| With the rapid development of molecular biology and the completion of Human Genome Project, gene therapy offers a new approach for the treatment of many diseases including inherited disorders, malignant tumor and cardiovascular diseases. Gene therapy involves the insertation of normal DNA or a functional gene into target cells to correct a genetic defect or achieve a therapeutic effect. However, naked nucleic acids are often prone to degradation before reaching their target sites and show low transfection efficiency. Therefore, it is necessary to develop an effective gene delivery carrier which is able to deliver gene into target cells without degradation and then enhance gene transfection efficiency. Gene delivery carriers include viral and non-viral vectors. Compared with viral vectors, non-viral vectors are safer, less immunogenic, much more reproducible in large scale and do not present DNA size limit. However, non-viral vectors often suffer from low transfection efficiency. With the deep understanding of gene delivery process, it is acknowledged that efficient gene delivery asks for multifunctional gene vectors which should possess long circulation time, cellular or tissue targeting, endosomal escape and nuclear targeting. Obviously, it is difficult to combine these functions in a single delivery system. Therefore, how to integrate functional materials into a single delivery system is becoming a hot topic and a difficulty for designing non-viral gene vectors.Layer-by-layer (LbL) technique based on electrostatic or other intermolecular forces presents a new approach to the formation of supramolecular architectures. It allows the control of assembly process. It is also an easy method without use of organic solvents and complicated chemical modification which helps to maintain biological activity of DNA. Therefore, in our research, a multifuctional gene nano-vector was successfully constructed by LbL technique with various kinds of functional electrolytes. Protamine (Pro) with nuclear targeting capability was used to condense DNA into a cationic core which was used as assembly template. Then additional layers of DNA, LHLN-liposomes for endosomal escape and pH- sensitive O-Carboxymethyl-chitosan (CMCS) for long circulation time were sequentially assembled onto the template. Finally, a multifunctional gene nanovector which offered long circulation time, efficient transmembrane transport, endosomal escape, high DNA loading dose and nuclear targeting was constructed. The main methods and results were as follows:1. Studies on polycationic multilayered nanoparticlesDuring LbL assembly process, the net surface charge of templates, the dose of polyelectrolytes, ionic strength of solution greatly influence the structure and functions of the assembly vectors. In our study, we prepared polycationic multilayered nanoparticles (PMN) and investigated the influence of different factors in the assembly process of PMN, which provided experimental basis for the application of LbL to assemble multifunctional gene vectors. Protamine (Pro) with nuclear targeting capability was used to condense DNA into a cationic core which was used as assembly template. Then additional layers of DNA and cationic PEI were sequentially assembled onto the template respectively and the PEI/DNA/Pro/DNA quaternary nanoparticles were obtained. The quaternary nanoparticles showed similar spherical morphology, narrow size distribution with average particle size of 150.1±2.9 nm and zeta potential of 14.9±2.1 mV. With the assembly of different layer, zeta porential reversed and the particle size increased. The condensed nanoparticles formed with Pro and DNA possesed high density of positive charge, which could be successfully used as template for LbL assembly. PEI/DNA/Pro/DNA was able to protect DNA from nuclease degradation. Most importantly, it showed lower cytotoxicity than commercial PEI/DNA (P<0.05) but exhibited higher transfection efficiency (P<0.05) in HepG2 and HeLa cells. Therefore, LbL technique might have enormous potential to construct high efficient and low cytotoxic gene vectors for successful gene delivery in the future.2. Studies on polycation/lipid multilayered nanoparticlesApart from various kinds of polyelectrolytes, charged liposomes can also be used to construct the gene vectors via LbL technique. Compared with polyelectrolytes, liposomes have similar lipid bilayered structure to the membrane, which often exhibit excellent cellular affinity and histocompatibility. They can fuse with cells and enhance the cellular entry of vectors. It is necessary to study the assembly behaviour of liposomes since the surface charge, hydrophilicity and hydrophobicity of them are very different from polyelectrolytes. In our research, Lipo/PEI/DNA ternary nanoparticles were prepared by the encapsulation of PEI/DNA complexes with anionic liposomes. The ternary nanoparticles were approximately spherical in shape with average particle size of 234.2±6.9 nm and Zeta potential of -20.7±2.5 mV. They could protect DNA from nuclease degradation and stabilized in plasma. It was shown that the release behavior of DNA from nanoparticles was in accordance with Ritger-Peppas equation without burst effect:lnQ=0.76711n;+2.9238(r=0.9942). They showed low cytotoxicity and had serum stability. They could successfully transfect HeLa cells in the presence of serum, which was better than PEI/DNA (P<0.05). Therefore, Lipo/PEI/DNA with