Dissertations / Theses on the topic 'Cationic Vector'

La thérapie génique est une forme de thérapie pour traiter les maladies génétiques héréditaires ou acquises tels que les cancers ou la mucoviscidose. L’introduction d’un polynucléotide, par voie systémique ou locale (orale ou nasale par exemple), au sein des cellules malades permet de corriger les défauts à l’origine des mutations génétiques. Néanmoins, le franchissement des différentes barrières biologiques nécessaire à l’internalisation de l’ADN ne peut se faire que par l’intermédiaire d’un vecteur qui va le protéger et lui permettre d’atteindre le noyau de la cellule où il sera transcrit. Divers vecteurs (viraux ou synthétiques) ont vu le jour, tels que des vecteurs polymères cationiques à base de PEI. Cependant, bien qu’efficaces, ces vecteurs sont immunogènes à forte dose. Des fonctionnalisations pour réduire cette toxicité, telle que la PEGylation, ont été mises au point et permettent de renforcer les vecteurs en apportant de la furtivité aux polyplexes finaux. Cependant, ces stratégies montrent des limites nécessitant la synthèse de nouveaux types de polymères. La POxylation représente une bonne alternative à l’utilisation du PEG pour former de nouveaux polyplexes par l’ajout d’un bloc formé d’une ou plusieurs poly(2-alkyl-2-oxazoline)s. Les copolymères sont synthétisés par hydrolyse sélective d’un copolymère triblocs PEtOx-b-PnPrOx-b-PMeOx en utilisant les propriétés thermosensibles des blocs hydrophobes et un sel kosmotrope afin de former des systèmes cœur-couronne permettant l’hydrolyse du bloc PMeOx en PEI. Les systèmes ont ensuite été formulés selon une formulation standard et une méthode par « micro-extrusion ». Les polyplexes ont ensuite été utilisés in vitro par déposition ou par une méthode de nébulisation, idéale pour le traitement de maladies pulmonaires. De très bons résultats de transfection ont été obtenus, résultats qui dépendent de différents paramètres (Mn, PEI, architecture polymère, rapport de charge +/-)
Gene therapy is a form of therapy used to treat hereditary or acquired genetic diseases such as cancer or cystic fibrosis. Introducing a polynucleotide into diseased cells, either via the systmeic route or the local route (oral or nasal inhalation), corrects the defects causing the genetic mutations. However, DNA can only be internalized using a vector that protects it and enables it to reach the cell nucleus, where it will be transcribed. Various vectors (viral or synthetic) have been developed, such as PEI-based cationic polymer vectors. However, although effective, these PEI-based vectors are immunogenic at high doses. Functionalizations to reduce this toxicity, such as PEGylation, have been developed, making it possible to reinforce vectors by adding stealthiness to the final polyplexes. However, these strategies have their limitations, necessitating the synthesis of new types of polymer. POxylation represents a good alternative to PEG usage to form new polyplexes by adding a block formed from one or more poly(2-alkyl-2-oxazoline)s. The copolymers are synthesized by selective hydrolysis of a PEtOx-b-PnPrOx-b-PMeOx triblock copolymer using the thermosensitive properties of the hydrophobic blocks and a kosmotropic salt to form core-shell systems enabling hydrolysis of the PMeOx block to PEI. Then, the systems were formulated using a standard formulation and a "micro-extrusion" method. The polyplexes obtained were used in vitro experiments, by deposition or by a nebulization method, ideal for the treatment of pulmonary diseases. Very good transfection results were obtained, depending on various parameters (Mn, PEI, polymer architecture, +/- charge ratio)

Non–viral gene vectors exhibit several desirable properties, over their viral counterparts. This has generated vast interest in the development of efficient non–viral vectors for DNA in gene therapy. The first generation lipid/peptide/DNA (LID) vector was comprised of Lipofectin™, a 1:1 mixture of DOTMA and DOPE, together with a targeting peptide. The resulting LID vector exhibited significant advantages over cationic liposome vectors (lipoplex systems) for gene delivery, transfecting a range of cells with high transfection efficiencies. The LID vector is introduced, and its advantages are outlined. The results and discussion starts in Chapter 2, as we begin our investigation to understand the structural requirements that may lead to enhanced transfection efficiency. The lipid component in the LID vector is described, together with a systematic strategy to enhance transfection activity in the LID vector via new lipid design. The synthesis of a range of C14:1 cationic lipids is described. We have also synthesised asymmetric cationic lipids and lipids containing a short poly(ethylene glycol) (PEG) chain, outlined in Chapter 3. Furthermore, we have synthesised a series of lipids containing a reducible disulfide bridge attached to a PEG moiety, as our strategy is to enhance solubility and shielding during the early stages of cell entry. Once the complex is internalised into the cell, the protective PEG units are shedded, as described in Chapter 4. In addition, the cationic thiol lipid synthesised was coupled to a fluorescent label, to afford a fluorescently–labelled lipid, which has enabled us to track the movement of the lipoplex (lipid:DNA) during transfection. The synthesis towards a cyclic head group for the attachment of different lipid chains is described in Chapter 5. We have utilised a range of synthetic methodologies, including metathesis strategies for the formation of the cyclic lipid precursor. To grasp a greater understanding of how the lipids influence the packaging of DNA, we have studied the biophysical properties of the liposomes and lipoplexes generated at different lipid:DNA mixing ratios, using sizing and zeta potential measurements, given in Chapter 6. The mixing ratios are later used in the design of transfection experiments. Finally, the cationic lipids synthesised in the earlier chapters are formulated into LID complexes, and assessed in a range of different cell lines. The effect of the lipids on transfection efficiency is discussed in Chapter 7. The potential for future work in this area of research is discussed in Chapter 8. A formal description of the experimental procedures and analytical data is presented in Chapter 9.

