摘要
纳米载体在药物/基因传递中具有特殊的价值和意义。纳米载体粒径大小在10～100nn,可将药物分子包裹其中或吸附在其表面,通过靶向分子与细胞表面特异性受体结合,在细胞摄取作用下进入细胞内,实现安全有效的靶向药物输送和基因治疗。无机纳米粒载体因具有生物安全、高效、价廉等优点,已成为近年来新型药物/基因传递系统研究的热点。本论文重点围绕新型无机纳米粒载体的构建及其在药物/基因传递中的应用开展研究,主要分为四个部分：
第一部分纳米载体在药物/基因传递中的应用研究进展
对纳米药物载体及基因载体的国内外研究现状进行了综述,主要内容包括：纳米载体的特点及制备新技术、纳米药物缓控释系统的优点、基因载体的分类与特点、非病毒纳米基因传递系统的优点、新型纳米载体在药物/基因传递系统中的应用进展、基于组织工程的三维支架以及软骨种子细胞的定向诱导分化研究进展等。文献综述为论文后续实验工作的开展奠定了基础。
第二部分新型多孔二氧化硅纳米粒在药物传递中的应用研究
以生物安全的硅胶为载体材料,运用纳米技术,自行研制出具有高效增溶、长效缓释作用的新型多孔二氧化硅纳米粒(Porous silica nanoparticles, PSNs)。分别选择水溶性药物水飞蓟宾葡甲铵(Silybin meglumine, SLBM和难溶性药物水飞蓟宾(Silybin, SLB)为模型药物,研究PSNs新载体在水溶性药物和难溶性药物72h高效长效制剂开发中的应用可能,为3天给药1次的高效长效新制剂开发提供技术支撑。
1.水溶性药物72h高效长效新制剂的研制：以水溶性药物水飞蓟宾葡甲铵)为模型药物,PSNs为载体,制备了水飞蓟宾葡甲铵72h高效长效制剂(3d-SLBM)。体外评价研究结果表明：3d-SLBM的主药含量为29.46 mg/50mg,载药量为58.91±0.39%,包封率为58.43±0.62%；体外释药特性研究结果显示,3d-SLBM中SLBM的释放适宜在低浓度的碳酸钠溶液中进行,72 h累积释放大于80%；体内药动学研究结果显示：比格犬口服3d-SLBM,高效液相法测定犬体内血药浓度,3d-SLBM的Tmax 24 h与参比制剂Tmax 0.5 h相比大大增加,3d-SLBM的MRT 38.89 h与参比制剂的MRT 7.31 h相比显著延长,显示出长效缓释特征；3d-SLBM中游离药物的相对生物利用度达到803.99%；体外溶出结果与体内吸收具有良好的相关性,体外溶出结果可以间接反映其体内质量；药效实验结果表明：3d-SLBM组小鼠血浆中AST(291.83±76.03 IU/L)和ALT(774.92±223.85 IU/L)值明显低于CCl4对照组(AST：901.00±174.32 IU/L,ALT：1997.58±335.08 IU/L),具有统计学意义(P<0.01)。说明3d-SLBM的生物利用度高、保肝效果好。
2.难溶性药物72h高效长效新制剂的研制：以难溶性药物水飞蓟宾为模型药物,综合运用固体分散速释技术、亲水凝胶骨架缓释技术和PSNs长效缓释技术制备了水飞蓟宾72h高效长效制剂(3d-SLB)。体外评价研究结果表明：3d-SLB中SLB的释放适宜在低浓度的碳酸钠溶液中进行,72 h累积释放大于80%；体内药动学研究结果显示：比格犬口服3d-SLB,高效液相法测定犬体内血药浓度,3d-SLB的Tmax 24h与参比制剂Tmax 1h相比大大增加,3d-SLB的MRT 32.15 h与参比制剂的MRT 6.11 h相比显著延长,显示出长效缓释特征；3d-SLB中游离药物的相对生物利用度达到458.98%,体外溶出结果与体内吸收具有良好的相关性,体外溶出结果可以间接反映其体内质量；药效实验结果表明：3d-SLB小鼠血浆中AST(149.90±28.14 IU/L)和ALT(843.33±169.18 IU/L)值明显低于CCl4对照组(AST:901.00±174.32 IU/L,ALT：1997.58±335.08 IU/L),具有统计学意义(P<0.01)。说明3d-SLB生物利用度高、保肝性能优越。
第三部分载基因纳米粒的制备及其在基因传递中的应用研究
选择生物安全的磷酸钙纳米粒(Calcium phosphate nanoparticles, CPNPs)和PSNs,制备载基因DNA-CPNPs和DNA-PSNs;通过对载基因纳米粒进行修饰,成功制备了多糖修饰的DNA-CPNPs和钙离子化DNA-PSNs;着重研究了DNA-CPNPs、多糖修饰的DNA-CPNPs、钙离子化DNA-PSNs 3 (?)中新型载基因纳米粒对干细胞的传递效果。