stability in human plasma was hoped to be used for in vivo transfection test. Also, the understanding of the behavior of liposomes during assembly process would offer foundation for the use of liposomes in LbL technique.3. Studies on a novel cationic LHLN-liposomeAmong various kinds of polycations used for LbL assembly, cationic liposomes which offer high DNA packaging capacity, low immunogenicity and large-scale production have been widely investigated. They can efficiently deliver DNA into cells via endocytosis pathway and most cationic liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have endosomal escape capability, which make them very suitable for the construction of multifunctional gene vectors. However, cytotoxic effects induced by cationic liposomes limited their widely application. In our research,6-lauroxyhexyl lysinate (LHLN) previously synthesized by our group and DOPE was mixtured to prepare cationic LHLN-liposomes by film dispersion-ultrasonication method and then the in vitro and in vivo transfection efficiency were estimated. LHLN-liposomes and LHLN-liposome/DNA complexes both had spherical or ellipsoidal shapes but LHLN-liposome/DNA complexes displayed small cavities in their interior. The average particle size was 120.6±2.4 nm and 226.3±6.8 nm, respectively. Zeta potential of the liposomes and the complexes was 38.9±1.9 mV and 20.5±2.1 mV, respectively. LHLN-liposomes could efficiently condense DNA, protect DNA from nuclease degradation and showed lower cytotoxicity than commercial liposomal transfection agent Lipofectamine 2000 at the transfection dose (P<0.05) but with similar transfection efficiency in HepG2 and A549 cells. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P<0.05). Therefore, LHLN-liposome was a novel high efficient but low cytotoxic cationic gene vector, which might substitute Lipofectamine2000 and be widely used for gene transfection test. Aslo, it would provide a new kind of functional material for the construction of multifunctional gene nano-vectors with caitonic lipsomes.4. Studies on CMCS modified multifunctional gene vectorAfter the studies of the assembly characteristics of different functional electrolytes and LHLN-liposomes, a novel multifunctional gene nano-vector was constructed via LbL technique. Pro with nuclear targeting capability was used to condense DNA into a cationic core which was used as assembly template. Then additional layers of DNA for higher loading, LHLN-liposomes for endosomal escape and pH-sensitive O-Carboxymethyl-chitosan (CMCS) for long circulation time were sequentially assembled onto the template via LbL technique. The anionic DNA was absorbed onto the cationic template to form double DNA loaded DNA/Pro/DNA ternary complex (DPD). Then, cationic liposomes composed of LHLN and DOPE were deposited onto DPD via LbL technique to form cationic liposomes coated DPD (CLDPD). Finally, a multifunctional gene vector (CMCS-CLDPD) was successfully constructed by assembling the anionic CMCS on the surface of the CLDPD under physiology pH. The outmost layer of CMCS with pH sensitivity was able to fall off at pH 6.5 and the exposed cationic CLDPD could enhance the transfection efficiency. During the assembly process, zeta potential reversed and apparent layered structure was found on the surface of DPD, CLDPD and CMCS-CLDPD, which both demonstrated the formation of nanoparticles with multilayered structure. CMCS-CLDPD had spherical or ellipsoidal shapes and showed "core-shell" structure. The average particle size was 210.9±6.8 nm and zeta potential was -8.9±2.7 mV. They could effectively protect DNA from nuclease degradation and stabilized in plasma. It was shown that DNA from CMCS-CLDPD released more quickly in acid medium than other medium. When the pH of medium was below 6.5, zeta potential of CMCS-CLDPD reversed, which indicated that CMCS had pH-sensitivity and it could fall off from CMCS-CLDPD when pH was below 6.5. CMCS-CLDPD showed low cytotoxicity and could efficiently transfect HepG2 cells. When pH of the transfection medium without surum was 7.4, the transfection efficiency of different vectors followed the relationship:CLDPD> PEI/DNA> CMCS-CLDPD (P<0.05). However, when pH of the transfection medium without surum was 6.5, both CMCS-CLDPD and CLDPD showed higher transfection efficiency than PEI/DNA (P<0.05) and CMCS-CLDPD obtained almost the same value as CLDPD (P>0.05). It was demostrated that the outmost layer of CMCS was able to fall off at pH=6.5 and then cationic CLDPD exposed which enhanced transfection efficiency. In the transfection medium with 20% serum (pH7.4), CMCS-CLDPD displayed the highest tranfection efficiency, which showed that CMCS-CLDPD had the best stability in serum and could highly tranfect HepG2 cells even in the presence of serum. When administered by introtumoral injection into tumor beared mice, CMCS-CLDPD displayed similar transfection efficiency to CLDPD. It was demonstrated that CMCS-CLDPD had pH-sensitivity in tumor tissues and could transfect tumor cells. Therefore, the multifunctional gene nanovectors with pH-sensitivity would provide a new approach for the construction of anti-tumor gene vectors.In conclusion, LbL technique was an efficient method with easy operation for the assembly of multifunctional gene carriers. It may furthermore be of great application value for the preparation of high efficient and low cytotoxic non-viral gene vectors. |
PDF Full Text Request