Gene therapy is a promising technique that offers great potential for the treatment of inherited and acquired disorders. However, one of the major obstacles is identifying suitable vectors with high targeting and transfection efficiency. A literature review on gene therapy and studies on the developments of vectors in gene therapy is presented in Chapter 1. The results and discussion for the development of novel targeting peptides is outlined in Chapter 2. Biophysical analyses of the peptides were performed in PD (peptide-DNA) and LPD (lipid-peptide-DNA) formulations, and found to be stable over time. The protection and release of DNA within the PD and LPD complexes was studied by using gel assays. The DNA binding affinity and transfection efficiencies are presented and discussed. Chapter 3 outlines the synthetic procedures to generate novel glycerol-based cationic lipids. The cationic headgroup was shielded with n-(ethylene glycol) (nEG) chains attached through non degradable and biodegradable linkers. It was noted that the transfection efficiency of formulations of lipid-nEG conjugate with DOPE was increasing with time. Further investigations using mass spectroscopy indicated the transacylation of an oleoyl group from DOPE to the terminal hydroxyl group of lipid-nEG conjugate, resulting in the formation of a new product compromised of three acyl chains (tri-chain lipids). This finding led to the synthesis of several series of novel tri-chain lipids. It was noted that most tri-chained analogues offered an enhanced transfection efficiency and DNA packaging compared to their original di-chained analogues. The synthetic routes towards the formation of novel glycerol-based lipids bearing a disulfide linker are given in Chapter 4. An overall summary and possible areas of future research are discussed in Chapter 5. A formal description of the experimental methods and procedures is presented in Chapter 6.

La mucoviscidose est une maladie monogénique, caractérisée par des mutations survenant au niveau du gène CFTR (Cystic Fibrosis Transmembrane Conductance Regulator). Le clonage en 1989 du gène CFTR a permis d’envisager de traiter cette maladie par thérapie génique. Cela consiste à transférer à l’aide d’un vecteur, une version normale du gène CFTR dans les cellules atteintes des patients. En raison de la gravité des complications pulmonaires, c’est l’épithélium respiratoire qui constitue aujourd’hui le tissu cible pour le transfert de gènes. Le principe de la thérapie génique est évidemment très séduisant et un certain nombre d’essais cliniques ont d’ores et déjà été réalisés. La thérapie génique nécessite des outils de vectorisation efficaces et compatibles avec une utilisation répétée en clinique.Mon sujet de thèse a porté donc sur le développement, la biodistribution et l’optimisation de vecteurs synthétiques (lipides cationiques) pour le transfert de gènes dans l’épithélium respiratoire. Au cours de mes travaux, nous avons donc pu mettre au point des lipophosphoramidates KLN47 fluorescents utiles pour les études de biodistribution in vivo. Comparés au KLN47 non fluorescent, ces nouveaux composés présentent les mêmes propriétés physicochimiques, à savoir une taille relativement petite et un potentiel zêta positif. Sur lignées cellulaires, nous avons montré que les nouvelles formulations étaient aussi efficaces que le KLN47, et pas ou peu toxiques. Ensuite, sur modèle animal, les profils de biodistribution de lipoplexes pégylés et non-pégylés ont été comparés après injection systémique. Les profils de biodistribution des lipoplexes pégylés et non-pégylés étaient similaires, cependant, la pégylation des complexes a conduit à une circulation prolongée dans la circulation sanguine, alors que l’expression du transgène (luciférase) était équivalente dans les deux cas. De plus, l’activité luciférase était similaire à celle obtenue avec le KLN47 non fluorescent. Nous avons ainsi démontré que l’ajout des sondes lipidiques fluorescentes dans la solution liposomale du KLN47, ne modifie pas ses propriétés physicochimiques et transfectantes. L’ensemble des résultats montre que nous disposons d’outils prometteurs pour les études de biodistribution in vivo. D’autres molécules ont également été testées avec succès
Cystic fibrosis is a monogenic disease characterized by mutations occurring at the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene. The clonining in 1989 of the CFTR gene has enabled to consider treating this disease by gene therapy. This consists of transferring a normal version of the CFTR gene in the affected patients’ cells, using a vector. Due to the severity of pulmonary complications, it is obvious that the respiratory epithelium constitutes the target tissue for the gene transfer. The principle of gene therapy is indeed very attractive and a number of clinical trials have already been made. Gene therapy requires vectorization tools that are efficient and compatible with repeated clinical use.My thesis has focused on the development, biodistribution and optimization of synthetic vectors (cationic lipids) for gene transfer in the respiratory epithelium. During my work, we were able to develop useful fluorescent KLN47 lipophosphoramidates for in vivo biodistribution studies. Compared to non fluorescent KLN47, these new compounds exhibit the same physicochemical properties: a relatively small size and a positive zeta potential. On cell lines, we found that the new formulations were as effective as the KLN47, with little or no toxicity. Then, in animal models, the biodistribution profiles of pegylated and non-pegylated lipoplexes were compared after systemic injection. The biodistribution profiles of pegylated and non-pegylated lipoplexes were similar. However, the pegylation of the complex resulted in prolonged circulation in the bloodstream, whereas transgene expression (luciferase) was equivalent in both cases. In addition, luciferase activity was similar to that obtained with the non-fluorescent KLN47. We have demonstrated that the addition of fluorescent lipid probes in the liposomal solution KLN47, does not change its physicochemical and transfectant properties. The overall results show that we have promising tools for in vivo biodistribution studies. Other molecules have also been tested successfully

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This work was dedicated to classify all the global phase portraits of the quadratic polynomial vector fields having a rational first integral of degree three. For this, techniques were used as blow-up, classification of singular points, invariant curves for a system of ordinary diferential equations and vector fields induced on the sphere.
Este trabalho foi dedicado a determinação global dos retratos de fase, no disco de Poincaré, dos campos vetoriais polinomiais quadráticos que possuem integral primeira racional de grau três. Para determinar o retrato de fase, utilizamos técnicas como blow-up, classi- ficação dos pontos singulares, curvas invariantes para um sistema de equações diferenciais ordinárias e a indução de campos vetoriais sobre a esfera.

