delivery[J]. Pharm Res. 2006, 23(5): 1008-1019.
50. Wu XM, Todo H, Sugibayashi K. Effects of pretreatment of needle puncture and sandpaper abrasion on the in vitro skin permeation of fluorescein isothiocyanate (FITC)-dextran[J]. Int J Pharm, 2006, 316(1-2): 102-108.
51. Pearton M, Allender C, Brain K, et al. Gene Delivery to the Epidermal Cells of Human Skin Explants Using Microfabricated Microneedles and Hydrogel Formμlations[J]. Pharm Res, 2007, 25(2): 407-416.
52. Wang J, Hu JH, Li FQ, Liu GZ, et al. Strong cellular and humoral immune responses induced by transcutaneous immunization with HBsAg DNA-cationic deformable liposome complex[J]. Exp Dermatol, 2007, 16(9):724-729.
53. Mahor S, Rawat A, Dubey PK, et al. Cationic transfersomes based topical genetic vaccine against hepatitis B[J]. Int J Pharm, 2007, 340(1-2): 13-19.
54. McCloskey PS, Salo RJ. Flow cytometric analysis of group B streptococci phagocytosis and oxidative burst in human neutrophils and monocytes[J]. FEMS Immunol Med Microbiol, 2000, 27(1):59-65.
55. DeYulia GJ Jr, Carcamo JM, B6rquez-Ojeda O, et al. Hydrogen peroxide generated extracellularly by receptor-ligand interaction facilitates cell signaling[J]. Proc Natl Acad Sci U S A, 2005, 102(14):5044-5049
56. Pachuk CJ, Mccallus DE, Weiner DB, et al. DNA vaccines-challenges in delivery[J]. Curr Opin Mol Ther, 2000, 2(2):188-198.
57. Dubensky TW Jr, Liu MA, Mlmer JB. Delivery systems for gene-based vaccines[J]. Mol Med, 2000,6(9):723-732.
58. Coulman SA, Barrow D, Anstey A, et al. Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles[J]. Curr Drug Deliv, 2006, 3(1):65-75.
59. Belakova J, Horynova M, Krupka M, et al. DNA vaccines: are they still just a powerful tool for the future[J]? Arch Immunol Ther Exp, 2007, 55(6):387-398
60. Lu S, Wang S, Grimes-Serrano JM. Current progress of DNA vaccine studies in humans[J]. Expert Rev Vaccines, 2008, 7(2): 175-191.
61. Nchinda G, Kuroiwa J, Oks M, et al. The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells[J]. J Clin Invest, 2008,118(4): 1427-1436.
62. Foged C, Sundblad A, Hovgaard L. Targeting vaccines to dendritic cells[J]. Pharm Res, 2002, 19(3):229-238.
63. Debenedictis C, Joubeh S, Zhang G, et al. Immune functions of the skin[J]. Clin Dermatol, 2001, 19(5):573-585.
64. Berger CL, Edelson R. The life cycle of cutaneous T cell lymphoma reveals opportunities for targeted drug therapy[J]. Curr Cancer Drug Targets, 2004,4(7):609-619.
65. Li L, Hoffman RM. The feasibility of targeted selective gene therapy of the hair follicle[J]. Nat Med, 1995, 1:705-706.
66. Fan H, Lin Q, Morrissey GM, et al. Immunization via hair follicles by topical application of naked DNA to normal skin[J]. Nat Biotechnol, 1999,17:870-872.
67. Shi Z, Curiel DT, Tang DC. DNA-based non-invasive vaccination onto the skin[J]. Vaccine, 1999, 17(17):2136-2141.
68. Watabe S, Xin KQ, Ihata A, et al. Protection against influenza virus challenge by topical application of influenza DNA vaccine[J]. Vaccine, 2001,19(31):4434-4444.
69. Mahor S, Rawat A, Dubey PK, et al. Cationic transfersomes based topical genetic vaccine against hepatitis B[J]. Int J Pharm, 2007,340(1-2):13-19.
70. Wang J, Hu JH, Li FQ, et al. Strong cellular and humoral immune responses induced by transcutaneous immunization with HBsAg DNA-cationic deformable liposome complex[J]. Exp Dermatol, 2007, 16(9):724-729.
71. Nasseri B. Efect of cholesterol and temperature on the elastic properties of niosomal membranes[J]. Int J Pharm, 2005, 300(1-2): 95-101
72. Vyas SP, Singh RP, Jain S, et al. Non-ionic surfactant based vesicles (niosomes) for non-invasive topical genetic immunization against hepatitis B[J]. Int J Pharm, 2005,296(1-2):80-6.
73. Wu H, Ramachandran C, Bielinska AU, et al. Topical transfection using plasmid DNA in a water-in-oil nanoemulsion[J]. Int J Pharm, 2001, 221(1-2):23-34.
74. Cui Z, Fountain W, Clark M, et al. Novel ethanol-in-fluorocarbon microemulsions for topical genetic immunization[J]. Pharm Res, 2003, 20(1): 16-23.
75. Cui Z, Mumper RJ. Chitosan-based nanoparticles for topical genetic immunization[J]. J Control Release, 2001,75(3):409-419.
76. Cui Z, Mumper RJ. Topical immunization using nanoengineered genetic vaccines[J]. J Control Release, 2002, 81(1-2):173-84.
