Resumo Em diversas formulações industriais, encontram-se, facilmente, vários exemplos da utilização de biopolímeros derivados de celulose em conjunto com tensoativos. Entretanto, na literatura, há divergências se ocorre realmente uma interação entre um polímero não iônico, como os derivados aquo-solúveis de celulose, em conjunto com tensoativos catiônicos. Desta forma, neste projeto de Mestrado, foram investigadas as interações entre três derivados de celulose (2-hidroxietil celulose, 2-HEC; hidroxipropil celulose, HPC e hidroximetilpropil celulose, HMPC) e três tensoativos catiônicos (cloreto de benzil-hexadecildimetilamônio, CBz; cloreto de fenil-hexadecildimetilamônio, Fenil e cloreto de 2-feniletil hexadecildimetilamônio, 2-Feniletil) obtendo-se informações sobre a estrutura dos agregados formados por este sistema através de medidas de condutividade, fluorescência no estado estacionário e viscosidade. Estas técnicas forneceram os parâmetros de concentração de agregação crítica (cac), concentração micelar crítica (cmc), grau de dissociação micelar e viscosidade das soluções. Em uma segunda etapa, foi investigada a variação da cmc do tensoativo zwitteriônico, coco amido propil betaína (CAPB), em soluções aquosas com diferentes pHs, visando futuros estudos de interação deste compostos com os biopolímeros derivados ou não de celulose. / There are many industrial formulations that are consisted of a biopolymer, such as cellulose derivatives, and surfactants. However, in literature, there are some divergences if there is a cooperative interaction between non-ionic polymers, and cationic surfactants. For this reason, in this project, the interactions between three cellulose derivatives (2- hydroxyethyl cellulose, 2-HEC, hydroxypropyl cellulose, HPC, and hydroxymethylpropyl cellulose, HPMC), and cationic surfactants (benzyl hexadecyldimethylammonium chloride, CBz, phenylhexadecyldimethylammonium chloride, Fenil, and 2-phenylethyl hexadecyl dimethylammonium chloride, 2-Feniletil) were studied through conductivity, steady-state fluorescence and viscosity measurements. The critical aggregation concentration (cac), critical micelle concentration (cmc), micelle dissociation degree, and viscosity were determined for each polymer surfactant system. In addition, the cmc of a zwitterionic surfactant, coco amido propyl betaine (CAPB), was measured in aqueous solutions of different pHs. This last experiment was performed as a preliminary essay to study the interaction of zwitterionic surfactant with several types of biopolymers.

A beta-alumina de sódio é uma cerâmica condutora de íons Na+ utilizada como eletrólito sólido em baterias de sódio para armazenamento de energias intermitentes como energia solar e eólica. Devido ao alto teor de sódio, esse material é instável a altas temperaturas, podendo sofrer variações de composição durante a etapa de sinterização convencional que utiliza altas temperaturas por longos períodos de tempo. A sinterização flash é uma técnica de sinterização ativada por corrente elétrica que proporciona a densificação de compactos cerâmicos em poucos segundos, a temperaturas notavelmente mais baixas que as convencionais. Uma vez obrigatória a passagem de corrente elétrica através da amostra, a sinterização flash de qualquer material condutor parece bastante razoável. Não obstante, até o presente momento a maioria dos trabalhos publicados sobre o assunto aborda apenas condutores de vacância de oxigênio ou semicondutores, materiais compatíveis com eletrodos de platina (Pt). Nesse trabalho a sinterização flash de um condutor catiônico foi estudada utilizando-se a beta-alumina como material modelo. A beta-alumina foi sintetizada pelo método dos precursores poliméricos, caracterizada e então submetida à sinterização flash. O material de eletrodo padrão (platina) provou ser um eletrodo bloqueador em contato com a beta-alumina. O sucesso da sinterização flash foi determinado pela troca do material de eletrodo por prata (Ag), o que possibilitou uma reação eletroquímica reversível nas interfaces eletrodo-cerâmica e possibilitou a obtenção de um material densificado com morfologia e composição química homogêneas. Devido à metaestabilidade da beta-alumina, a atmosfera dos experimentos precisou ser alterada para manter a integridade desse material rico em um metal alcalino (Na+). A sinterização flash de um condutor catiônico é apresentada pela primeira vez na literatura e ressalta a importância da reação de eletrodo, que é um fator limitante para o sucesso da sinterização flash e precisa ser estudada e adaptada para cada tipo de material. / Sodium beta-alumina is a Na+ ions conductor ceramic used as solid electrolyte in sodium batteries for energy storage in solar and eolic plants. Due to its high content of sodium, this