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Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex? 355; Tween? 80; Spam? 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q?). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way
A terapia g?nica ? um dos maiores desafios propostos pela pesquisa p?s-gen?mica e se baseia na transfer?ncia de material gen?tico a uma c?lula, tecido ou ?rg?o com o intuito de curar ou melhorar o estado cl?nico do paciente. Em sua forma mais simples, a terapia g?nica consiste na inser??o de genes funcionais em c?lulas com genes defeituosos objetivando substituir, complementar ou inibir esses genes causadores de doen?as. Para que o DNA ex?geno seja expresso em uma popula??o celular faz-se necess?ria a sua transfer?ncia at? o local de a??o. Assim, ? necess?rio criar ve?culos, que transportem e protejam o DNA at? que este chegue a uma popula??o celular alvo. Os obst?culos encontrados com a utiliza??o de vetores virais t?m proporcionado o interesse no desenvolvimento de vetores n?o-virais, por serem f?ceis de produzir, apresentarem estabilidade control?vel e facilitarem a transfec??o g?nica. O objetivo deste trabalho foi avaliar dois diferentes vetores n?o virais, lipossomas e nanoemuls?es cati?nicos, e sua poss?vel utiliza??o na terapia g?nica. Para isso, foram utilizados lip?deos cati?nicos e co-tensoativos na produ??o dos dois sistemas. As nanoemuls?es foram produzidas pelo m?todo de sonica??o e compostas por Captex? 355; Tween? 80; Spam? 80; lip?deo cati?nico, Estearilamina (EA) ou N-[1-(2,3-Dioleoiloxi)propil]-N,N,Ntrimetilamonio metilsulfato (DOTAP); e ?gua ultra-pura (Milli-Q?). Estes sistemas foram caracterizados quanto ao tamanho m?dio de got?cula, ?ndice de polidispers?o (PI) e potencial zeta. Avaliou-se ainda a estabilidade dos sistemas e suas capacidades de compacta??o do material gen?tico. Os lipossomas foram preparados a partir do m?todo de hidrata??o do filme e compostos por DOTAP, Dioleilfosfatidiletanolamina (DOPE), na presen?a ou aus?ncia de Rodaminafosfatidiletanolamina (PE-Rodamina) e do conjugado ?cido Hialur?nico DOPE (HA-DOPE). Estes sistemas foram caracterizados da mesma forma que as nanoemuls?es e tamb?m foram avaliados estabilidade, influ?ncia do tempo, tamanho de material gen?tico e presen?a ou aus?ncia de endotoxinas na forma??o dos lipoplexos. Os resultados obtidos permitem afirmar que os sistemas s?o promissores para posterior utiliza??o na terapia g?nica e que esta ?rea promete ser uma ?rea f?rtil de pesquisa cient?fica e cl?nica por muitos anos, e provavelmente se tornar? uma pr?tica cl?nica importante neste s?culo. No entanto, da possibilidade ? pr?tica existe um longo caminho a percorrer

La grande majorité des essais cliniques de transfert de gènes in vivo utilise des vecteurs viraux. Si ces derniers sont efficaces, ils présentent des risques immunogènes, toxiques, voire mutagènes avérés. Les vecteurs synthétiques (non viraux), par leur grande modularité et leur faible toxicité représentent une alternative très prometteuse. Le principal frein à leur utilisation est leur manque d’efficacité. L’objectif majeur de ce travail de thèse a été de comprendre le mécanisme de transfert de gènes associé à différents complexes vecteurs synthétiques/ADN plasmidique, ce qui est indispensable pour une conception rationnelle de nouveaux vecteurs. Nous avons étudié, sur cellules en culture, le mécanisme de transfert de gènes associé à deux lipides cationiques ; le BGTC (bis(guanidinium)-tren-cholesterol) et la DOSP (DiOleylamine A-Succinyl-Paromomycine) qui sont connus pour être des vecteurs efficaces in vitro. Nous avons ainsi pu visualiser par microscopie électronique leurs voies d’entrée, leurs remaniements structuraux ainsi que leur échappement endosomal qui représente une étape clé du processus de transfert de gènes. L’identification non ambigüe des lipoplexes tout au long de leur trafic intracellulaire a été rendue possible grâce au marquage de l’ADN par des nanoparticules de silice dotées d’un cœur de maghémite (Fe2O3) dense aux électrons. Cette stratégie de marquage a également été appliquée à l’étude du mécanisme d’action d’un autre vecteur synthétique de type polymère, le copolymère à blocs non ionique P188 ou Lutrol. Contrairement à la plupart des vecteurs synthétiques, celui-ci présente une efficacité de transfection in vivo chez la souris par injection in situ pour le tissu musculaire ou en intra trachéale dans le poumon. En revanche, il est totalement inefficace in vitro. Nous avons montré que le Lutrol permet une augmentation de l’internalisation d’ADN par les cellules mais n’induit pas son échappement endosomal, ce qui expliquerait son absence d’efficacité in vitro. D’autres voies d’entrée sont alors à envisager in vivo pour comprendre son mécanisme d’action
The vast majority of clinical trials of gene transfer in vivo use viral vectors. Although they are effective, they induce immunogenic, toxic or mutagenic risks. Due to their high modularity and low toxicity, synthetic vectors (non viral), represent a promising alternative despite their lack of effectiveness. The major objective of this work was to understand the mechanism of gene transfer using two prototypic synthetic vectors, in the context of a rational design of new vectors. We studied on cultured cells, the mechanism of action of two cationic lipids; BGTC (bis(guanidinium)-tren-cholesterol) and DOSP (DiOleylamine A-Succinyl-Paromomycine) formulated with plasmid DNA (lipoplexes) which are in vitro efficient vectors. We have been able to visualize by electron microscopy, their intracellular pathways, their structural alterations and their endosomal escape, the latter being a key step in the process of gene transfer. The unambiguous identification of lipoplexes throughout their intracellular trafficking has been made possible thanks to the labelling of DNA by core-shell silica nanoparticles with an electron dense maghemite core (Fe2O3). The labeling strategy has also been applied to study the mechanism of action of a nonionic block copolymer (P188 or Lutrol). Interestingly, these synthetic vectors have an in vivo transfection efficiency in mice lung and muscle tissue while they are totally inefficient in vitro. We have shown that Lutrol induces an increase of DNA internalization into cells and fails to trigger endosomal escape, which would explain the lack of in vitro efficacy. These findings suggest that the in vivo mechanism of action of Lutrol would involve other internalization pathways