1.DNA-磷酸钙纳米粒的制备及其干细胞的传递研究：用反相微乳法制备了DNA-CPNPs,对其进行表征,并将其应用于干细胞的转染。研究结果表明：DNA-CPNPs的粒径为20-50 nm；琼脂糖电泳结果表明：磷酸钙：质粒质量比为2：1时,CPNPs携带质粒量最大,可有效阻滞DNA的迁移；活细胞工作站测定结果显示：游离DNA因没有纳米粒载体的作用,细胞未显红色,说明游离DNA未进入细胞内,而DNA-CPNPs 2 h开始有少数细胞显红色,随着时间的推移,越来越多的外源基因进入细胞,说明纳米粒可有效携带基因进入细胞；激光共聚焦显微镜观察核转运结果显示：2 h未见细胞核内显红色,说明DNA-CPNPs此时尚未进入细胞核,4 h进核亦不明显,8 h有少量进核,18h可见细胞核内呈明显的红色,表明此时外源DNA在纳米粒作用下进核明显；活细胞工作站和共聚焦显微镜测定结果均显示纳米粒可被干细胞吞噬。MTT法测定细胞毒性,结果显示：DNA-CPNPs的毒性明显低于市售转染试剂Lipofectamine2000 (P<0.01);ELISA测定结果表明,DNA-CPNPs在干细胞中的转染效果与转染试剂Lipofectamine2000相当(P>0.05)。实验研究结果表明：DNA-CPNPs转染效率高、毒性低,可以作为一种生物安全的非病毒基因载体。
2.多糖修饰的DNA-磷酸钙纳米粒的制备及其干细胞的传递研究：选择DNA-CPNPs,通过对载基因纳米粒进行修饰,成功制备了多糖修饰的DNA-CPNPs,初步研究了多糖修饰的DNA-CPNPs在干细胞中的转运情况。透射电镜测定结果表明：多糖修饰的DNA-CPNPs呈球型、粒径分布稳定、分散均一,粒径在50nm左右；ELISA测定结果表明：与市售脂质体lipofectamine2000相比,多糖修饰的DNA-CPNPs在干细胞中具有更高的转染效率。
3.钙离子化DNA-多孔二氧化硅纳米粒的制备及其性能评价：选择PSNs为载体,通过对其进行修饰,成功制备了钙离子化DNA-PSNs,初步研究了钙离子化DNA-PSNs在干细胞中的转运。琼脂糖电泳结果表明：钙离子化的PSNs能够与质粒DNA较好的结合,阻滞其电泳迁移,而未修饰的PSNs与DNA结合能力弱,不能有效阻滞DNA迁移；EDS能谱分析结果表明：钙离子有效结合到PSNs上；倒置荧光显微结果表明：与DNA-PSNs相比,钙离子化DNA-PSNs能够转染更多的细胞,说明钙离子化DNA-PSNs可以作为一种有效的非病毒基因载体。
第四部分三维纳米基因传递系统的构建及其在干细胞诱导分化中的应用研究
将细胞外基质修饰纳米粒技术与基于组织工程的三维支架技术相结合,构建非病毒三维纳米基因传递系统。以细胞外基质(ECM)成分为支架材料,制备嵌合有DNA-CPNPs的三维支架,成功构建了兼具高效、缓释特征的非病毒三维纳米基因传递系统(3 dimensional nanoparticles gene delivery system,3D-NGDS)。
1.三维纳米基因传递系统的构建及其性能评价：以细胞外基质黏附实验为基础,选择对干细胞黏附及增殖效果好的纤维粘结蛋白、胶原为支架材料,制备胶原/FN/壳聚糖三维支架；在此基础上,采用冷冻干燥法制备嵌合DNA—CPNPs的三维纳米基因传递系统(3D-NGDS),并观测其孔径、孔隙率、吸水率、保水率,结果表明：3D-NGDS中存在较大孔径(≥100μm)的孔道,吸水率和保水率与支架材料的配比有关。研究结果表明：3D-NGDS多孔、生物安全,适宜干细胞生长。
