O uso de genes em terapias é um conceito originado em 1970 em consequência do crescimento exponencial de novas tecnologias para liberação de DNA, também pela capacidade de expressão de genes exógenos em células de mamíferos. Propomos, então, a síntese de polímeros catiônicos, em dois grupos, por meio da aminólise da poli(succinimida) (PSI): grupo 1An, polímeros catiônicos com arcabouço poli (ácido aspártico) com cadeias laterais contendo aminas protonáveis; grupo 2An, polímeros catiônicos anfifílicos, contenho o poli (ácido aspártico) como arcabouço, com aminas protonáveis e cadeias dodecilamina. Estudos de SEC mostraram que derivados dodecilados 2An tiveram tamanho menor que os polímeros do grupo 1An, não dodecilados. A capacidade tamponante para todos os polímeros sintetizados foi maior que o bPEI 25, e o grupo 2An apresentou as maiores capacidades tamponantes. Derivados 2An com as aminas A1 a A4 apresentaram menor CMC do que o grupo 1An. Citotoxicidade dos policátions foi dependente das suas concentrações e, entre todos os polímeros, aqueles com aminas A5 e A6 não foram citotóxicos. A presença da cadeia dodecilamina na PSI não diminuiu a viabilidade celular até 250 ?L-1, sugerindo que a porção hidrofóbica não é citotóxica na faixa de concentrações testada. A complexação do pEGFP-N3 com os derivados de PSI foi realizada, bem como a transfecção dos poliplexos em células HeLa. A expressão de GFP dos complexos obtidos com bPEI 25 foi quantificada e comparada com os poliplexos preparados com os derivados da PSI. Ensaios de transfecção mostraram que os derivados dodecilados da PSI apresentaram expressão negligenciável de GFP em células HeLa, sugerindo uma ligação forte entre plasmídeo e os derivados sintetizados e não-liberação do material genético nas células, ou dano celular causado pela cadeia hidrofóbica nas células. Os maiores valores de GFP quantificados foram encontrados nos poliplexos contendo polímeros não-dodecilados e com as aminas A3 e A4, nas razoes N:P 5 a 20 para A3 e N:P 5 para A4. Ambas as estruturas A3 e A4 fazem parte do core do bPEI 25. / Genes as drugs for human therapy is a concept originally conceived around 1970, a consequence of the exponential growth in knowledge of human gene function, the more effective technologies for DNA delivery, and the ability to transfer and express exogenous genes in mammalian cells. Here we propose synthesizing two small library groups of cationic polymers via aminolysis of poly(succinimide) (PSI) backbone: group 1An, polycationic polymers with a degradable amide of poly(aspartic) acid backbone, protonable oligoamine side chains into the main polymer structure, and group 2An, amphiphilic cationic polymers with a degradable amide of poly(aspartic) acid backbone, protonable oligoamine side chains into the main polymer structure and dodecyl side chain moieties. SEC showed that dodecylated derivatives 2An had lower size than 1An group, non-dodecylated polyelectrolytes. Buffering capacity of all synthesized polymers was higher than the standard bPEI 25, and the dodecylated 2An group had the highest buffering capacities values. 2An derivatives with amines A1 to A4 showed lower CMC than their non-dodecylated pairs. Cytotoxicity of all polycations was dependent on the concentrations, and among all polymers, those with amines A5 and A6 had lower cytotoxicity than bPEI 25. Moreover, the presence of the hydrophobic dodecyl side chain in the PSI backbone did not decrease the cell viability until 250 ?g mL-1 polymer concentration, thus suggesting the hydrophobic moiety is not cytotoxic in this range. Complexation of pEGFP-N3 plasmid with PSI derivatives grafted with amines A1 to A4 was performed, as well as the transfection of polyplexes into HeLa cells. GFP expression of bPEI25 polyplexes in different complex volumes was quantified and compared with PSI derivatives/pEGFP-N3 polyplexes. Transfection assays showed that dodecylated PSI derivatives had negligible or no GFP expression in HeLa cells, thus suggesting a strong interaction between polycations and pDNA or a cellular damage caused by the hydrophobic moiety, although cytotoxicity assay of polyplexes showed low cytotoxicity of polyplexes. The highest GFP expression values were found for polycations 1A3 and 1A4, both without the dodecylamine side chain, in the N:P ratios 5 to 20 for 1A3, and N:P ratio 5 for 1A4. Both amines A3 and A4 used for the PSI grafting are core structures of bPEI 25.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