碩士
高雄醫學大學
醫藥暨應用化學研究所
100
In this study, amphiphilic star-shaped cationic copolymers were synthesized of hydrophobic monomer and hydrophilic cationic monomer. The synthesized polymer was determined by 1H-NMR spectroscopy and gel permeation chromatography (GPC). The synthesized star-shaped cationic copolymer micelles and hybrid micelles contained PEG are formed by oil-in-water solvent evaporation method. The polyplex binding ability, transfection efficiency and cytotoxicity of micelles are all compared by gel electrophoresis performance, luciferase assay and MTT assay.

博士
國立臺灣大學
生物化學暨分子生物學研究所
91
During the past years, non-viral gene delivery vector was emerging as a new modality as well as a technology in laboratory and in medical practice. A promising area of research is the design of targeted lipid-DNA complexes. It is clear that the physicochemical properties of lipid-DNA complexes are very important in the design of the ideal lipidic vector. The requirements for such an ideal vector can be summarized as follows: (1) the particles should be small enough to be taken up by organs and surface protected by polyethylene glycol (PEG), to avoid protein interaction in the serum and thus extend their circulation life time into the blood; (2) furthermore, the addition of a targeting ligand is necessary to allow specific recognition by target cells, and (3) the DNA should be condensed and protected from nuclease degradation; (4) finally, DNA should be sufficiently inert to the immune cells to avoid a high level of cytokine production. The results in this report demonstrate the feasibility of improvement in target-specific gene delivery to HER-2-overexpressing cells by insertion of lipid-modified anti-HER-2-Fab’ into the preformed liposomes; in another hand, the large polyethylenimine (PEI) could synergistically increase the transfection efficiency when combined with the cationic liposomes.

碩士
國立臺灣大學
藥學系
86
Abstract Gene therapy could represent an important advance in the treatment of both inherited and acquired disease. Successful of human gene therapy depends upon the development of delivery vehicles or vectors which can selectively deliver therapeutic genes to the target cells with efficiency and safety. Various methods of nonviral vectors have been proposed. Cationic liposomes are particularly attractive due to their favorable characteristics such as biodegradability, minimal toxicity, nonimmunogenicity, and relati ve ease of large scale production. However, the composition of the liposome, phospholipids, were very unstable during the period of storage The purposes of the present study were: a)to evaluate the shelf- life of AF-cationic liposome stored under different conditions, and b) to examine the gene delivery efficiency of AF-cationic liposome in vivo using experimental hepatoma rats. Asialofetuin(AF), a glycoprotein with triantennary galactose terminated sugar chain was selectively recognized by the specific receptor on the plasma membrane of the hepatocytes. AF labelled cationic liposomes have been reported to be able to increase transfection efficiency in hepatoma cell lines in the receptor mediated manner. DC-Chol (3b[N-(N*,N*-dimethylaminoethane)- carbamoyl]cholesterol) and DOPE (Dioleoyl phosphatidyl ethanolamine)(1/1 molar ratio) were selected for preparing the cationic liposome. AF-DC liposome was prepared by the detergent dialysis method. The freshly prepared liposomes were dived into two portion. One was stored at 4℃and another, after lyophylization, was stored at -20℃. They were sampled on the schedule time for the gene transfection experiment, the reporter gene was fluorescence gene (pGreen Lantern-1 and the cell lines were HepG2, GP7TB, NIH3T3. The transfection efficiency was examined by the fluorescence microscope and quantitated by a flow cytometry. Adult male rats (Fischer) were used for all in vivo experiment. GP7TB (5×106~107cell/ml) 300ml was injected into subcutis of Fischer rats. After 21~30 days, the tumor was transferred to liver. As the tumor grew up to about 1~2 cm, rat*s hepatoma was exposed upon abdominal incision, and the DNA-liposome mixture (DNA/liposome =1/5, w/w) was injected around and into the hepatoma, then the abdominal incision was sutured. At 24, 48 and 72 hr following injection, liver was excised for histologic examination unde the fluorescence microscope. The result of in vitro study showed that AF-DC liposome exhibited better transfection efficiency in HepG2 and GP7TB cell lines than NIH3T3, and the lyophilized liposomes stored at -20℃kept longer shelf-life than the liposome stored at 4℃. The thermograms of DSC suggests that degradation of liposomal lipid may be the primary factor for loss of transfection activity. In animal experiment, no evidence of successful gene transfection was observed. In summary, the lyophilization was a better storage method for AF-DC liposome. Much effort should be devoted to further study the expression of the vector, AF-DC liposome, in vivo.