2.三维纳米基因传递系统的基因转运研究：以碘化丙啶标记基因,通过活细胞工作站观测,结合免疫组化的方法,对3D-NGDS中基因的细胞内转运进行了研究；运用Picogreen Dye法测定不同支架不同时间培养液中DNA的含量,绘制DNA累积释放曲线,结果显示3D-NGDS中DNA累积释放率3d达到33.4%,21d达到69.7%；而游离DNA-三维支架3d DNA累积释放率达到87.7%,3D-NGDS对DNA具有良好的缓释作用。ELISA法测定二维体系及3D-NGDS 3-15天细胞表达的TGF-β1浓度,结果表明：3D-NGDS在3-15天内,蛋白表达水平维持在10 ng/ml左右,其中第6天表达浓度达到12.6 ng/ml,显著高于二维转染体系(P<0.01),达到了外加TGF-β1因子有效浓度,显示出明显的高效特征。通过观测干细胞在二维及3D-NGDS中转染15天的甲苯胺蓝(GAG)染色图及Ⅱ型胶原免疫组化染色图,结果表明：二维单层培养细胞的GAG染色和Ⅱ型胶原免疫组化染色均为阴性,干细胞在3D-NGDS中培养15天后,GAG染色和Ⅱ型胶原免疫组化染色均为阳性,说明种子细胞已成功分化成软骨细胞,分泌软骨细胞特征性的细胞外基质：Ⅱ型胶原和GAG。三维纳米基因传递系统作为一种新型的非病毒基因载体,可以实现外源基因在种子细胞中的高效持续表达,为后续组织工程和再生医学研究提供新技术、新思路。
Nano-scaled carriers have special value and significance in drug/gene delivery. The particle size of nano-carriers ranges from 10 to 500 nm. Drug molecules could be encapsulated in the carrier or absorbed on the surface. Safe and effective targeted drug delivery and gene therapy could be achieved by the combination of targeting molecules with the specific receptors on cell surface followed by entering into cells via cellular uptake. Inorganic nanoparticles enjoying such advantages as biosafety, high-efficacy and low price have become the hot spot of new drug/gene delivery system study in recent years. This paper focuses on the construction of novel inorganic nanoparticles and the investigation of their application in drug/gene delivery. There are mainly four parts in this thesis.
Part One Resent Advances on the Application of Nano-Scaled Carriers for Drug/Gene Delivery
This part has summarized the research status of nano-scaled drug and gene vectors over the world, including characteristics and new preparation technology of nano-scaled vectors, advantages of nano drug-sustained-release system, classification and characteristics of gene vectors, merits of nonviral nano gene delivery system, the application of new nano vectors in drug/gene delivery system, and progress of study on three-dimensional scaffold based on tissue engineering and inducing oriented-differentiation of seeded chondrocyte. The literature review has laid a foundation for the development of subsequent work in this study.
