Abstract Many hydrophilic drugs do not easily cross biological membranes. One approach to improve intestinal absorption of hydrophilic compounds is to co-administer these drug molecules with absorption enhancers to optimize their physico-chemical properties. In this project, novel liposaccharides were used to modulate the aqueous solubility as well as increase the lipophilicity, improve the intestinal permeability and hence, oral bioavailability of hydrophilic molecules. However, the solubility of the drug/liposaccharide mixtures in aqueous system might be poor, therefore, introducing a hydrophilic component such as quaternization of amine derivatives or sodium salt of carboxylic acid is required to modulate hydrophilic/lipophilic balance and in the same time and promote surfactant and ion pairing characteristics of these derivatives. This dissertation illustrates the development of novel liposaccharide absorption enhancers to be used for oral delivery of hydrophilic drugs. This research includes chemical synthesis of new ionic liposaccharides, examination of their physico-chemical properties, and their in vitro and in vivo biological examinations. The work consists of two parts; cationic and anionic liposaccharide absorption enhancers. a) Cationic Liposaccharide Absorption Enhancers The focus of this work was on the molecular design, synthesis, and evaluation of novel cationic liposaccharide derivatives as drug delivery agents to improve the oral bioavailability of piperacillin as a model hydrophilic drug. These derivatives were designed to possess surfactant as well as ion pairing properties, and were synthesized from biocompatible and biodegradable materials such as glucose, lipoamino acids and lipophilic amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Minimum inhibitory concentrations of piperacillin/liposaccharide conjugates were studied to investigate the effect of liposaccharides on piperacillin activity. The usefulness of these derivatives as absorption enhancers for the oral delivery of piperacillin was assessed in vitro (Caco-2 cells) and in vivo (rat oral absorption). It was concluded that these derivatives did show hemolytic activity in low concentration (suitable for enhancing activity), while they increased permeability of piperacillin through Caco-2 cells. However, these promising results obtained from in vitro assay were not confirmed in vivo. viii b) Anionic Liposaccharide Absorption Enhancers In this thesis, molecular design, synthesis, and evaluation of novel anionic liposaccharide derivatives as absorption enhancers to improve the intestinal permeability of a model hydrophilic drug such as tobramycin were carried out. These derivatives were designed to have surfactant as well as ion pairing properties, and were synthesized from biocompatible materials such as sugar, lipoamino acids and amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Many absorption enhancers have (to some extent) cytotoxic activity, therefore, cytotoxic activity of the novel anionic liposaccharides were examined. The usefulness of these derivatives as absorption enhancers to increase the lipophilicity of tobramycin and hence, its oral bioavailability, was evaluated through carrying out partition coefficient examination. It was concluded that these derivatives did not show haemolytic and cytotoxic activities in low concentration (suitable for enhancing activity). In addition, the permeability of tobramycin into the organic phase (n-octanol) was increased when liposaccharide derivatives were used. The obtained results are promising and encouraging to continue the work to include Caco-2 cells assay (in vitro assay) and oral absorption in rats (in vivo assay) as a prospective work in the future.

Abstract Many hydrophilic drugs do not easily cross biological membranes. One approach to improve intestinal absorption of hydrophilic compounds is to co-administer these drug molecules with absorption enhancers to optimize their physico-chemical properties. In this project, novel liposaccharides were used to modulate the aqueous solubility as well as increase the lipophilicity, improve the intestinal permeability and hence, oral bioavailability of hydrophilic molecules. However, the solubility of the drug/liposaccharide mixtures in aqueous system might be poor, therefore, introducing a hydrophilic component such as quaternization of amine derivatives or sodium salt of carboxylic acid is required to modulate hydrophilic/lipophilic balance and in the same time and promote surfactant and ion pairing characteristics of these derivatives. This dissertation illustrates the development of novel liposaccharide absorption enhancers to be used for oral delivery of hydrophilic drugs. This research includes chemical synthesis of new ionic liposaccharides, examination of their physico-chemical properties, and their in vitro and in vivo biological examinations. The work consists of two parts; cationic and anionic liposaccharide