碩士
國立臺灣大學
藥學研究所
89
Abstract The main purpose of the present study was to evaluate the influence on the transfection efficiency of Gal-lipoosme/DNA complexes after freeze-thawing or freeze-drying in the presense of sucrose as cryoprotectant. Meanwhile, the possibility of two cholesterol derivatives to prepare cationic liposomes was also evaluated. The mouse embryonic normal liver cell line (BNL CL.2) was used to be the transfection target. DC-chol (3β[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol) and DOPE (dioleoyl phosphatidyl ethanolamine) (1/1 molar ratio) were selected for preparing the cationc DC-liposomes. DC-liposomes were modified by galactosyllipid which was synthesized in our laboratory (Gal-liposomes) and applied as a gene delivery vector. BNL CL.2 cells were incubated with fresh, freeze-thawed, or freeze-dried Gal-liposome/DNA complexes consisting of a reporter gene (pGreen Lantern-1) for 5 hrs in an incubator. Then the medium was replaced by fresh DMEM. After 48 hrs, the transfection efficiency was quantified by flow cytometery. To evaluate the stability of rapid freeze-dried Gal-liposomes and Gal-liposome/DNA complexes which were stored at —20℃ and 4℃, the transfection efficiency on the 3rd and 7th day after freeze-drying were also quantfied by flow cytometery. Pip-liposomes or Gua-liposomes were prepared by Pip-chol (4-methyl-piperazine-1-carboxy cholesterol)/DOPE or Gua-chol ((2-guanidino-ethyl)-carbamoyl cholesterol)/DOPE (1/1 molar ratio). Their ability to present on liposomes was evaluated by zeta potential values. The results showed that freeze-thawed and freeze-dried Gal-liposome/DNA complexes were lyoprotected by 1mmole per o.268 μmole total lipid. The transfection efficiencies of rapid freeze-dried Gal-liposomes and Gal-liposome/DNA complexes stored at —20℃ and 4℃ after 28 days were not significant different from that of fresh Gal-liposome/DNA complexes. And according to zeta potential values, there was not net positive charge on the surface of Pip-liposomes or Gua-liposomes. In summary, the rapid freeze-dried Gal-liposomes and Gal-liposome/DNA complexes could be stored under 4℃ for 28 days with entire transfection efficiency but the stability of extended storage period should be further studied. And Pip-chol and Gua-chol were not suitable to be the lipid content of cationic liposomes.

碩士
嘉南藥理科技大學
生物科技系暨研究所
96
Efficient transfect ion and nontoxicity were two of the most important requirements of an idea gene delivery vector. The main objective of this study was the development of new carrier system by blending. These polyesters were obtained in high through a Michael-type conjugated addition of diacrylate monomers with amine monomers. We identified the structure and molecular weights of two cationic polymers: PEDP and PE-3D by NMR、FT-IR and GPC. PEDP and PE-3D had less cytotoxicity in the different cells. PEDP and PE-3D were able to combine with DNA yield complexes at a mass ratio of 15/1 to 20/1 and 10/1 to 15/1. We used the PEI to modify the PEDP and PE-3D with different mass ratios by blending, and then react with plasmid DNA. The results were showed that the polymers blending with PEI were better than others.

碩士
嘉南藥理科技大學
生物科技系暨研究所
93
The goal of this project is to design and synthesize the biodegradable cationic polymer with low cytotoxicity that can be used as vector for gene delivery. To optimize the relation between transfection efficiency, the characterizations of molecular weight, buffering capacity, the structure of the polyurethane, and complexes formed by the plasmid DNA with polycation. In this assay show the protection effect of polycations to plasmid DNA against endonucleases and bovine albumin by agarose gel band assay. The acid-base titration profile was obtained for polymers resulting in a proton buffering effect within the endosomal/lysosomal compartments of the cell. New Cationic Polyurethane as Potential Non-Viral Vector: Synthesis and Characterizations of Molecular Weight To study the characterizations of molecular weight of LGEA-PU on transfection, polymers with four different molecular weights were prepared. In the particle size and zeta-potential assays, it was found that LGEA-PU with higher molecular weight possessed DNA condensing with results required for transfection. The LGEA-PU59/DNA complexes were able to transfect COS-7 cells in vitro with higher transfection efficiency than the PEI and other LGEA-PU systems. The introduction of PEG into the backbone of polyurethane can provide better cytotoxicity profiles, and increased the hydrophilicity and flexibility of the polymer, promoting the susceptibility to hydrolysis. Relation between Transfection Efficiency and Structure Characterization of Amino Groups in the Cationic Polyurethanes The aim of the study is that to optimize the structure in the side chain or backbone of the polyurethane (e.g., targeting groups) so that the transfection efficiency and characterizations of particles formed by the plasmid DNA with polycation can be improved. We synthesized novel water-soluble amino acid-based polyurethanes, PEG-PU bearing tertiary amines in the side chain and PPA-PU bearing tertiary amines in the backbone and side chain. Although the PEG-PU and DNA was able to form complexes, the interpolyelectrolyte force between PEG-PU and DNA was poor, not stable to bovine albumin. It was found that bovine albumin is unable to release any DNA from the PPA-PU/DNA complexes. That PPA-PU has stronger electrostatic force between polymer and DNA and higher pH buffering abilities. Structural Characterization and Buffering Capacity in Relation to the Transfection Efficiency of Biodegradable Polyurethane The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than Poly 2 and Poly 1. This study reveals that the amine groups in polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Effect of Side Chain Bearing Tertiary Amino Group on Transfection Efficiencies of Cationic Polyurethanes as Gene Vectors To explore the side chain bearing tertiary amino groups-transfection efficiency relationships of the polyurethanes, we synthesized a variety of polyurethanes (Poly 1-6). These data showed that Poly 6 demonstrated the most transfection efficiency, and suggested that transfection abilities of cationic polyurethanes were improved by increasing the number of amino unit. It was found that the numbers of side chain bearing tertiary amino groups can significantly improve the DNA condensing abilities, buffering capacities, and transfection efficiencies of the cationic polyurethanes.