Part Two Novel Porous Silica Nanoparticles and the Application in Sustained Drug Delivery
A new porous silica nanoparticle with high solubilization efficient and long-term sustained-release effect has been prepared in this study based on nanotechnology using silica as the carrier materials which is biologically safe. The water-soluble drug silybin meglumine and poorly water-soluble drug silybin are employed as model drugs respectively to investigate the possibility of the novel porous silica nanoparticles to develop long-term sustained release system for 72h of both water-soluble and poorly water-soluble drugs, which will provide promising future for the development of high-efficacy and long-acting preparations after oral administration once every 3 days.
1. Development of 72h long-lasting and high-efficacy preparation for water-soluble drugs.
Taking the water-soluble drug silybin meglumine (SLBM) as the model drug and porous silica nanoparticles as carriers, we prepared SLBM-loaded nanoparticles (3d-SLBM). The results of in vitro evaluation revealed that the amount of drug in the drug-loaded nanoparticles was 29.46 mg/50mg, the drug loading rate was 58.91±0.39%, and encapsulating rate was 58.43±0.62%. The investigation of in vitro dissolution showed that low concentration sodium carbonate solution was most suitable for the release of SLBM from 3d-SLBM, with the 72h accumulative release rate over 80%. For the in vivo pharmacokinetics study, drug was administrated orally to beagle dogs and high performance liquid chromatography (HPLC) was used to determine the plasma concentration in beagle dogs. The results of in vivo study demonstrated that the Tmax of 3d-SLBM was 24h, significantly increased compared with that of the reference preparation (Tmax 0.5 h). Furthermore, compared with the MRT 7.3 1h of the reference preparation, the MRT of 3d-SLBM was noticeably prolonged to 38.89h, revealing long-lasting sustained release characteristic. Additionally, the relative bioavailability of the free drug from 3d-SLBM reached 803.99%. Interestingly, the in vitro dissolution and in vivo absorption showed a good correlation, that is, the in vivo absorption profile can be reflected indirectly from the in vitro dissolution profile. The results of hepatic protection showed that AST(291.83±76.03 IU/L) and ALT(774.92±223.85 IU/L) level in the plasma of mice which were given 3d-SLBM were significantly lower than that of mice in CCl4 treated group (AST:901.00±174.32 IU/L, ALT:1997.58±335.08 IU/L), with statistical significance (p<0.01). All these results indicated that 3d-SLBM possessed high bioavailability and excellent hepatic protection effect.
2. Development of 72h long-lasting and high-efficacy preparation for poorly water-soluble drugs.
A 3-day release formulation of silybin (3d-SLB) with high-efficacy, long-acting, and slow-release properties was prepared in this study by combined use of silybin solid dispersion, silybin loaded silica nanoparticles and slow-release matrix material. Silybin was employed as the model drug. The result of in vitro evaluation demonstrated that SLB was apt to release from 3d-SLB in low concentration sodium carbonate solution, with the accumulated release rate over 80%. The results of in vivo pharmacokinetics research showed that Tmax of 3d-SLB was 24h, which was much later than that of the reference preparation (Tmax 1h). The MRT of 3d-SLB was 32.15 h, greatly prolonged than the MRT 6.11h of the reference preparation, indicating the long-lasting sustained release profile. Moreover, the bioavailability of the free drug released from 3d-SLB reached 458.98%; there is a good correlation between the in vitro dissolution and in vivo absorption, that is, the in vivo absorption profile can be reflected indirectly from the in vitro dissolution profile. The results of drug effect showed that AST(149.90±28.14 IU/L) and ALT(843.33±169.18 IU/L) levels in the plasma of mice which were administered 3d-SLBM were significantly lower than that of mice in CCl4 treated group (AST:901.00±174.32 IU/L, ALT:1997.58±335.08 IU/L), with statistical significance (p<0.01). All these results indicated that 3d-SLB possessed high bioavailability and excellent hepatic protection effect.
