The Application Of Non-ionic Surfactants In Colloidal Gene Delivery Systems

Since the first human clinical gene therapy was approved by FDA in 1989, there have been a 16-year development thus far. In this period the clinical applications of gene therapy have expanded from the treatment of genetic diseases to acquired diseases and gene therapy is believed to be a medicinal revolution in this millennium. To date, there have been nearly 1,000 clinical trials approved. However the adverse effect of human adenovirus vector brought about the first death case in clinic, which was a great blow to gene therapy. Thereafter the hopes and the setbacks have been coexistence with some good clinical results or conversely. It is gradually realized that exploring gene delivery systems with high efficiency and safety is the bottleneck for human gene therapy. Compared with viruses, non-viral vectors, represented by cationic liposomes and cationic polymers, have attracted a great deal of interest due to its advantageous safety profile and mature preparation methods. Compared with virus vectors, the lower transfection efficiency of non-viral vectors is a main obstacle for clinical application however. A number of studies have been carried out to solve this problem and some progresses have been made. However there still is a long way to go for clinical therapy.This project carried out an investigation of non-ionic surfactants for cationic lipids based gene delivery systems. Non-ionic surfactants modified cationic liposomes and cationic niosomes(non-ionic surfactants vesicles) were prepared and used for gene delivery. This project intended to investigate effects of non-ionic surfactants on gene delivery and develop new gene carriers with safety and efficiency.In this project, gene delivery systems were constructed using non-ionic surfactants -sorbitan monoesters (Span(?)) modified cationic liposomes. For cationic colloidal systems, gene transfer efficiency could be optimized through modulation of surfactants category and concentration. Antisense oligonucleotides/surfactantmodified cationic liposomes self-assemble complexes were prepared by auto-coacervation through electrostatic effect. Span-cationic liposomes were characterized using transmission electron microscope, particle and zeta potential sizer, and turbidity. And OND/cationic liposmes self-assembles complexes were characterized by agarose electrophoretic retarding and fluorescence spectra to evaluate the binding efficiency and binding process, respectively. A fluorescent analysis method was built to evaluate distribution of OND. A pH gradient method was used to prepare the liposomes with pH gradient intra/extra-liposomes. Labeled with pH-dependent fluorescent probe(5-carboxyfluorescein), OND was studied with its distribution extra/intra-liposomes. Furthermore, the cellular uptake studies of antisense oligonucleotides mediated by different Span modified cationic liposomes in COS7, Hela & RKO cell lines were carried out. The effects such as category and concentration of Span, endocytosis inhibitors, serum concentration, particle size of liposomes and etc. on gene transfer efficiency were investigated. The HLB value of Span related to cellular uptake efficiency was study.The results showed that the concentration of span must be controlled under a level of 16.7%(molar ratio) for a higher level could be destablized and destroyed the liposomes structures. Under this level, Span-CL showed good stability in 4 weeks with slight changes of particles size. Particles size of The Span cationic liposomes were between 100-200 nm and zeta potential of those were between +30-+45 mV. OND in gel electrophoresis was retarded when +/- charge ratio was 4 or above, which showed completely and efficiently binding was achieved.Results also showed that most of OND were absorbed on cationic liposomes and a small part entered intraliposomes.Span modified cationic liposome is an efficiency gene vector mediating OND cellular uptake. Span40 is the most efficient modifier among the test spans which HLB value was from 1.8 to 8.6. It may be attributed to the proper HLB value (HLB 6.7). A supplementary evidence was the cellular uptake study of span80-20 (mixedHLB 6.45) co-modified cationic liposomes, which achieved the similar efficiency compared with span40 cationic liposomes. And both of them showed significant enhancement compared with cationic liposomes without span. It showed that modifiers of span with HLB value 6-7 could promote the cellular uptake. It was supposed that surfactants with proper HLB value could modulate the surface property of microparticular colloidal systems to facilitate cellular endocytosis for gene delivery.In this study, several endocytosis inhibitors such as Chloroquine, N-ethylmaleimide and sodium azide were proved to be able to drastically lower the cellular uptake efficiency;additionally the study was undertaken at 4°C and the similar result was obtained. Endocytosis is energy-dependent process and proper temperature is required. At 10°C or below, therefore, endocytosis is inhibited. Endocytosis is a main pathway for OND/Span-cationic liposomes complexes.Liposomes with some different particle sizes (123.8, 221.1, 420.9 and 644.1 nm) were prepared by extruding through filter membranes with different pore size. It was found that there was size-dependent cellular uptake efficiency in these size distribution. The smaller size were, the higher cellular uptake efficiency. Endocytosis plays a key role on Lipoplexes internalization into cells. Due to the size limit of endosomes with mean diameter of 100-200 nm, lipoplexes with too large size are hard to be cellular internalized. Considering the size limit of endosomes, the