Studies On Biodegradable Nanoparticles Based Gene Vectors

At present the serious problems in clinical medicine such as malignant tumor, cardiovascular and cerebrovascular diseases, nervous system diseases and virulence epidemic diseases and so on will be solved by gene therapy with the development of molecular biology and technique for gene engineering. However, the naked DNA mediated gene transfection has some disadvantages such as low efficiency, easy degradation by nuclease in vivo and short expression time et al, which lead to the appearance of gene delivery systems. Therefore it's an interesting trend to explore a safe and efficient gene delivery system in gene therapy research area.An ideal gene vectors should have some properties as follows: less toxic, less immunogenic, much safer and easy to large-scale production, stable structures, protecting the loaded gene from degradation, selectively delivering gene into target cells, efficient transfection and expression in target cells and so on. Gene transfer vectors can be divided into two categories: viral and non-viral delivery systems. Among the viral delivery systems, adenovirus is the common vector with the most high transfecion efficiency. However, it tends to cause inflammatory reaction, and the length of loaded gene in adenovirus is limited. In contrast, the non-viral gene vectors are received more and more attentions because of their higher safety, easy to large-scale production, less immunogenicity and so on. Among the non-viral gene vectors, the nano gene delivery systems based on biodegradable and biocompatible materials such as PLGA or PLA have gained increasing attentions because of their advantages such as safety, biocompatibility, flexible structure, controlled particle size, stability, sustained release and avoidance of polymer accumulation in vivo after repeated administration. In the present study, pEGFP was chosen as reporter gene to be carried by biodegradable nanoparticles composed of biodegradable and biocompatible materials, such as PLGA and PLA-PEG. The four biodegradable nanoparticles based gene vectors were prepared or assembled by different technologies: 1) anionic and encapsulated gene loaded PLGA nanoparticles; 2) cationic gene loaded PLGA nanoparticles; 3) cationic gene loaded PLA-PEG nanoparticles; 4) anionic and bioadhesive gene loaded PLGA nanoparticles. The method of determining entrapment efficiency of DNA loaded nanoparticles by PicoGreen fluorospectrophotometry was established and the four biodegradable nanoparticles based gene vectors were evaluated in terms of pharmaceutical characterizations and biocharacteristics, respectively. The main methods and results were as follows:1. Determination of entrapment efficiency of DNA loaded nanoparticles by PicoGreen fluorospectrophotometryPicoGreen fluorospectrophotometry was established for determining the entrapment efficiency of DNA loaded nanoparticles. The entrapment efficiency of DNA loaded nanoparticles was calculated by determining the fluorescence intensity of the mixture of PicoGreen and free DNA in the supernatant of DNA loaded nanoparticles after centrifugation. Effects of time and temperature in the process and interferents on the fluorescent quantitation of DNA were investigated, respectively. The results were listed as follows: the linear range was 5 ng·ml"^-1～2000 ng·ml^-1, r=0.999 4. There were no significant influences of time and temperature in the process or most of interferents on the fluorescent quantitation of DNA. The intra-day RSD at the low, middle and high concentrations were 3.64%, 2.09% and 1.32%, respectively (n=5) and the inter-day RSD were 4.03%, 2.47% and 1.71%, respectively (n=5). The recovery were 99.4%, 99.5% and 99.7%, respectively, RSD were 2.15%, 1.74% and 0.97%, respectively (n=3). It could be concluded that PicoGreen fluorospectrophotometry is a convenient, accurate, sensitive and specific method for determining the entrapment efficiency and in vitro release of many kinds of DNA loaded nanoparticles ormicroparticles.2. Studies on anionic and encapsulated gene loaded PLGA nanoparticles In view of the drawbacks of the common double emulsion evaporation method in preparing gene loaded nanoparticles, a modified nanoprecipitation method was established to formulate anionic and encapsulated gene loaded PLGA nanoparticles. The resulted PLGA nanoparticles by this method had uniform spherical shape, narrow size distribution with average particles size of 197.0 run, negative zeta potential of -12.6 mV at pH 7.4. The PLGA nanoparticles obtained by the modified nanoprecipitation method exhibited greater loading efficiency (>95%) and better structural integrity compared to those prepared by W/O/W double emulsion/solvent evaporation method. The DNA loaded PLGA nanoparticles fabricated by the modified nanoprecipitation method showed sustained-release property in vitro within 30 days, and the release behavior was accorded with Weibull equation (r=0.988 0). The PLGA nanoparticles could protect the encapsulated plasmid DNA from nucleases digestion. It was found that the PLGA nanoparticles were much safer to A549 cell compared to commercial Lipofectamine 2000 and could successfully transfer pEGFP into A549 cells, though