Part Three Preparation of Gene Loaded Nanoparticles and the Application in Gene Delivery
Biologically safe calcium phosphate nanoparticles and porous silica nanoparticles were used to encapsulate gene to prepare DNA- calcium phosphate nanoparticles and DNA- porous silica nanoparticles. Polysaccharide modified DNA- calcium phosphate nanoparticles and calcium-ionized DNA- porous silica nanoparticles were also successfully prepared by modification of gene-loaded nanoparticles. This part focuses on the transfection effect of three kinds of new gene-loaded nanoparticles on mesenchymal stem cells, including DNA- calcium phosphate nanoparticles, polysaccharide modified DNA- calcium phosphate nanoparticles and calcium-ionized DNA- porous silica nanoparticles.
1. Preparation of DNA- calcium phosphate nanoparticles and the application in gene delivery to mesenchymal stem cells.
DNA-calcium phosphate nanoparticles were prepared by reverse microemulsion method, characterized, and applied in transfection of mesenchymal stem cells. The result showed that the particle size of DNA-calcium phosphate nanoparticles ranged from 20 to 50 nm. Agarose gel electrophoresis found that calcium phosphate nanoparticles could load the maximum amount of DNA when the weight ratio of calcium phosphate to plasmid DNA is 2:1. Additionally, DNA- calcium phosphate nanoparticles with this weight ratio could effectively retard plasmid DNA migration. The determination of live cell imaging revealed that free plasmid had almost no transfection effect with no staining in the cells, indicating that free DNA could not enter into cells. For the DNA- calcium phosphate nanoparticles transfected cells, a few cells were stained at 2h post transfection. With time went by, more and more exogenous gene entered into cells, demonstrating that DNA- calcium phosphate nanoparticles could effectively carry gene into cells. Laser confocal fluorescence microscopy displayed that no red florescence was observed in the nuclei at 2h post transfection, indicating that DNA- calcium phosphate nanoparticles were not in nuclei at this time. At 4h post transfection, there was still no obvious red staining in the nuclei. At 8h post transfection, a few DNA- calcium phosphate nanoparticles showed in the nuclei. At 18h post transfection, obvious red florescence was observed in nuclei, indicating that exogenous gene had entered into cells with the help of calcium phosphate nanoparticles. The results of both live cell imaging and laser confocal fluorescence microscopy indicated that DNA- calcium phosphate nanoparticles could be phagocytosed by mesenchymal stem cells. Cytotoxicity determined by MTT assay showed that DNA- calcium phosphate nanoparticles had lower cytotoxicity than commercially available transfection agent Lipofectamine2000 (P<0.01). The result of ELISA test revealed that DNA- calcium phosphate nanoparticles had comparable transfection effect with transfection agent Lipofectamine2000 (P>0.05), which was obviously higher than standard calcium phosphate transfection agent (P<0.01). Altogether, DNA- calcium phosphate nanoparticles with high transfection efficiency and low cytotoxicity could be developed into a kind of biologically safe nonviral gene vector.
2. Preparation of polysaccharide modified DNA- calcium phosphate nanoparticles and the application in gene delivery to mesenchymal stem cells.
Modified by polysaccharide, DNA- calcium phosphate nanoparticles were employed successfully to prepare polysaccharide modified DNA- calcium phosphate nanoparticles. Primary research has been carried out about the transportation of polysaccharide modified DNA- calcium phosphate nanoparticles in the nuclei. Transmission electron microscopy displayed that the polysaccharide modified DNA-calcium phosphate nanoparticles were of spherical shape, and the particle size was about 50 nm which were uniformly distributed within a narrow range. The result of ELISA test showed that polysaccharide modified DNA- calcium phosphate nanoparticles possessed higher transfection efficiency in mesenchymal stem cells than the commercially used Lipofectamine2000.