particles size control is an important procedure to develop an efficient gene delivery system.Non-specific binding was brought out between many components in serum including serum proteins and cationic liposomes by electrostatic absorption. After binding with serum proteins, positive charge of lipoplex could be masked, thus the gene transfer ability was hampered. Span40-cationic liposomes maintained high cellular uptake efficiency in 10% serum. It showed span40 modifier hinder the binding between lipoplexes and serum proteins to a certain extent. In 50% serum, however, gene transfer efficiency of span-cationic liposomes drastically decreased asthe cationic liposomes without Span did. And Span did make little attribution to gene transfer in high concentration of serum.Niosomes are self-assemble closed vesicles with bilayer membrane. The major components in niosomes were non-ionic surfactants and cholesterol. In this study, non-ionic surfactants such as Span?, Tween? and Pluronic? were used as well as the cationic cholesterol derivative - 3 ￡ -[N-( N\ Ar'-dimethylaminoethane)carbamoyl] cholesterol (DC-Choi) to prepare cationic niosomes.The cationic niosomes made of non-ionic (Span?, Tween? or Pluronic?) and DC-Choi were prepared successfully with regular morphology and 100~200 nm particles size. Zeta potentials were +3O-+5O mV due to the incorporation of DC-Choi;thus vesicles were positively charged. The binding efficiency of cationic niosomes and OND can be evaluated by gel electrophoresis method. When +/- charge ratio was 4 or above, OND can be binding by niosomes completely and the complexes formed. The gene delivery study showed cationic niosomes had high efficiency for mediating cellular uptake;the reporter gene expression was also in a relative high level. Therefore, cationic niosomes were the potential gene delivery systems.Quaternary ammonium salt cationic surfactant -cetyltrimethylammonium bromide (CTAB) can bind with DNA efficiently. It can compress DNA into microparticls and its quaternary ammonium group can facilitate DNA escaping from endosomes to free them into cytoplasm. Considering its positive charge and helping endosomes escape, the cationic niosomes containing CTAB, non-ionic surfactant and cholesterol were prepared and this system for gene delivery was evaluated. CTAB has cytotoxicity. Compared with CTAB aqueous solution, however, cytotoxicity of CTAB incorporated into niosomes drastically decreased in this cytotoxicity study using MTT method. It was well worth notice that niosomes containing CTAB had comparable high gene transfectionion efficiency for the reporter gene with the marked transfectionion reagents of DOPE/DC-Chol liposomes.Sterically stabilized microparticles drug delivery systems are hotting up inpharmaceutical research and industries. Poly(ethylene glycol) modification is the most mature technology for the preparation of sterically stabilized microparticles drug delivery systems. Taking in account of the similar features between liposomes and niosomes, sterically stabilized niosomes were successfully prepared according to the methods using in liposomes.The turbidity was observed little changes for sterically stabilized cationic niosomes in 10% serum PBS in 6 h while the turbidity of cationic niosomes increased drastically. It proved that the hydrophilic layer constructed by PEG prevented cationic vectors and serum proteins from binding each other. Additionally vesicles aggregation and fusion were inhibited. In the serum proteins binding study, the proteins binding efficiency ability showed cationic niosomes > sterically stabilized cationic niosomes significantly. It further proved that PEG exhibited sterically stabilized effect. Hydrophilic polymer PEG can form a hydration layer of sterical conformation around niosomes and prevent DNA from degrading by nuclease and stabilize cationic niosomal systems in serum.DNA / sterically stabilized cationic niosomes complexes were prepared by two methods: "pre-coating" and " post-coating". In cellular uptake study under serum condition, the two samples prepared by "pre-coating" and " post-coating" methods were compared for their efficiency. The two samples both exhibited superior to the cationic niosomes with significant higher gene delivery efficiency in serum. The samples prepared by " post-coating" method showed the better superiority. The result showed that the preparation method was a key factor to gene delivery ability. It could be attributed to the structures difference induced by the preparation methods. And the complexes structures affected on the DNA protection and cellular internalization;thus brought out the difference of gene delivery ability.In conclusions, this project was carried out an investigation on non-ionic surfactants applied in colloidal gene delivery systems - Span modified cationic liposomes and cationic noisomes were constructed and served as gene carriers. Bothof them were efficient to gene delivery. These trials were new attempts for gene delivery. The study found HLB value of Span using to modify cationic liposomes could affect the cellular uptake efficiency and the optima value was about 6-7. A pH gradient method was used to prepare the liposomes with pH gradient intra/extra-liposomes. A fluorescent method was built to o study OND distribution extra/intra-liposomes. The cationic cholesterol derivative was firstly used to prepared cationic niosomes with the non-ionic surfactants of Span?, Tween? or Pluronic?. These cationic niosomal systems were firstly served as new gene carriers and achieved satisfactory efficiency. Further, a new gene delivery system of sterically stabilized cationic niosomes was prepared. These gene carriers showed positive results.