material is instable at high temperatures and can suffer decomposition during conventional sintering (high temperatures for long periods of time). Flash sintering is an electric current activated sintering technique which promotes the densification of ceramic powder compacts in a few seconds at significantly lower temperatures than conventional sintering. Since in flash sintering the electric current flows through the sample, the flash sintering of any conductor material seems to be reasonable. Nevertheless, up to this date all published papers concern flash sintering of oxygen vacancies conductors or semi-conductors, materials which are compatible with platinum (Pt) electrodes. In this work the flash sintering of the cationic conductor betaalumina was studied. Beta-alumina was synthetized by the polymeric precursors method, characterized and then it was submitted to flash sintering. Platinum electrode was shown to be a blocking electrode for beta-alumina. Flash sintering success was determined by the change in electrode\'s material to silver (Ag), which made possible the reversible electrochemical reaction at the electrode-electrolyte interfaces and preserved chemical composition and morphology after flash. Due to this electrolyte metastability, atmosphere had to be changed in order to preserve the chemical composition of an alkali-rich (Na+) material such as beta-alumina. The flash sintering of a cationic conductor is shown for the first time in the literature and highlights the relevance of electrode reaction, which is a limiting point to the success of a flash sintering and needs to be well studied and adapted to each type of material.

Embora menos eficientes que os vetores virais, lipídios catiônicos têm sido cada vez mais usados como transportadores de material genético. Eles têm como vantagens em relação aos transportadores virais, a baixa imunidade, controle de qualidade e produção em grande quantidade, entre outros. Todavia, a interação entre lipídios catiônicos e material genético ainda é pouco compreendida. No presente trabalho, estudamos a interação entre o anfifílico catiônico brometo de dioctadecildimetilamônio (DODAB) e o nucleotídeo 2´-desoxiadenosina 5´-monofosfato (dAMP). Para tanto, utilizamos a técnica de Ressonância Paramagnética Eletrônica (RPE) aplicada a marcadores de spin derivados do ácido esteárico incorporados em vesículas de DODAB. Os resultados mostraram que na fase gel, na região próxima à superfície das bicamadas de DODAB, o dAMP causa muito pouca alteração na fluidez da membrana, enquanto que, na região do centro da bicamada, o dAMP diminui a fluidez da membrana, o que pode ser um efeito apenas de cargas, isto é, devido à blindagem eletrostática. Já na fase fluida, em ambas as regiões o dAMP aumenta a fluidez da membrana, sendo que, no centro das bicamadas induz um acréscimo significativo na polaridade do meio. A transição de fase gel-fluida do DODAB não sofre modificações com o acréscimo de dAMP. De uma maneira geral, nossos resultados mostraram que o dAMP não causa grandes modificações na estrutura do DODAB. Concluímos que o dAMP se localiza na região da superfície das bicamadas do DODAB e se comporta como um íon grande. Este resultado deverá ser considerado na análise estrutural da interação DNA/vesículas lipídicas em nível molecular. / Though less efficient than viral vectors, cationic lipids have been used as carriers in gene therapy. They offer several advantages over viral vectors, including the low immunogenic and inflammatory responses, the potential transfer of unlimited-size expression units, and the possibility for engineered cell-specific targeting. However, the interaction between cationic lipids and genetic material needs to be better understood for the optimization of the transfection protocol. In the present work, we study the interaction between the nucleotide 2´-deoxyadenosine 5´-monophosphate (dAMP) and cationic liposomes of dioctadecyl dimethylammonium (DODAB), through the analysis of the electron spin resonance (ESR) spectra of spin labels incorporated in the bilayers. DODAB gel phase structure is not much affected by dAMP. The observed packing effect at the bilayer core seems to be related to the expected electrostatic screening effect at the bilayer surface, bringing the headgroups closer together, due to the presence of anions at the surface. In DODAB fluid phase, above 44 oC, ESR results strongly suggest that the relatively large double charged molecule of DMP is localized at the bilayer surface, but incorporated in the membrane, such that it is able of somehow space the headgroups, turning the bilayer core less packed and more hydrated. This result is certainly relevant for the structural understanding of the DNA-cationic membrane interaction on a molecular level.