The 21st century has been called the age of genomic medicine, yet gene therapy for medicinal use remains a theory. One reason that there are no safe and effective treatments for human disease is the lack of a vehicle capable of delivering genetic material to a specific target. In nature we observe gene pathology by viral vectors, which deliver their own genetic material to specific host cells efficient at spreading the viral blueprint throughout the organism. The aim of my research into gene therapy has been to develop a synthetic vector with the delivery capability of viral vectors found in nature. This includes the ability to protect genetic cargo from modification and degradation in vivo, target to a desired cell type within a specific tissue, facilitating absorption into the cell, and delivery to the nucleus, where expression of genetic material occurs. The goal of this thesis project was to synthesize a novel vector which would selectively target the dendritic cell SIGN receptor, mirroring the method of pathogens such as HIV, which target this receptor and subsequently the immune system, resulting in chronic infection. The vector we designed contains two major components, the high mannose N-glycan Man9GlcNAc2Asn, and a peptide composed of nine amino acids: four lysine spacing residues, four lysines derivatized with acridine on the epsilon amine of their side chains, and a cysteine for conjugation to the glycan. This compound, the Man9-AcrLys Glycopeptide, was engineered to intercalate into plasmid DNA via the acridine functional groups and to bind the DC-SIGN receptor through the glycan's mannose residues. The vehicle was tested in vitro in CHO cells bearing a recombinant DC-SIGN receptor in the context of luciferase reporter gene delivery. We found that under equal treatment conditions, DC-SIGN (+) CHO cells expressed more luciferase and were 100-fold more luminescent than control DC-SIGN (-) CHO cells. My delivery method was further analyzed in a cell-sorting FACS experiment. I covalently labeled pGL3 reporter plasmid with a fluorophore, and transfected the CHO cells under typical transfection conditions. The experimental results confirmed preferential DC-SIGN mediated gene delivery.

Gene therapy is an approach for the treatment of acquired cancers, infectious disease, degenerative disease, and inherited genetic indications. Developments in the fields of immunotherapies and CRISPR/Cas9 genome editing are revitalizing the efforts to move gene therapy to the forefront of modern medicine. However, slow progress and poor clinical outcomes have plagued the field due to regulatory and safety concerns associated with the flagship delivery vector, the recombinant virus. Immunogenicity and poor transduction in certain cell types severely limits the utility of viruses as a delivery agent of nucleic acids. As a result, significant efforts are being made to develop non-viral delivery systems that perform mechanistically similarly to viral delivery but lack immunogenic factors. Though safer, existing agents lack the efficacy inherent in the natural design of viral vectors. Clinical relevance of non-viral vectors will therefore depend on the ability to engineer optimized systems for cellular delivery in physiological environments. Progress in non-viral vector design for gene delivery requires a deep understanding of the various barriers associated with nucleic acid delivery, including cell surface interaction, internalization, endosomal escape, cytosolic transport, nuclear localization, unpackaging, etc. Further, it requires a knowledge of vector design properties (surface chemistry, charge, size, shape, etc.) and how these physical parameters affect interactions with the cellular environment. Of these interactions, charge is shown to govern how particles are internalized and subsequently processed, thereby affecting the intracellular fate and efficacy of delivery. Charge also affects the in-serum stability where negative zeta potential improves stability and circulation time. Therefore, it is important to understand the effects of polyplex charge and other parameters on the internalization and intracellular fate of polyplexes for gene therapy. In chapter 2, studies are performed to delineate the effects of polyplex charge on the cellular internalization and intracellular processing of polymer-mediated gene delivery. Charge is shown to affect the endocytic pathway involved in internalization, and the caveolin-dependent and macropinocytosis pathways lead to higher gene delivery efficacy, likely due to avoidance of acidified compartments such as late endosomes and lysosomes. In chapters 3-4, novel nanoparticles carrying DNA, RNA, and antioxidants are assessed for therapeutic effect with an emphasis on studying the internalization mechanisms and resulting effect on efficacy. Novel RNA delivery agents are shown to benefit from EGFR-targeting aptamer and nanoceria/PEI hybrids are demonstrated to provide simultaneous antioxidant and gene therapy. Finally, chapter 5 demonstrates the use of silencing RNA and CRISPR/Cas9 genome editing to study the prevalence of gene targets in vivo. The overall goal of this work is to contribute to the design and application of novel nanoparticles for gene delivery and offer insight into the engineering of novel polyplexes. It remains clear that route of internalization is key to successful gene delivery, and designing polyplexes to enter through non-acidified endocytic pathways is highly beneficial to transgene expression. This can be achieved through incorporation of surface chemistries that trigger internalization through targeted pathways and is the source of further work in the lab.