77. Heckert RA, Elankumaran S, Oshop GL, et al. A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization[J]. Vet Immunol Immunopathol, 2002, 89(1-2):67-81.
78. Lori F, Trocio J, Bakare N, et al. DermaVir, a novel HIV immunisation technology[J]. Vaccine, 2005, 23(17-18):2030-2034.
79. Calarota SA, Weiner DB, Lori F, et al. Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine[J]. Vaccine, 2007,25(16):3070-3074.
80. Drabick JJ, Glasspool-Malone J, King A, et al. Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization[J]. Mol Ther, 2001, 3(2):249-255.
81. Luxembourg A, Hannaman D, Ellefsen B, et al. Enhancement of immune responses to an HBV DNA vaccine by electroporation[J]. Vaccine, 2006,24(21):4490-4493
82. Birchall J, Coulman S, Anstey A, et al. Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation[J]. Int J Pharm, 2006, 312(1-2):15-23.
83. Fang JY, Lee WR, Shen SC, et al. Transdermal delivery of macromolecules by erbium:YAG laser[J]. J Control Release, 2004, 100(1):75-85.
84. Lee WR, Shen SC, Liu CR, et al. Erbium: YAG laser-mediated oligonucleotide and DNA delivery via the skin: an animal study[J]. J Control Release, 2006, 115(3):344-353.
85. Liu LJ, Watabe S, Yang J, et al. Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses[J]. Vaccine, 2001,20(1-2):42-8.
86. Gregersen JP. DNA vaccines[J]. Naturwissenschaften, 2001, 88(12):504-513.
87. Steele KE, Stabler K, VanderZanden L. Cutaneous DNA vaccination against Ebola virus by particle bombardment: histopathology and alteration of CD3-positive dendritic epidermal cells[J]. Vet Pathol,2001,38(2):203-15.
88. Raju PA, McSloy N, Truong NK, et al. Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles[J]. Vaccine, 2006 , 24(21):4644-7.
89. Braun RP, Babiuk LA, Loehr BI, et al. Particle-mediated DNA immunization of cattle confers long-lasting immunity against bovine herpesvirus-1. Virology, 1999,265(1):46-56
90. Gaffal E, Schweichel D, Tormo D, et al. Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells[J]. Eur J Cell Biol, 2007, 86(11-12):817-26.
91. Stoecklinger A, Grieshuber I, Scheiblhofer S, et al. Epidermal langerhans cells are dispensable for humoral and cell-mediated immunity elicited by gene gun immunization[J]. J Immunol, 2007, 179(2):886-93
92. Walther W, Stein U, Fichtner I, et al. Intratumoral low-volume jet-injection for efficient nonviral gene transfer[J]. Mol Biotechnol, 2002, 21(2):105-l 15.
93. Cui Z, Baizer L, Mumper RJ. Intradermal immunization with novel plasmid DNA-coated nanoparticles via a needle-free injection device. J Biotechnol, 2003, 102(2): 105-115
94. Henry S, McAllister DV, Allen MG, et al. Microfabricated microneedles: A novel approach to transdermal drug delivery[J]. J Pharm Sci, 1999, 88(9):948.
95. McAllister DV, Wang PM, Davis SP, et al. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: Fabrication methods and transport studies[J]. Proc Nat Acad Sci, 2003,100(24): 13755-13760
96. Cormier M, Johnson B, Ameri M, et al. Transdermal delivery of desmopressin using a coated microneedle array patch system[J]. J Control Release, 2004, 97 (3): 503-511.
97. Coulman SA, Barrow D, Anstey A, et al. Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles[J]. Curr Drug Deliv, 2006, 3(1):65-75
98. Chabri F, Bouris K, Jones T, et al. Microfabricated silicon microneedles for nonviral cutaneous gene delivery[J]. Br J Dermatol, 2004, 150(5):869-877.
99. Mikszta JA, Alarcon JB, Brittingham JM, et al. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery[J]. Nature, 2002,8(4):415-419.
100. Alarcon JB, Hartley AW, Harvey NG, et al. Preclinical evaluation of microneedle technology for intradermal delivery of influenza vaccines. Clin Vaccine Immunol, 2007, 14(4):375-381.
101. Gill HS, Prausnitz MR. Coated microneedles for transdermal delivery[J]. J Control Release, 2007, 117(2):227-237.
102. Shirkhanzadeh M. Microneedles coated with porous calcium phosphate ceramics: effective vehicles for transdermal delivery of solid trehalose[J]. J Mater Sci Mater Med, 2005, 16(1):37-45.
103. Matriano JA, Cormier M, Johnson J, et al. Macroflux microprojection array patch technology: a new and efficient approach for intracutaneous immunization[J]. Pharm Res, 2002, 19(1):63-70.
104. Lin W, Cormier M, Samiee A, et al. Transdermal delivery of antisense oligonucleotides with microprojection patch (Macroflux)technology[J], Pharm Res, 2001,18 : 1789-1793.
105. Hooper JW, Golden JW, Ferro AM, et al. Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge[J]. Vaccine, 2007, 25(10):1814-1823.
106. Pearton M, Allender C, Brain K, et al. Gene delivery to the epidermal cells of human skin explants using microfabricated microneedles and hydrogel formulations[J]. Pharm Res, 2008, 25(2):407-16.