This thesis explores the capabilities of cationic polymers modified with lipids of different carbon chain length to deliver DNA molecules to primary cells and transformed cell lines. Our studies focus on two different polymers: polyethylenimine (PEI) and poly(L-lysine) (PLL). Firstly, PEI and PLL were conjugated to palmitic acid (C16). The delivery of plasmid DNA to rat bone marrow stromal cells (rat-BMSC) was evaluated by using a Green Fluorescent Protein gene expressing plasmid (pEGFP-N2) as a reporter system. The rationale for lipid substitution is to give the polymer an amphiphilic character so as to improve the transfection efficiency of native polymers by improving the DNA/polymer translocation through the phospholipid-rich cell membranes. In the case of PLL-C16, transfection efficiency was significantly increased (5 fold) as compared to native PLL, and it was significantly higher than commercially available cationic lipids (LipofectamineTM 2000 and FugeneTM). We further explore the use of other lipids with variable chain lengths (carbon chain length ranging from 8 to 18 saturated and unsaturated) in order to identify other candidates to enhance the gene delivery properties of the PLL. Lipid-modified PLL of high molecular weight (25 vs. 4 kDa) was found to be more effective in delivering plasmid DNA in rat-BMSC. We noted that C14-, C16- and C18-substituted PLL gave the most effective DNA delivery. Moreover, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was found Additionally, transgene expression by BMSC significantly increased when amphiphilic PLLs were used as compared to native PLL. The modified polymers were able to transfect the cells up to 7 days, after which the expression decreased. Encouraged by the successful transgene expression agents obtained by modifying low molecular weight PEI with the same series of lipids described above, we explored the possibility of modifying low molecular weight PEI (2 kDa) with longer lipids; saturated fatty acid (C22), trans fat (C18:1T) and essential fatty acids (C22:1, C22:6 and C18:3). Transfection efficiency proved to be cell dependent. Only the transformed 293T cells were able to express GFP compared to human-derived BMSC. The highest transfection efficiency was found with highly unsaturated lipid-substituted PEI (C18:3 and C22:6) and were able to increase transgene expression overtime (6 days). Furthermore, internalization studies indicated that effective transfection of these carries do not follow any known endocytosis pathway instead the DNA/carrier penetrates the plasma membrane directly.
Pharmaceutical Sciences