Non-viral gene delivery is plagued by low transfection levels compared to viral delivery. The nuclear envelope presents a significant obstacle for non-viral vectors. A peptide-based nuclear localizing sequence has been incorporated into non-viral vectors to traverse the nuclear envelope. Here, we selected a photo-chemical method for covalently labeling the peptide onto plasmid DNA. The hypothesis of this work was to incorporate a nuclear localizing sequence into a non-viral delivery vector, demonstrate increased nuclear uptake and show a subsequent increase in transgene expression both in vitro and in vivo. We focused on pursuing in vitro and in vivo methods by which to test non-viral vectors for increases in gene expression based on the nuclear localizing sequence. Hydrodynamic dosing and intramuscular dosing (followed by electroporation) are two efficient delivery routes for dosing DNA in vivo. Through preliminary experiments, we became confident that whole animal bioluminescent imaging was a reliable and quantitative method by which to detect luciferase expression by either delivery route. Moving forward, both hydrodynamic and intramuscular dosing would be used to test formulations for nuclear localizing ability in vivo. Nuclear localizing peptides containing a photo-activatable functionality were synthesized and characterized. We quantitatively explored the photo-labeling capabilities on plasmid DNA via a radioactive peptide. In vitro, tissue culture-based experiments were carried out to show increased nuclear uptake by confocal microscopy as well as increased transgene expression. Throughout the literature, achieving an increase in expression by incorporating a nuclear localizing sequence into a non-viral vector has been elusive. The complexity of achieving this goal is increased when considering an in vivo system for improving gene transfer efficiency. Several strategies have been explored to demonstrate an increase in reporter gene expression from this type of non-viral vector, and the methods developed herein can be applied to other nuclear localizing vectors.

Gene therapy is the process of delivering genetic material, such as DNA (encoding for an important protein) into a patient’s cells in order to treat a particular disease such as a genetic disorder or heart disease. This process of DNA delivery into cells is known as “transfection” and it is important that the efficiency of transfection be optimized such that a patient can obtain maximum therapeutic benefit from such a treatment. DNA is susceptible to being destroyed by harsh physiological environments prior to reaching its target. This problem can be diminished with the use of vectors that not only protect against harsh conditions but also encourage entry into cells. By mixing 1) DNA with 2) positively charged polymers, “polyplexes” form which protect DNA from degradation and increase transfection efficiency. The development of effective polyplex formulations requires optimization. In the work presented here, it was discovered that when polyplexes contained specific sequences within the DNA called “CpG”, this lowered transfection efficiencies and increased inflammatory responses compared to DNA without CpG, as measured using a mouse lungs model. Thus, DNA composition played an important role in influencing DNA transfection efficiency of polyplexes. Another aspect to take into account is the degree of positive charge of the polymer. We tested a new polymer called poly(galactaramidoamine) or PGAA. We found that this PGAA can form polyplexes with DNA and could be used in gene therapy. At the present time, mechanisms by which the polyplexes get inside and transfect the cells are still unclear. We also introduced a new system called high-content screening to the gene delivery field. This system offers automated measurements of transfection efficiency and cytotoxicity and could be used to reveal the polyplexes trafficking inside cells.

<p>In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA)<i> in vitro</i> and after lung administration <i>in vivo</i>. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of<i> in vivo</i> gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing <i>in vitro</i>. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution<i> in vivo</i> compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.</p>