absorption enhancers. a) Cationic Liposaccharide Absorption Enhancers The focus of this work was on the molecular design, synthesis, and evaluation of novel cationic liposaccharide derivatives as drug delivery agents to improve the oral bioavailability of piperacillin as a model hydrophilic drug. These derivatives were designed to possess surfactant as well as ion pairing properties, and were synthesized from biocompatible and biodegradable materials such as glucose, lipoamino acids and lipophilic amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Minimum inhibitory concentrations of piperacillin/liposaccharide conjugates were studied to investigate the effect of liposaccharides on piperacillin activity. The usefulness of these derivatives as absorption enhancers for the oral delivery of piperacillin was assessed in vitro (Caco-2 cells) and in vivo (rat oral absorption). It was concluded that these derivatives did show hemolytic activity in low concentration (suitable for enhancing activity), while they increased permeability of piperacillin through Caco-2 cells. However, these promising results obtained from in vitro assay were not confirmed in vivo. viii b) Anionic Liposaccharide Absorption Enhancers In this thesis, molecular design, synthesis, and evaluation of novel anionic liposaccharide derivatives as absorption enhancers to improve the intestinal permeability of a model hydrophilic drug such as tobramycin were carried out. These derivatives were designed to have surfactant as well as ion pairing properties, and were synthesized from biocompatible materials such as sugar, lipoamino acids and amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Many absorption enhancers have (to some extent) cytotoxic activity, therefore, cytotoxic activity of the novel anionic liposaccharides were examined. The usefulness of these derivatives as absorption enhancers to increase the lipophilicity of tobramycin and hence, its oral bioavailability, was evaluated through carrying out partition coefficient examination. It was concluded that these derivatives did not show haemolytic and cytotoxic activities in low concentration (suitable for enhancing activity). In addition, the permeability of tobramycin into the organic phase (n-octanol) was increased when liposaccharide derivatives were used. The obtained results are promising and encouraging to continue the work to include Caco-2 cells assay (in vitro assay) and oral absorption in rats (in vivo assay) as a prospective work in the future.

Abstract Many hydrophilic drugs do not easily cross biological membranes. One approach to improve intestinal absorption of hydrophilic compounds is to co-administer these drug molecules with absorption enhancers to optimize their physico-chemical properties. In this project, novel liposaccharides were used to modulate the aqueous solubility as well as increase the lipophilicity, improve the intestinal permeability and hence, oral bioavailability of hydrophilic molecules. However, the solubility of the drug/liposaccharide mixtures in aqueous system might be poor, therefore, introducing a hydrophilic component such as quaternization of amine derivatives or sodium salt of carboxylic acid is required to modulate hydrophilic/lipophilic balance and in the same time and promote surfactant and ion pairing characteristics of these derivatives. This dissertation illustrates the development of novel liposaccharide absorption enhancers to be used for oral delivery of hydrophilic drugs. This research includes chemical synthesis of new ionic liposaccharides, examination of their physico-chemical properties, and their in vitro and in vivo biological examinations. The work consists of two parts; cationic and anionic liposaccharide absorption enhancers. a) Cationic Liposaccharide Absorption Enhancers The focus of this work was on the molecular design, synthesis, and evaluation of novel cationic liposaccharide derivatives as drug delivery agents to improve the oral bioavailability of piperacillin as a model hydrophilic drug. These derivatives were designed to possess surfactant as well as ion pairing properties, and were synthesized from biocompatible and biodegradable materials such as glucose, lipoamino acids and lipophilic amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Minimum inhibitory concentrations of piperacillin/liposaccharide conjugates were studied to investigate the effect of liposaccharides on piperacillin activity. The usefulness of these derivatives as absorption enhancers for the oral delivery of piperacillin was assessed in vitro (Caco-2 cells) and in vivo (rat oral absorption). It was concluded that these derivatives did show hemolytic activity in low concentration (suitable for enhancing activity), while they increased permeability of piperacillin through Caco-2 cells. However, these promising results obtained from in vitro assay were not confirmed in vivo. viii b) Anionic Liposaccharide Absorption Enhancers