Tese de doutoramento em Ciências (área de especialização em Física)
Throughout the last decades, several liposomal formulations have been developed with the goal of condensing, transporting and releasing complementary DNA (cDNA) into cells, thus allowing the treatment of several genetic diseases via DNA replacement therapy. Although this technology has been partially substituted with the more contemporary use of small interference RNA (siRNA) therapy, the intracellular delivery of cDNA still remains the only viable gene therapy approach for stable host genome modification instead of transient transfection. The success of lipofection technique greatly depends on the structural and physicochemical properties of the liposomal carrier and ultimately, on the molecular attributes of the components of the lipid mixture. The empirical testing of diverse families of lipids such as cationic surfactants, pH-sensitive tensioactives or other lipid conjugates has allowed the development of highly efficient nanoparticles that stably condense the genetic material and withstand the harsh destabilizing conditions of the biological environment. However, it is this very same increased carrier stability that is responsible for limiting the gene release from the nucleic acid (NA)/cationic liposome complex (lipoplex) and reducing its final cell transfection efficiency. One recent approach to overcome this problem has come with the inclusion of non-lamellar forming lipids (also called helper lipids) in the liposomal formulation, which potentiate the formation of membrane fusion intermediates that disrupt the lamellar organization of the lipoplexes and favour the release of the genetic content. Molecules such as dioleoylphosphatidyl ethanolamine (DOPE) and cholesterol (Chol) have been employed with success as helper lipids in several liposomal formulations, enhancing the lipofection efficiency through the formation of inverted hexagonal structures (HII). Adopting a similar strategy, the work plan for this thesis was focused on the development of a new nucleic acid (NA)/cationic liposome formulation for gene delivery purposes comprising the synthetic cationic lipid Dioctadecyl Dimethylammonium Bromide (DODAB) and the non-ionic and non-lamellar forming lipid Monooleoyl-rac-glycerol (monoolein, MO). The structural and physicochemical characterization of DODAB:MO liposomes and DNA/DODAB:MO lipoplexes has been done through light scattering, microscopy, spectroscopy (with emphasis in UV/Visible fluorescence emission) and calorimetry techniques. The lipoplex formation process has been studied through the same techniques referred above at varying molar fractions of cationic lipid/DNA and cationic lipid/neutral lipid, while lipoplex destabilization was assessed by incubation of the pre-formed lipoplexes in several physiological simulation environments (salt, serum, temperature, pH, anionic lipids). Finally, the cell transfection efficiency and cytotoxicity profile of these non-viral gene delivery vectors was assessed by β-galactosidase reporter gene expression and Lactate Dehydrogenase (LDH) cell viability assays, respectively, in the 293T human embryonic kidney cell line. The results attained substantiate the high potential for DNA/DODAB:MO lipoplexes to be used as nonviral vectors in gene delivery, and validate the application of MO as helper lipid in cationic liposome formulations, being a viable alternative to classic DOPE and Chol molecules.
Ao longo das últimas décadas, várias formulações lipossomais têm sido desenvolvidas com o intuito de condensar, transportar e libertar DNA complementar (cDNA) em células, permitindo o tratamento de várias doenças genéticas através da terapêutica de substituição de DNA. Embora esta tecnologia tenha sido parcialmente substituída pelo uso mais contemporâneo da terapêutica de RNA de interferência (microRNA), a entrega intracelular de cDNA permanece ainda como a única alternativa viável para a modificação genómica definitiva do hospedeiro em vez da transfeção transiente. O sucesso da técnica de lipofeção depende grandemente das propriedades estruturais e físico-químicas do vetor lipossomal e, em última instância, das propriedades moleculares dos constituintes da formulação lipídica. O teste empírico de diversas famílias de lípidos tais como os surfatantes catiónicos, os tensioativos sensíveis a pH ou outros conjugados lipídicos permitiu o desenvolvimento de nanopartículas altamente eficientes capazes de condensar estavelmente o material genético e suportarem as agressivas condições desestabilizantes do microambiente biológico. Contudo, é esta mesma estabilidade acrescida dos vetores que é responsável por limitar a libertação de genes dos complexos DNA/lipossomas catiónicos (lipoplexos) e reduzir a eficiência final de transfeção celular. Uma abordagem recente para ultrapassar este problema proveio da inclusão de lípidos nãolamelares (também chamados de lípidos adjuvantes) na formulação lipossomal, que potenciam a formação de intermediários membranares fusogénicos que desestabilizam a organização lamelar dos lipoplexos e favorecem a libertação do conteúdo genético. Moléculas tais como dioleoilfosfatidil etanolamina (DOPE) e colesterol (Chol) têm sido utilizadas com sucesso como adjuvantes em várias formulações lipossomais, aumentando a eficiência de lipofeção através da formação de estruturas hexagonais invertidas (HII). Adotando uma estratégia similar, o plano de trabalhos para esta tese contemplou o desenvolvimento de uma nova formulação ácido nucleico (NA)/lipossoma catiónico para efeitos de entrega genética, composta pelo lípido catiónico sintético Brometo de Dioctadecil Dimetilamónio (DODAB) e o lípido não-iónico e não-lamelar Monooleína (MO). A caracterização estrutural e físico química dos lipossomas DODAB:MO e lipoplexos DNA/DODAB:MO foi feita através das técnicas de dispersão de luz, microscopia, espetroscopia (com ênfase na emissão de fluorescência UV/Visível) e calorimetria. O processo de formação dos lipoplexos foi estudado através das mesmas técnicas referidas anteriormente em frações variáveis de lipossomas catiónicos/DNA e de lípido catiónico/lípido neutral, enquanto a desestabilização dos lipoplexos foi avaliada pela incubação dos lipoplexos recém-formados em diversos microambientes fisiológicos simulados (sal, soro, temperatura, pH, lípidos aniónicos). Finalmente, a eficiência de transfeção celular e o perfil citotóxico destes vetores genéticos não-virais foi avaliada por ensaios de expressão do gene repórter β-galactosidase e ensaios de viabilidade celular com a enzima Lactato Desidrogenase (LDH), respetivamente, na linha celular 293T derivada de rim embrionário humano. Os resultados obtidos substanciam o elevado potencial dos lipoplexos DNA/DODAB:MO para serem usados como vetores não-virais em entrega genética, e validam a aplicação de MO como lípido adjuvante em formulações de lipossomas catiónicos, apresentando-se como uma alternativa viável às moléculas clássicas DOPE e Chol.

碩士
國立中正大學
化學暨生物化學研究所
101
Over the past few years, a number of studies have been conducted to focus on the hydrolysis property of imine groups. Research on cationic polymers contain imine groups in related to their capability of gene delivery has not yet been investigated. In this research, a new series of poly(L-lysine) – grafted – polyethylenimine（PLI）were successfully synthesized. The grafted polymer contains poly(L-lysine) as a backbone with and low-molecular-weight polyethylenimine（LMW-PEI）in the side chains via imine linkages, was examined for their potentials as non-viral gene vectors which deliver plasmid DNA into cells in vitro. The electrophoretic mobility on agarose gels, dynamic light scattering and zeta potential analyses of polymer∕DNA complexes confirmed that PLIs were able to self-assemble with plasmid DNA to form nano-scaled（around 200 nm）and positively charged complexes（~ + 25 mV）. PLIs hardly showed any cytotoxicity on COS-7 cells duo to their biodegradability and biocompatibility. Most importantly, in vitro transfection results showed that PLIs displayed a far better transfection efficiency in comparison to LMW-PEI and poly(L-lysine). Moreover gene delivery results even better than the commercially available high-molecular-weight polyethylenimine (PEI25K). Therefore, these new cationic polymers with imines having low cytotoxicity and high transfection efficiency may be applied as safe and efficient non-viral gene delivery vectors.

Gene therapy is a potential strategy for treatment of diseases caused by a gene defect. Recent studies are involved particulary in the cure of diseases caused by single gene defect (cystic fibrosis, haemophilia, muscular dystrophy etc.). Our work is part of a project aiming at developing ex vivo non-viral gene delivery systems that could be used for the treatment of ocular and cardiovascular diseases. The gene vectors are biodegradable polymeric carriers based on poly-α-amino acids. These polyplexes should transfect target cells which are supposed to be seeded on polyimide membranes. The biodegradable polymer membrane will be implanted into the retina or used as a coating for cardiovascular prosthesis. As a cover of the implantable membranes we used polymerized methacrylamide-modified gelatin forming hydrogels and mediating a growth support for transfected cells. We focus on material bio- and immunocompatibility/immunoacceptability. The results indicated a very good bio- and immunocompatibility of the gelatin B hydrogel both in vitro and in vivo. The gelatin B hydrogel did not cause erythrocytes lysis, stimulation of proliferation (spontaneous or mitogen-induced) of mouse or human lymphoid cells, neither production of cytokines or NO in vitro. Histological examination following subcutaneous...