The discovery that cationic lipids can independently stimulate the immune system has generated interest in their potential as vaccine adjuvants. Here, we show that the cationic lipid R-DOTAP can independently stimulate type 1 interferon production in dendritic cells in both primary culture and immortalized cell culture. Levels of type 1 interferon production are cell line-dependent and limited in vitro by lipid-induced cell death. We show that cationic lipids can independently activate TLR-7 and TLR-9, suggesting a mechanism for type 1 interferon induction. This TLR-stimulatory activity is not restricted to R-DOTAP and can be extended to other similar cationic lipids in a lipid-specific and TLR-specific manner.

Photopolymerizations offer many advantages (such as temporal and spatial control of initiation, cost efficiency, and solvent-free systems) over traditional thermopolymerization. While they are now well-established as the preferred option for a variety of films and coating applications, they are limited from many applications due to problems such as oxygen inhibition, light attenuation, additive interference, or the creation of shadow regions and oxygen pockets due to complex shapes. These problems can be solved by using an underutilized form of photopolymerization--cationic photopolymerization. Cationic photopolymerizations have unique active centers which are essentially non-terminating causing extremely long active center lifetimes. In this contribution, the unique characteristics of cationic active centers are explored for their ability to be used in many new applications where previous photopolymerization techniques failed. It was found that the long lifetimes of the active centers permitted them to be very mobile, allowing them to migrate into and polymerize regions that were never illuminated in a process termed shadow cure. The mobility of cationic active centers provides a very efficient means of photopolymerizing of thick and pigmented systems. The long lifetimes of the cationic active centers can be used in the creation of a sequential stage curable polymer system and in the development of novel methods to cure complex shapes, two applications previously unattainable by photopolymerization. The termination of the cationic active centers was found to be reversible and can be used as a technique for external temporal control of the photopolymerization after the illumination has ceased. These abilities have great potential and will allow cationic photopolymerization to be used in many new applications where previous photopolymerization techniques failed, expanding their influence and benefits.

Orientador: Cristiane Duque / Coorientador: Luciano Tavares Angelo Cintra / Banca: João Eduardo Gomes Filho / Banca: Thais Marchini de Oliveira Valarelli / Banca: Christine Men Martins / Resumo: Este trabalho foi dividido em dois capítulos que objetivou avaliar: 1) o efeito isolado ou combinado do flavonoide epigallocatechin-3-gallate (EGCG) em associação com o peptídeo LL-37 e seu análogo KR-12-a5 sobre a viabilidade celular de fibroblastos e sobre cultura planctônica, biofilme simples, dual-espécies e túbulos dentináios e 2) as interações sinérgicas do EGCG e proantocianidina do oxicoco (A-type cranberry proanthocyanidins, AC-PAC), quando usado em combinação com LL-37 ou KR-12-a5 sobre a viabilidade celular, a capacidade de migração e inibição das citocinas em cultura de fibroblastos (HGF-1), quando estimuladas ou não pelo lipopolissacarídeo de A. actinomycetencomitans (LPS). No capítulo 1, a concentração inibitória mínima (MIC), a concentração bactericida mínima (MBC) e concentração inibitória fracionária (FIC) de EGCG, LL-37 e KR-12-a5 foram determinadas a partir de valores decrescentes dos compostos por meio dos métodos de microdiluição e checkerboard contra Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Fusobacterium nucleatum após 24 horas de tratamento. Fibroblastos da linhagem L-929 foram expostos a combinações de EGCG com peptídeos em diferentes concentrações e o metabolismo celular avaliado por ensaios de MTT. Os compostos com melhor efeito antimicrobiano e citotóxico foram avaliados por 24-36h, isoladamente ou em combinação, em biofilmes individuais ou biofilmes de dual-espécies com E. faecalis formados em placas de poliestireno por 4... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This study was divided in two chapters that aimed to evaluate: 1) the effect of flavonoid epigallocatechin-3-gallate (EGCG), cationic peptide LL-37 peptide and its analogue KR-12-a5, alone or in combination, on fibroblast cell viability and on bacteria in planktonic and single/dual-species biofilms/dentin tubules; 2) the synergistic interactions of EGCG and cranberry proanthocyanidins (A-type cranberry proanthocyanidins, AC-PAC), when used in combination with LL-37 or KR-12-a5 on cell viability, the ability to induce cell migration and inhibit cytokines in culture of fibroblasts (HGF-1) when stimulated or not by the lipopolysaccharide of A. actinomycetencomitans (LPS). For the chapter 1, Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and fractional inhibitory concentration (FIC) of EGCG, LL-37 and KR-12-a5 were determined from decreasing values of the compounds by Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii and Fusobacterium nucleatum against microdilution and checkerboard after 24 hours of treatment. L-929 fibroblasts were exposed to combinations of EGCG with peptides at different concentrations and cell metabolism assessed by MTT assays. The compounds if the best antimicrobial and cytotoxic effect were also evaluated for 24-36h, alone or in combination, in 48h single- or dual-species biofilms with E. faecalis formed on polystyrene plates by bacterial counting. E. faecalis biofilms were also cultured in dentin tubules f... (Complete abstract click electronic access below) / Doutor

Neste trabalho foram analisadas sob o ponto de vista físico-químico, as interações entre DNA e lipossomos catiônicos de brometo de dioctadecildimetilamônio (DODAB). Foram utilizados os seguintes modelos: 1) 2\'-desoxiadenosina 5\'-monofosfato (DMP), um modelo de unidade monomérica do polímero que é o DNA; 2) DNAs de bacteriófagos T2, T4, T5, T7 e λ; 3) vesículas pequenas de DODAB preparadas por sonicação; 4) vesículas grandes de DODAB preparadas por aquecimento (56 ºC/30 minutos) ou vaporização clorofórmica. A interação entre 2\'-desoxiadenosina 5\'-monofosfato (DMP) e lipossomos de DODAB resultou em adsorção máxima de DMP ao lipossomo catiônico com uma proporção molar DODAB : DMP de 2:1 e mobilidade eletroforética (ME) mínima mas positiva dos lipossomos. Em força iônica de 5 mM, a adsorção máxima de DMP sobre os lipossomos aproxima-se de zero, mostrando que a formação do complexo DODAB/DMP é essencialmente dirigida pela atração eletrostática. A adição de nucleotídeo na faixa de milimolar (0,4 - 1,5 mM) induz a um aumento de turbidez da dispersão de lipossomos (DODAB 0,08 mM) em função do tempo. Ocorrem velocidades de floculação muito maiores que as obtidas por adição de NaCl (40 120 mM). O nucleotídeo comporta-se como um ânion hidrofóbico com uma afinidade pela membrana muito maior que a exibida por um ânion simples como o cloreto. DMP induz ruptura dos lipossomos contendo [14C]sacarose, sugerindo que a interação DMP/bicamada não é superficial. Embora a interação preserve a carga positiva do liposomo, a integridade lipossomal não é preservada. Na adsorção máxima, a inserção de DMP na bicamada é a explicação mais razoável para manutenção da carga positiva da vesícula, proporção molar DODAB : DMP de 2:1 e extravasamento de conteúdo interno lipossomal. A interação DODAB/DNA também é dirigida por atração eletrostática entre o DNA e a bicamada catiônica. Marcadores incorporados ao DNA ou a sítios da bicamada são deslocados para a água devido à interação. Sob condições de excesso de DODAB, na situação de adsorção máxima de DODAB sobre DNA, existem cerca de 70 moléculas de DODAB por nucleotídeo de DNA e esta proporção não depende do tipo de DNA. A interação DODAB/DNA leva à formação de glóbulos visíveis por microscopia óptica de campo escuro e ocorrência de uma dependência linear entre turbidez e 1/λ2, onde λ é o comprimento de onda da luz incidente. Na condição de adsorção máxima de DODAB, a formação de complexos globulares DODAB/DNA causa perda de hipocromismo da dupla fita de DNA conforme detectado por efeitos de temperatura sobre absorbância em 260 nm de misturas DNA/DODAB. Em suma, a interação de DODAB/DNA não é superficial: o lipossomo perde sua integridade e o DNA perde sua estrutura em dupla hélice, tornando-se fita simples. A atração hidrofóbica entre bases nitrogenadas de DNA e cadeias hidrocarbônicas dos lipídios catiônicos tem papel importante na determinação da estrutura do complexo. / In this work, interactions between DNA and cationic