In this thesis, molecular design, synthesis, and evaluation of novel anionic liposaccharide derivatives as absorption enhancers to improve the intestinal permeability of a model hydrophilic drug such as tobramycin were carried out. These derivatives were designed to have surfactant as well as ion pairing properties, and were synthesized from biocompatible materials such as sugar, lipoamino acids and amino acids. Thermodynamic profiles of these derivatives as well as haemolytic activity were examined. Many absorption enhancers have (to some extent) cytotoxic activity, therefore, cytotoxic activity of the novel anionic liposaccharides were examined. The usefulness of these derivatives as absorption enhancers to increase the lipophilicity of tobramycin and hence, its oral bioavailability, was evaluated through carrying out partition coefficient examination. It was concluded that these derivatives did not show haemolytic and cytotoxic activities in low concentration (suitable for enhancing activity). In addition, the permeability of tobramycin into the organic phase (n-octanol) was increased when liposaccharide derivatives were used. The obtained results are promising and encouraging to continue the work to include Caco-2 cells assay (in vitro assay) and oral absorption in rats (in vivo assay) as a prospective work in the future.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

Orientador: Robson Frederico Cunha / Coorientadora: Cristiane Duque / Coorientadora: Sandra Maria Herondina Ávila de Aguiar / Banca: Ana Cláudia Okamoto / Banca: Marcelle Danellon / Banca: Juliana Campos Junqueira / Banca: Cássia Cilene Dezan Garbelini / Resumo: O uso de agentes antimicrobianos naturais que reduzam a adesão e proliferação de S. mutans no biofilme poderia ser uma estratégia interessante para o controle da cárie dentária. No entanto, a estabilidade química e física de alguns desses agentes, como os peptídeos catiônicos antimicrobianos e fragmentos de peptídeos, pode ser comprometida por fatores externos, como temperatura e pH, reduzindo sua ação antimicrobiana. Com isso, os objetivos deste estudo foram desenvolver e caracterizar sistemas de liberação de fármaco nanoestruturados bioadesivos para a incorporação dos fragmentos peptídicos D1-23 e P1025 e avaliar seu efeito citotóxico e atividade contra biofilme de S. mutans. A primeira formulação (F1), composta de ácido oleico, polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol (PPCA), Carbopol® 974P e Carbopol® 971P, foi analisada por microscopia de luz polarizada (MLP), reologia e bioadesão in vitro. A concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) de D1-23 foram determinadas contra S. mutans para posterior avaliação da atividade sobre biofilme formado após 4h e 24h de tratamento. A segunda formulação (F2) foi selecionada a partir de três diferentes concentrações de ácido oleico, PPCA e Carbopol® 974P. Cada formulação foi analisada por MLP, espalhamento de raios x a baixo ângulo (SAXS), reologia e bioadesão. CIM e CBM de P1025 sobre S. mutans e seu efeito quando incorporado ou não em F2 sobre biofilme de S. mutans em formação foram anal... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of natural antimicrobial agents for reducing the adhesion and proliferation of S. mutans in the biofilm could be an interesting strategy for the control of dental caries. However, the chemical and physical stability of some natural antimicrobials, such as cationic antimicrobial peptides and peptide fragments, can be compromised by external factors such as temperature and pH, reducing their antimicrobial action. Thus, the objectives of this study were to develop and characterize nanostructured bioadhesive drug delivery systems for the incorporation of D1-23 and P1025 peptide fragments and to evaluate their citotoxicy and activity against S. mutans biofilm. The first formulation (F1) was composed of oleic acid, polyoxypropylene- (5) -polyoxyethylene- (20) -cetyl alcohol (PPCA), Carbopol® 974P and Carbopol® 971P and analyzed by polarized light microscopy (PLM), rheology and in vitro bioadhesion. Minimum inhibitory concentration (MIC) and minimal bacterial concentration (MBC) of D1-23 were determined against S. mutans for further evaluation of activity against S. mutans biofilm after 4h and 24h of treatment. The second formulation was selected from three different concentrations of oleic acid, PPCA and Carbopol® 974P. Each formulation was analyzed by PLM, small-angle x-ray scattering (SAXS), rheology and bioadhesion. MIC and MBC of P1025 were determined against S. mutans. Thus, P1025 was incorporated in the best formulation (F2). The effect of P1025 incorporated or not in... (Complete abstract click electronic access below) / Doutor