3. Preparation and evaluation of calcium-ionized DNA- porous silica nanoparticles.
Calcium-ionized DNA- porous silica nanoparticles were successfully prepared based on the modification of DNA- porous silica nanoparticles. Primary investigation has been conducted to study the transportation of calcium-ionized DNA- porous silica nanoparticles inside mesenchymal stem cells. The result of agarose gel electrophoresis demonstrated that calcium-ionized DNA- porous silica nanoparticles could be well combined with plasmid DNA and retard DNA migration. However, the unmodified DNA- porous silica nanoparticles had a weak ability to combine plasmid DNA and could not effectively retard DNA migration. The result of EDS revealed that calcium ion could effectively combine with porous silica nanoparticles. Fluorescent microscopy showed that calcium-ionized DNA- porous silica nanoparticles could transfect more cells than DNA- porous silica nanoparticles, indicating that calcium-ionized DNA- porous silica nanoparticles could be a kind of effective nonviral gene vector.
Part Four Construction of Three-Dimensional Nano Gene Delivery System and Its Application in the Differentiation of Mesenchymal Stem Cells
Combining the technology of preparing extracellular matrix (ECM) modified nanoparticles and three-dimensional scaffold based on tissue engineering; we constructed a nonviral 3 dimensional nano gene delivery system (3D-NGDS). Following ECM ingredients as scaffold material, DNA- calcium phosphate nanoparticles were encapsulated in three-dimensional scaffold to fabricate 3D-NGDS, and the characteristics were then evaluated such as DNA release kinetics, cell uptake, gene transfection efficiency, MSCs differentiation.
1. Construction and evaluation of three-dimensional nano gene delivery system.
Choosing materials Based on the results of ECM adhesion experiment, FN and collagen (C), which possess good adhesion and proliferation ability on mesenchymal stem cells, a three-dimensional scaffold was prepared with collagen, FN, and chitosan. 3D-NGDS was produced by encapsulating DNA- calcium phosphate nanoparticles in scaffold using freeze drying technique. The results of its pore size, porosity rate, water absorption rate, and water retaining rate showed that most of the scaffold pore size was about 100μm in addition to some pores with larger pore size (>100μm); the water absorption and water retaining abilities were related to the proportioning of scaffold materials. In a word, the three-dimensional nano scaffold was porous, biosafe and suitable for the growth of mesenchymal stem cells.
2. Gene delivery via the three-dimensional nano gene delivery system.
By using propidium iodide to mark gene, cellular uptake of exogenous gene in the three-dimensional system was investigated via combined employment of live cell imaging and immunohistochemistry. The DNA contents in different three-dimensional scaffolds were determined by Picogreen Dye method at different time points. DNA accumulative release curves in different scaffolds showed that the DNA accumulative release rate of DNA encapsulated three-dimensional scaffold was 33.4% at 3d, and 69.7% at 21d; for free DNA- three-dimensional scaffold, the DNA accumulative release rate was 87.7% at 3d. The result indicated that 3D-NGDS had good sustained-release effect on DNA delivery. TGF-β1 concentration expressed by cells in 3D-NGDS from day 3 to day 15 was determined by ELISA, and the result revealed that during the period of day 3 to day 15, the TGF-β1 protein expression level could maintain at about lOng/ml. Particularly, the expression concentration at day 6 reached 12.6 ng/ml, which was significantly higher than that of the two-dimensional transfection system (P<0.01). This protein expression level reached the effective concentration of TGF-β1 protein normally added in medium, showing obviously high efficiency of 3D-NGDS. Mesenchymal stem cells in two-and three-dimensional nano scaffolds at 15 days post transfection were observed by GAG staining and typeⅡcollagen immunohistochemistry staining, and the results found that the GAG staining and typeⅡcollagen immunohistochemistry staining of the cells cultured in two-dimensional monolayer condition were all negative, while for the mesenchymal stem cells cultured in 3D-NGDS for 15 days, the GAG staining and typeⅡcollagen immunohistochemistry staining were positive. These results indicate that the seeded cells have successfully differentiated into chondrocytes, which could secrete ECM with chondrocyte characteristics:typeⅡcollagen and GAG. As a kind of new nonviral gene delivery carrier,3D-NGDS could achieve high efficient and long-lasting expression of exogenous gene in the seeded cells, which provided a new idea and technology for the subsequent study on tissue engineering and regenerative medicine.
引文
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