| 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 DeliveryThis 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 DeliveryA 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 DeliveryBiologically 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 CellsCombining 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. |
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