Dissertação de mestrado em Biofísica e Bionanossistemas
Gene therapy based on gene silencing with siRNA has evolved and gained great importance over the last few years as a method with great potential in the treatment of genetic diseases. However, a major challenge to the use of RNAi-based therapies is their efficient delivery through an appropriate vector. Cationic liposomes are an example of the most versatile nonviral vectors for the delivery of nucleic acids and have been researched in the past few years as a strategy to promote effective transfection of genetic material. This project has focused on the development and characterization of novel vectors for therapeutic siRNA delivery based in three different gemini amino acid-based surfactant derived from serine with an helper lipid called monoolein (MO). Monoolein lipid is a known promoter of inverted nonbilayer structures such as inverted cubic mesophases and could enhance the fusogenicity of serine-based gemini aggregates while modulating the properties of the final aggregates. The use of these vectors aims at therapeutic purposes, and thus requires a full characterization of the formed systems. The vectors must have robust properties and the ability to ensure efficient and reliable performance. Therein, it is shown that the chemical nature of the spacer linker (amine, amide and ester) in serine-based gemini surfactant influences the properties of the aggregates formed and their complexes with nucleic acids. On the other hand, the inclusion of MO in liposomal or micellar formulations, as helper lipid, induces modifications on the morphology of the aggregates. Typically, this leads to a reduction in the mean diameter of particles, without the need of mechanic processes such as sonication or extrusion to increase their stability over time. The three gemini:MO systems tested have good ability to complex efficiently siRNA and they did not show significant levels of cytotoxicity. This compactation of siRNA leads to liposomal/micellar-siRNA complexes with optimal mean diameters, which could be used in vitro and in vivo. Lastly, the capacity to deliver siRNA and thus silence the target gene was also evaluated. The results obtained showed high percentages of down regulations, indicating that the gemini:MO formulations studied in this work are good candidates as lipofection vectors for RNAi-therapies.
A terapia génica baseada no silenciamento de genes com siRNA foi evoluindo e ganhando grande importância nos últimos anos como um método com grande potencial no tratamento de doenças genéticas. No entanto, um dos grandes desafios no uso de terapias à base de RNA de interferência é a sua entrega eficiente através de um vetor apropriado. Os lipossomas catiónicos são um exemplo de vetores não virais dos mais versáteis para a entrega de ácidos nucleicos e têm sido estudados nos últimos anos como uma estratégia para promover a transfeção eficaz do material genético. Este projeto focou-se no desenvolvimento e caracterização de novos vetores para entrega de siRNA baseados em misturas de surfactantes gemini derivados do aminoácido serina com um lípido co-adjuvante - monoleína (MO). Os surfactantes gemini diferem entre si somente pelo tipo de ligações ao nível do espaçador, que podem ser ligações éster, amida e amina. A monoleína é um conhecido promotor de estruturas invertidas como fases cúbicas e pode aumentar a fusogenicidade de agregados, enquanto modula as propriedades dos agregados finais. Uma vez que estes vetores são projetados para uso terapêutico, é imperativo caracterizar completamente os sistemas formados, que devem ter propriedades consistentes e capacidade de assegurar o desempenho eficiente e confiável. Este trabalho mostrou que a natureza química do espaçador dos surfactantes gemini derivados de serina (éster, amida e amina) tem influência nas propriedades dos agregados formados e, consequentemente, nos seus complexos com ácidos nucleicos. Por outro lado, a inclusão de MO, como lípido co-adjuvante, nas formulações lipossomais/micelares, induz modificações na morfologia dos agregados, levando à diminuição do seu diâmetro médio, sem a necessidade de se utilizar métodos mecânicos – como sonicação ou extrusão – para aumentar a estabilidade do agregado ao longo do tempo. Os três sistemas gemini:MO estudados não apresentam níveis de citotoxicidade significativos e têm elevada capacidade para complexar, eficientemente, siRNA. Esta compactação do siRNA origina complexos lipossomais/micelares-siRNA com diâmetros médios ótimos para aplicação, tanto in vitro como in vivo. Por último, foi avaliada a capacidade destes vetores para libertarem o siRNA e silenciar um genealvo. Os resultados obtidos mostram elevadas percentagens de silenciamento genético, indicando que as formulações Gemini:MO estudadas neste trabalho, são eficazes como vetores de lipofecção para terapias baseadas em RNA de interferência.

Gene therapy has gained increased attention over the last decades due to the possibility to treat a disease at its routes. Several vehicles intended to carry and deliver a functional copy of the deficient gene have been developed. Amongst these, viral vectors are highly effective systems, capable to deliver the genetic cargo to the nucleus. However, these carriers have raised safety concerns regarding to immunogenicity and insertional mutagenesis, creating the need to develop equally efficient vehicles with higher safety profiles. Therefore, non-viral vectors have been suggested as an alternative to viral gene transfer methods, as these overcome some of the drawbacks presented by viral vectors. The main goal of this project was to develop safe and effective non-viral gene carriers, using solid lipid nanoparticles (SLNs) with surface modulated properties. SLNs with surface modulated properties using polyethyleneimine (PEI) combined, or not, with protamine, were produced by hot high shear homogenization. The obtained particles possessed sizes <300 nm suitable for intravenous administration, and good physical stability for 3 months, under the different storage conditions tested (4ºC, room temperature and 37ºC). Moreover, these particles showed good plasmid condensation levels and were able to deliver the gene into the nucleus. Additionally, no cytotoxic effects concerning membrane integrity and metabolic activity of HEK 293-T cells were observed after 24 h of exposition. In conclusion, the developed nanoparticles presented suitable properties for gene delivery, with high capacity to condense DNA and transfect cells without cytotoxicity.