the transfection efficiency was not comparable to Lipofectamine 2000. In the further study, the formulation of PLGA nanoparticles was needed to optimized to improve the transfection efficiency in vitro.3. Studies on cationic gene loaded PLGA nanoparticlesCompared with the conventional anionic PLGA nanoparticles, cationic PLGA nanoparticles could combine with DNA by electrostatic attraction, leading to cationic gene loaded PLGA nanoparticles. The stability of DNA loaded in this nanoparticles will be improved and the cationic PLGA nanoparticles tend to interact with cell membrane with negative charge, to promote internalization and then to deliver DNA into cell, increasing the transfection efficiency. In this chapter, blank cationic PLGA nanoparticles (CTAB-NPs) with smaller size and appropriate electropositivity was prepared using nanoprecipitation method with PLGA as the vector material and CTAB as cationic surfactant. And the report gene pEGFP was incubated with the blank cationic PLGA nanoparticles by means of static electro-adsorption instead of encapsulation, leading to the formation of the DNA loaded cationic PLGA nanoparticles with adsorption efficiency of nearly 100%. The obtained nanoparticles were approximately spherical in shape with average particle size of 175.5 nm,. zeta potentials of +12.54 mV. The CTAB-NPs could combine DNA thoroughly (>95%) and protect DNA from nuclease degradation. It manifested sustained-release of DNA in vitro without burst effect and the release behavior was in accordance with Higuchi equation (r=0.997 7). It showed low cytotoxicity to A549 cells and could transfer the loaded gene into A549 cells, and the gene could express well inside the cells. Therefore the cationic PLGA naonparticles could be prepared easily with excellent characteristics that could be a promising non-viral nano-device, which has the potential to make in vivo cancer gene therapy achievable.4. Studies on cationic gene loaded PLA-PEG nanoparticlesIn respect of the drawbacks of conventional gene loaded PLA nanoparticles, in this chapter, a relatively desirable non-viral nano gene delivery was established to improve the common gene loaded PLA nanoparticles. Blank cationic PLA-PEG nanoparticles with smaller size and appropriate electropositivity was prepared using nanoprecipitation method with PLA-PEG as the vector material, CTAB as cationic surfactant and Tween 80 as coemulsifier. And the report gene pEGFP was incubated with the blank cationic PLA-PEG nanoparticles by means of charge attraction, leading to the formation of the gene loaded cationic PLA-PEG nanoparticles. The obtained cationic PLA-PEG nanopariticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 nm and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 mV and +16.8 mV, respectively. The nanopariticles with a higher DNA binding efficiency (>95%) could protect DNA from nuclease degradation and stabilized in plasma. The nanopariticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity and higher transfection efficiency in Hela cells compared with Lipofectamine 2000, even in presence of serum in medium. In conclusion, the gene loaded cationic PLA-PEG nanoparticles could be prepared easily with excellent properties and the anticipant design goal was achieved.5. Studies on anionic and bioadhesive gene loaded PLGA nanoparticlesBased on the problems existed in anionic and encapsulated gene loaded PLGA nanoparticles and cationic nano gene vectors, a new idea on anionic and bioadhesive gene loaded PLGA nanoparticles was suggested at the first time by our group, that is, the bioadhesive agent and stabilizer, Carbopol 940 was chosen to establish bioadhesive PLGA nanoparticles for efficient gene delivery to lung cancer cells. Pluronic F68, Pluronic F127 stabilized PLGA nanopartilces were formulated as control. The effects of different surfactants on the physicochemical and biological characterizations of PLGA nanoparticles were compared. All the obtained nanoparticles showed negative surface charge, similar spherical morphology, a relatively narrow particle size distribution, and lower cytotoxicity to A549 cells compared with Lipofectamine 2000. Carbopol stabilized nanoparticles hold advantages in DNA binding efficiency (>80%) at an optimal Carbopol concentration, DNA protection from enzymatic degradation in vitro release and better buffering capacity. Most importantly, the transfection efficiency of Carbopol stabilized nanoparticles in A549 cells was observed higher comparing to Pluronics stabilized nanoparticles or naked DNA, which was similar to that of Lipofectamine 2000. These results revealed that the bioadhesive PLGA nanoparticles formulated with Carbopol might be a very attractive candidate as a nonviral vector for lung cancer gene therapy and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo. In the future study, the in vivo application of this gene vector needs further investigation.In conclusion, biodegradable gene loaded nanoparticles could be prepared easily with small particle sizes, low cytotoxicity and high protection to DNA. In vitro studies have showed that the nanoparticles could be a promising non-viral nano-device, which has the potential to make in vivo gene therapy achievable.