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Orientadora: Cristiane Duque / Banca: Rodrigo Alex Arthur / Banca: Rogério de Castilho Jacinto / Resumo: Os objetivos do estudo foram avaliar os efeitos citotóxico e antimicrobiano de análogos de peptídeos catiônicos e sua influência na expressão de marcadores fenotípicos e genotípicos de mineralização dentinária. Fibroblastos da linhagem L929 foram expostas a diluições seriadas dos peptídeos LL-37 e análogos hBD-3-1CV e KR-12-a5 e a viabilidade das células foi avaliada por ensaios de metil tiazol tetrazólio (MTT). Concentração inibitória mínima (CIM) e concentração letal mínima (CLM) para os peptídeos e controles (clorexidina - CHX) foram determinadas contra Streptococcus mutans, Actinomyces israelii, Enterococcus faecalis, Candida albicans, Fusobacterium nucleatum e Porphyromonas gingivalis, pelo método de microdiluição, após 4 e 24 horas. Biofilmes de E. faecalis e F. nucleatum foram formados sobre blocos de dentina radicular bovina e expostos a 5X e 10X MLC do peptídeo com melhor atividade antimicrobiana e CHX e analisadas por contagem de unidades formadoras de colônias/ml (UFC/ml) e por Microscopia de Varredura Confocal à Laser (CLSM). Células semelhantes à odontoblastos da linhagem MDPC-23 foram expostas a diluições seriadas de LL-37, hBD-3-1CV, KR-12- A5 por 24 horas, seguidas de trocas de meio osteogênico por 7 dias e avaliada a viabilidade celular, produção de proteína total (TP), atividade da fosfatase alcalina (ALP) e deposição de nódulos mineralizados. A expressão de genes de marcadores de mineralização (sialofosfoproteína dentinária - DSPP e fosfoproteína de matriz dentinária - DMP-1) foi realizada por PCR quantitativo, após 24 h de exposição aos peptídeos e de incubação durante 14 dias em meio osteogênico. LL-37/hBD-3-1CV e KR-12-a5 afetaram o metabolismo dos fibroblastos em concentrações acima de 500 e 250 μg/ml, respectivamente. KR-12-a5 teve os melhores valores... / Abstract: The objectives of the study were to evaluate the cytotoxic and antimicrobial effects of analogues of cationic peptides and their influence in the expression of phenotypic and genotypic markers of dentin mineralization. L929 fibroblast cells were exposed to serial dilutions of peptides LL-37, hBD-3-1CV and KR-12-a5 and cell metabolism was evaluated by methyl thiazol tetrazolium (MTT) assays. Minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of peptides and controls (chlorhexidine - CHX) were determined for Streptococcus mutans, Actinomyces israelii, Enterococcus faecalis, Candida albicans, Fusobacterium nucleatum and Porphyromonas gingivalis, by microdilution method, after 4 and 24h. E. faecalis and F. nucleatum biofilms were formed in blocks of bovine root dentin and exposed to 5X and 10X MLC of the peptide with the best antimicrobial activity and CHX and analyzed by colonies forming units/ml (CFU/ml) counts and by Confocal Laser Scanning Microscopy (CLSM). MDPC-23 odontoblast-like cells were exposed to serial dilutions of peptides LL-37, hBD-3-1CV and KR-12-a5 for 24 hours, followed by changes of osteogenic medium for 7 days and evaluated cell viability, total protein (TP) production, alkaline phosphatase (ALP) activity and mineralized nodule deposition. The gene expression of mineralization markers (Dentin Sialophosphoprotein - DSPP and Dentin matrix phosphoprotein 1 - DMP-1) was performed by quantitative PCR, after 24h of peptide exposure and incubation for 14 days in osteogenic medium. LL-37 and hBD-3-1CV affected cell metabolism at concentrations above 500μg/ml and KR-12-a5 above 250μg/ml. KR-12-a5 had the best MIC/MLC values against all microorganisms and both times of exposure. E. faecalis and C. albicans growth was affected only for KR-12-a5 and CHX. hBD-3-1CV and... / Mestre

Neste trabalho, são utilizados diversos métodos de química teórica para estudar as propriedades eletrônicas e o espectro de absorção de seis corantes catiônicos: laranja de acridina (LA), proflavina (PF), safranina (SF), vermelho neutro (VN), azul de metileno (AM) e tionina (TN). Inicialmente é realizado um estudo para verificar a influência do solvente na geometria e no espectro desses corantes utilizando diversos métodos de química quântica, o método de solvatação contínuo IEFPCM e a simulação de Monte Carlo (MC). A seguir são estudados a diprotonação, a dimerização e alguns modelos para a argila do tipo montmorilonita e para a interação desta com os corantes, fenômenos esses que explicam a metacromasia observada experimentalmente. / This work presents a theoretical study on the electronic properties and the absorption spectra of six cationic dyes: acridine orange, proflavine, safranine, neutral red, methylene blue and thionine. First of all, we have carried out calculations to verify the solvent effects on geometries and spectra employing methods of Quantum Chemistry and including solvent effects with the polarizable continnum model and Monte Carlo (MC) simulation. Also, we have studied diprotonation, dimerization and some models of the montmorillonite clay and its interaction with the cationic dyes under study here, phenomena that explain the experimental methacromatic behavior of these dyes.