liposomes made up of dioctadecyldimethylammonium bromide (DODAB) are evaluated from a physicochemical point of view. The following models were used: 1) 2\'-deoxyadenosine 5\'-monophosphate (DMP), a model of monomeric unity of polymer, the DNA; 2) DNAs of T2, T4, T5, T7 and λ bacteriophages; 3) small vesicles of DODAB prepared by sonication; 4) large vesicles of DODAB prepared by heating (56 ºC/30 minutes) or by chloroform vaporization. The interaction between 2\'deoxyadenosine 5\'-monophosphate (DMP) and cationic liposomes made up of dioctadecyldimethylammonium bromide (DODAB) in water is described. At maximal adsorption, the molar ratio DODAB/DMP is 2:1 and electrophoretic mobility for the liposomes attains a minimum at a positive value. At 5 mM ionic strength, maximal DMP adsorption on the liposome becomes close to zero, demonstrating that the electrostatic attraction essentially drives the DODAB/DMP complexation. Over the millimolar range of DMP concentrations (0.4-1.5 mM), upon nucleotide addition, turbidity of the liposome dispersion (0.08 mM DODAB) steeply increases as a function of time in contrast with the much smaller flocculation rates upon NaCl addition over a much higher range of NaCl concentrations (40-120 mM). The nucleotide behaves as a hydrophobic anion with an affinity for the membrane that is much higher than that exhibited by a simple anion as chloride. DMP-induced rupture of liposomes containing [14C]sacarose was evaluated from dialysis of DMP/liposomes mixtures. In water, DMP-induced leakage of radioactive liposomal contents suggests that the DMP/bilayer interaction is not superficial. Although the interaction preserves the positive liposome charge, it doesn\'t preserve its integrity. At maximal adsorption, DMP insertion in the cationic bilayer is the most reasonable explanation for the remaining positive charge on the vesicle, the 2:1 DODAB : DMP molar ratio, and leakage of internal contents from the liposome. The DODAB/DNA interaction is also driven by the electrostatic attraction between DNA and bilayer. Probes located on DNA or in the bilayer are displaced from their DNA or bilayer sites. Under conditions of DODAB excess, at maximal DODAB adsorption on DNA, there are ca. 70 DODAB molecules adsorbed per nucleotide on DNA, a molar proportion (MP) that does not depend on DNA type. The DODAB/DNA interaction led to formation of globules as visualized from dark-field optical microscopy and to occurrence of a linear dependence between turbidity for the mixture and 1/λ2, where λ is the wavelength of incident light. At maximal DODAB adsorption, the formation of DODAB/DNA globular complexes causes loss of doublestranded DNA hypochromism as detected from temperature effects on DNA absorbance at 260 nm in the presence or absence of DODAB. In summary, liposome loses its integrity and DNA loses its double helix becoming single-stranded. The hydrophobic attraction between nitrogenous bases on DNA and hydrocarbon chains on liposome bilayers plays an important role in determining structure of the complex.

Bacterial resistance to non-antibiotic agents is being increasingly studied. Plasmid-mediated resistance to cationic agents, which are important biocides, has been described in antibiotic-resistant Staphylococcus aureus. Multi-resistant Staphylococcus aureus (MRSA) are often found to express resistance to a range of cationic biocides including quaternary ammonium compounds (QACs), biguanides, diamidino compounds, cationic dyes and nuclear stains. Three resistance determinants, qacA, qacB and smr genes, have been identified that confer resistance to cationic biocides in staphylococci. These genes encode multi-drug efflux pumps that remove the cationic biocides from the cytoplasm using a membrane bound pumping mechanism dependent on the cell's proton-motive force (PMF). This prevents the build up of lethal concentrations of cationic compounds within the cytoplasm avoiding cell death.This research project has focused on the S. aureus strain WBG4364, a transcipient strain carrying the cationic biocide resistant plasmid pWBG1773. The plasmid encodes resistance to several QACs, including benzalkonium chloride and CTAB, and cationic dyes rhodamine 6G, crystal violet and safranin O but not to the dye ethidium bromide and therefore differing from other cationic biocide resistant plasmids previously identified in staphylococci (Emslie et al. 1986). This unique phenotype was