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

In this thesis, chitosan, a biocompatible polysaccharide that has been approved as a food additive was selected as a platform for the development of safe, efficient non-viral gene delivery systems to mammalian cells. Previously, chitosan-based gene formulations had been generally associated with high molecular weight chitosans, which were poorly characterised in terms of molecular weight distribution and degree of acetylation. Therefore, in order to improve the properties of chitosan-based gene formulations, the research associated with this thesis focused on establishing the structure-property relationships of well-defined, low molecular weight chitosans (chitosan oligomers) as delivery systems for nucleic acids (pDNA and siRNA) in vitro and after lung administration in vivo. pDNA dissociated more easily from chitosan oligomers than from conventional high molecular weight chitosans, resulting in a faster onset and higher levels of in vivo gene expression, comparable to those mediated by polyethyleneimine (PEI), one of the most efficient non-viral delivery systems. Coupling of a trisaccharide branch to the chitosan oligomers so as to target extracellular lectins resulted in a significant improvement in transfection efficiency because of enhanced cellular uptake and colloidal stability. In contrast to pDNA, longer linear chitosan oligomers were required to form physically-stable nanoparticles with siRNA that mediated efficient, sustained gene silencing in vitro. Finally, the use of an optimised catheter device for the nebulisation of small volumes of pDNA formulations resulted in improved dose precision and lung distribution in vivo compared with conventional intratracheal instillation. In conclusion, chitosan oligomers are interesting and viable alternatives to other non-viral gene delivery systems.

Research on self-assembling gemini surfactants and other amphiphiles for potential gene delivery applications in research as well as in clinical practice, and as alternatives to viral gene delivery vectors, is beginning to focus more on structureactivity relationships to address the current low gene delivery efficiencies of amphiphiles. Some underlying structureactivity relations are beginning to emerge. But, as a better understanding of the factors that govern the transfection abilities of amphiphile molecules emerges, development of improved non-viral vectors with clinical potential may also emerge.<p> The research conducted for this thesis was aimed at the design, synthesis and in vitro investigation of gemini surfactants as one of a family of novel amphiphiles being investigated for gene therapeutic applications. The properties of these compounds can be controlled as well as allowed to vary naturally. Gemini surfactant-based gene delivery systems were prepared and characterized for transfer of Luciferase plasmid (pMASIA.Luc) to both COS-7 and PAM 212 cells. Characterization was accomplished using microscopy, dynamic light scattering (DLS) and zeta (ζ) potential analysis. In vitro gene expression and toxicities were evaluated in COS-7 cell and PAM 212 keratinocyte cultures.<p> The level of in vitro transfection in general was found to correlate strongly with the structure of the gemini surfactants. Among the 12-spacer-12 surfactants, incorporation of a pH-sensitive aza (N-CH3) group, which is also steric hindrance-imposing, in the spacer chain yielded increased transfection, particularly for the 12-7N-12 surfactant. In comparison, the incorporation of the more pH-sensitive imino (N-H) group in the 12-7NH-12 surfactant yielded the highest increase in transfection among the 12-spacer-12 surfactants. The deleterious effect of steric hindrance due to the aza group is more evident when comparing the transfection efficiency of 12-5N-12 (1 × aza, higher) vs. 12-8N-12 (2 × aza, lower transfection). Another highlighted structural feature is provided by the fact that both the 12-7NH-12 and 12-7N-12 surfactants had higher transfection efficiencies than 12-5N-12 and 12-8N-12 surfactants; the first pair has trimethylene spacing, which constitutes an optimal separation between nitrogen centres, while the second pair has shorter dimethylene spacings.<p> After expanding the structure of surfactants, transfection efficiencies were found to increase in response to increase in hydrocarbon tail length, but were much lower for surfactants with no amino functional groups, those that lacked the optimal trimethylene spacing, or those having both of these limitations in the gemini surfactant spacer. The 18-7NH-18 surfactant had the highest overall transfection in both COS-7 and PAM 212 cells. Gemini surfactant-based gene delivery systems capable of adopting both polymorphic structural phases and which could undergo pH-induced structural transition demonstrated high transfection efficiencies. Gemini surfactants with both characteristics (e.g., 12-7NH-12-based complexes are both polymorphic and pH-sensitive) had higher transfection than gemini surfactants with only one (e.g., 12-3-12-based complexes are only polymorphic).<p> Overall, the m-7NH-m surfactants, the most efficient surfactants studied, had transfection efficiencies similar to that of the commercial Lipofectamine Plus reagent and imposed no higher toxicity on cells relative to the less efficient surfactants. Thus, the design of the m-7NH-m surfactants to enhance their transfection abilities also ensured that their toxicity to cells were kept minimal. Overall, the design, synthesis and in vitro transfection screening of gemini surfactant candidates has revealed that the m-7NH-m surfactants have the highest transfection efficiencies; they have emerged as suitable candidates for non-viral gene delivery in vivo or at higher levels. Gene delivery investigations for six of the gemini surfactant candidates are being reported for the first time.