The biggest hurdle in success of gene and drug therapy is designing and preparation of suitable bio-nanomaterials to carry the desired nucleic acid and drug to the targeted site. The work described in the present thesis encompasses two different approaches for the delivery of bioactive oligo/polynucleotides and drugs in vitro and in vivo using either cationic lipids or their nanocomposites with different carbon nanomaterials. The idea of using carriers for oligo/polynucleotides and drugs came into existence because of numerous physiological barriers in pathway of delivery of naked oligo/polynucleotides or drugs which reduces the overall activity of these bioactives in biological systems. These barriers trigger scientific research toward the preparation of appropriate biomaterials which can overcome the physiological barriers and improve the activity of bioactive oligo/polynucleotides and drugs in cellular systems. Toward this end, the design and synthesis of different cationic lipids and carbon nanomaterials were undertaken as described in seven chapters of the thesis. A series of novel cationic lipids with structural variability was prepared and used for gene delivery in vitro. They were further tuned chemically to sustain delivery efficiency in high serum percentage during in vitro transfection. These serum compatible lipids were used to perform transfection of reporter gene plasmid and found to be more efficient compared to the some well known commercial products for the same purpose. Another series of novel lipids were synthesized for the targeted gene delivery in vitro. These tryptophan based cholesteryl lipids were used to prepare mixed liposomes. These mixed liposomes were highly efficient in targeting sigma receptor rich HEK293T over sigma receptor negative HeLa cells. Mixed liposomes were also prepared for selective targeting of αvβ3 and αvβ5 integrins in gene transfection protocol using a palmitoyl-RAFT-RGD4 template. A mixed liposomal formulation was developed to carry out anti-sense siRNA mediated knockdown of Smad-2 protein with better efficiency compared to some of the best known commercial products for the same purpose. These mixed liposomes were also highly efficient for regression via induction of p53 mediated apoptosis in xenograft tumors developed in nude mice. Carbon nanomaterials have been extensively explored as nanoscale gene/drug carriers for potential applications. But the challenge is to solubilize these highly hydrophobic materials in aqueous medium for use in biological systems. Although there are reports for covalent modifications of such nanomaterials but it could be done only with the loss of some beneficial features of these materials. Herein a non-covalent technique has been efficiently used to suspend single walled carbon nanotubes in water using biocompatible cationic lipids. These nanosuspensions were used to complex plasmid DNA and transfect them in vitro. They proved to be highly serum compatible DNA carriers which did not drop the efficiency even in very high percentage of serum. Similarly exfoliated graphene was modified with cationic lipid and serum components to improve IC50 of Tamoxifen citrate and Methotrexate to a considerable extent in vitro. The improved Methotrexate formulations were highly efficient for regression in size of xenograft tumors developed in nude mice. Thus, the present thesis entails generation of cationic lipids and carbon nanomaterials based nanocomposites which were not only highly biocompatible themselves but their efficiency was found many fold better compare to some of the best commercial delivery agents. These were useful for the delivery of various bioactive oligo/polynucleotides and drugs in vitro and in vivo.

This thesis presents an investigation of cationic microgels based on poly(N-vinylformamide-co-glycidyl methacrylate) (PNVF-GMA) and poly(N-vinylformamide-co-2-(N-vinylformamido) ethyl ether) (PNVF-NVEE). They arestudied in the context of future heteroaggregated doubly crosslinked (DX) microgelsfor damaged heart tissue repair. The microgel particles were synthesised fromPNVF-GMA, which is also a water swellable microgel. The PNVF-GMA particleshad a core-shell structure in which PNVF provides the core and PGMA creates thecross-linked shell. The morphology of particles is that of a “cane-ball” like shape. There are interconnected ridges, and this unusual morphology can be controlled bythe weight fraction of GMA used during preparation. The hydrolysed PNVF-GMA(H-PNVF-GMA) particles were both positively and negatively charged. Moreover,charge patch aggregation occurred at low ionic strength. However, these microgelswere colloidally unstable after water rinsing due to shell fragmentation. PNVF microgel particles containing (N-Vinylformamido) ethyl ether (NVEE) as acrosslinking agent were also studied to avoid the fragmentation of the particles. Thismicrogel was hydrolysed in alkali conditions to provide poly(vinylamine-co-bis(ethyl vinylamine) ether) (PVAM-BEVAME), which contains primary aminegroups. It is proposed from the data presented that the content of hydrolysis was veryhigh and the particles were stable after hydrolysis owing to the stability of etherlinkage in NVEE. These microgels were able to swell upon decreasing pH. ThePVAM-BEVAME microgel with 9 mol% of BEVAME was then used to formdoubly crosslinked (DX) microgel. To form the inter-particles crosslinking, the vinylgroups were included by functionalisation using glycidyl methacrylate (GMA)monomer. The vinyl groups of neighbouring particles were linked together via freeradical reaction. The DX microgel formed under physiological temperature andshowed extensive porosity. These DX microgels had good mechanical propertiesconfirmed by high storage modulus (G’). Moreover, the precursor gels wereinjectable which is favourable for future biomaterial applications. The study providesa new family of cationic microgel that may be suitable for a future heteroaggregatedDX microgel for heart tissue repair.