further classified in this study alongside those strains carrying the qac gene families, qacA/B and smr.Plasmid pWBG1773 was cloned, sequenced and analysed to reveal a unique plasmid of 2,916 bp in length. Plasmid pWBG1773 was placed with the pC194 family of rolling-circle replicating plasmids. This family appear to be largely composed of interchangeable cassette structures.The plasmid was found to carry three ORFs, designated ORF1, ORF2 and ORF3. ORF1 was homologous to rep genes of small staphylococcal ++ / plasmids belonging to the pC194 rolling-circle replication family and has been redefined as repWBG1773. ORF2 was found to have no similarity to any proteins of known function in the GenBank database whereas ORF3 was found to have homology to the marR gene, a regulator of the multiple antibiotic resistance (mar) operon of Gram-negative organisms. MIC analysis of these ORFs found both ORF2 and ORF3 were essential for expression of resistance to cationic biocides. The exact ORF2 sequence required for resistance to be expressed was reduced to only 141 nt in size. This translated to a 47 aa sequence that contained a hydrophobic C-terminus indicating ORF2 to be a membrane-bound protein. The aa sequence of ORF3 contained a helix-turn-helix motif characteristic of the DNA binding domains of MarR-like proteins. Further analysis of pWBG1773 identified a putative 'marbox', a binding site for the homologous transcriptional activators of mar, within the ORF2 sequence. This indicated that ORF3 was binding to the 'marbox' sequence and activating transcription. Induction studies have not been able to ascertain any compounds capable of interacting with the ORF3 regulatory protein resulting in induction of cationic biocide resistance. Each ORF when analysed alone had no effect on the expression of cationic biocide resistance and it is thought that a efflux pump was not involved. This is further corroborated by the CCCP efflux experiments performed in an attempt to determine the mechanism of resistance. The unique ORFs of plasmid pWBG1773 appears to encode a novel cationic biocide resistance phenotype and mechanism.MRSA strains from all around the world were analysed to determine if they possessed sequences homologous to ORF2 and ORF3. Sequences sharing a high degree of homology to ORF2 and/or ORF3 were detected in several MRSA strains including strains sensitive to all cationic ++ / biocides tested. These findings suggest that the appearance of ORF2 and ORF3 sequences in MRSAs was not an isolated event and the fact that some MRSAs do not carry both ORF2 and ORF3 sequences simultaneously indicates that these genes have another role that does not involve expression of resistance to cationic biocides.Bacteria are noteworthy for their remarkable ability to adapt to changes in their environments and possess an impressive set of tools with which to adjust the blueprint of the cell to this change. The acquisition of a single system that may decrease a potential pathogenic organisms susceptibility to a wide range of cationic biocides, such as seen in pWBG1773, poses a clinical threat, one that needs to be thoroughly investigated.

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

Mit der vorliegenden Arbeit sollten mit Hilfe elektronenmikroskopischer Methoden verschiedene Liposomen-DNA-Komplexe zum Gentransfer charakterisiert sowie die Aufnahme und Verteilung in der Zellkultur untersucht werden. Dabei waren vor allem solche Präparationen von besonderem Interesse, die in unserer Arbeitsgruppe 'Drug Targeting' getestet oder entwickelt und verwendet wurden, wie Sendai-Virus Liposomen (HVJ-Liposomen), Virosomen sowie DAC-Chol und DOCSPER-Liposomen als Vertreter der kationischen Lipide.<br /> <br /> Im ersten Teil der Arbeit wurden fusogene Liposomen und Virosomen charakterisiert. Bei diesen Untersuchungen wurden folgende Ergebnisse erzielt:<br /> ·Sendai-Viren fusionieren mit Liposomen unterschiedlicher Lipidzusammensetzung. <br /> ·Die daraus resultierenden HVJ-Liposomen sind mit elektronenmikroskopischen Methoden identifizierbar.<br /> ·Die Spikes auf den HVJ-Liposomen besitzen fusogene Eigenschaften. <br /> ·HVJ-Liposomen eignen sich auf Grund der geringen Ausbeute sowie der geringen Transfektionseffizienz nicht zum in vitro Gentransfer.<br /> ·Virosomen stellen einen weiteren Typ fusogener Gentransfervesikel dar.<br /> ·Ihre Größe und fusogenen Eigenschaften sind abhängig von der externen Zugabe einer optimierten Lipidmischung.<br /> ·Im Innenraum der Virosomen kann mit Poly-L-Lysin vorkomplexierte DNA verkapselt werden.<br /> ·Die fusogenen Eigenschaften der Virosomen wurden mit Hilfe immunelektronenmikroskopischer Techniken und monoklonaler Antikörper gegen Hämagglutinin/Neuraminidase und das Fusionsprotein sowie mit polyklonalen Antiseren gezeigt.<br /> ·An Hand goldmarkierter DNA sind Virosomen nach der Transfektion in der Zelle nachweisbar.<br /> <br /> Da in unserer Arbeitsgruppe bevorzugt kationische Liposomen zum Gentransfer verwendet werden, wurde auch die Struktur der Liposomen untersucht und folgende Ergebnisse dokumentiert:<br /> ·Die Struktur und die Größe kationischer Liposomen werden hauptsächlich durch die Lipidzusammensetzung bestimmt. <br /> ·Die Bildung von Liposomen-DNA-Komplexen ist mit einer Größenzunahme der Komplexe gekoppelt.<br /> ·Die Anzahl gebundener Plasmide steigt mit der Größe der Lipoplexe. <br /> ·Gentransferaktive Lipopolyplexe (mit Protaminsulfat komplexierte DNA und DAC-Chol- Liposomen) sind kleiner als Lipoplexe. Ihre Struktur wird von der Zusammensetzung bestimmt. <br /> <br /> Eine weitere wichtige Frage betrifft den Weg der Gencarrier in der Zelle. Kenntnisse über diese Vorgänge sind vorteilhaft, um die einzelnen Schritte zu verstehen und möglichst gezielt zu verbessern.<br /> Bei der Untersuchung der Partikel im Hinblick auf zelluläre Barrieren beim Gentransfer konnten folgende Ergebnisse erzielt werden: <br /> ·Die Bindung der Partikel an die Zellmembran und Aufnahme sind abhängig von den eingesetzten Zellen und Komplexen sowie derInkubationszeit.<br /> ·Die Aufnahme erfolgt über endozytotische Mechanismen, wobei Lipopolyplexe schneller als Lipoplexe in die Zellen gelangen. Nicht alle gebundenen Komplexe werden aufgenommen.<br /> ·Die aufgenommenen Partikel befinden sich in Endosomen und werden ins Innere der Zelle transportiert. <br /> ·Freisetzung der DNA und Eintritt in den Zellkern über Kernporen konnte nicht beobachtet werden.<br /> ·DNA-haltige Vesikel in Kernnähe deuten auf einen weiteren Mechanismus hin (Vesikeltransfer zum Zellkern). / The aim of this work is the characterisation of several liposome DNA complexes for in vitro gene transfer and uptake as well as their distribution in cultured cell lines using electron microscopy. The particles used were fusogenic liposomes made from Sendai virus, virosomes and cationic liposomes. <br /> At first fusogenic liposomes and virosomes were characterised. The results obtained are summarised below:<br /> ·Sendai virus can fuse with liposomes made from different lipid composition.<br /> ·HVJ-liposomes are detectable using electron microscopy techniques. <br /> ·The spikes from HVJ-liposomes have fusogenic properties.<br /> ·HVJ-liposomes are not suitable for in vitro gene transfer due to the low amount of fusogenic liposomes leading to low transfection efficiency.<br /> ·Virosomes, reconstituted virus envelopes, are another type of fusogenic vesicles.<br /> ·Size and morphology of virosomes depends on the addition of an optimised lipid mixture. <br /> ·PLL treated DNA is entrapped into virosomes.<br /> ·Monoclonal and polyclonal antibodies and protein A gold technique can be used for the detection of viral glycoproteins on virosomes. ·Gold labelled DNA was used to show the distribution in cultured cells.<br /> <br /> In order to characterise the structure of cationic liposomes following results were obtained:<br /> ·Size and structure depends on the lipid composition.<br /> ·The formation of liposomes leads to an increase of the size.<br /> ·Larger lipoplexes contain more DNA.<br /> ·Lipopolyplexes composed of DNA complexed with protamine sulphate and DAC- Chol liposomes are smaller than lipoplexes. Their structure depends on the composition.<br /> <br /> To improve transfection ability examination of the cellular barriers is useful.<br /> With regard to the fate of lipoplexes following results were obtained.<br /> ·Binding depends on the cell line, kind of particles and incubation time.<br /> ·Uptake occurs through endocytosis. Lipopolyplexes enter the cells faster than larger lipoplexes.<br /> ·Lipoplexes are enclosed in endosomes and were carried into the centre of the cell.<br /> ·Escape of DNA from endosomes and entry into nucleus were not visible.<br /> ·Vesicles with DNA were observed near the nucleus. There is an opportunity for another pathway to the nucleus.