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Twaites, Beverley Ruth. "Polymer-biopolymer interactions : applications in gene delivery." Thesis, University of Portsmouth, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402281.
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Shaw, Paul Andrew. "Improving gene delivery for gene therapy and DNA vaccination applications." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614094.
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Cifuentes, Rius Anna. "Tailoring Carbon Nanotubes Properties for Gene Delivery Applications." Doctoral thesis, Universitat Ramon Llull, 2013. http://hdl.handle.net/10803/127706.
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La teràpia gènica s’està convertit en una tècnica innovadora per tal de curar una malaltia mitjançant la inserció de gens dins les cèl•lules i òrgans d’un individu. El repte recau en l’alliberació eficient I segura de l’àcid nucleic terapèutic a les cèl•lules i òrgans objectiu. De tots els vectors sintètics desenvolupats recentment, els nanotubs de carboni són una elecció interessant que ja ha demostrat prometre considerablement com a sistema alliberació, gràcies a la seva proporció amplada-alçada i la seva capacitat de traspassar la membrana cel•lular. El problema que sorgeix és la seva limitada solubilització i l’agregació espontània in vivo.
Amb l’objectiu de desenvolupar nous dissenys basats en nanotubs de carboni per a la formació de complexos capaços de transfectar ADN a les cèl•lules, amb un a bon registre de biocompatibilitat i viabilitat cel•lular, s’han desenvolupat diferents estratègies. En primer lloc, s’ha optimitzat la funcionalització covalent dels nanotubs per mitjà de tècniques de plasma. Aquest tipus de modificació permet aconseguir tan superfícies altament reactives capaces d’unir ADN a través d’una molècula enllaçant, com superfícies carregades positivament que permeten l’envolcall de l’àcid nucleic per interacció electrostàtica. En segon lloc, s’ha avaluat la dispersió de nanotubs de diferents mides per mitjà d’un agent estabilitzant incloent un surfactant, un polímer amfifílic i proteïnes. Aquesta naturalesa química de la superfície del nanotub, juntament amb altres propietats físiques com ara l’allargada o el diàmetre, té un efecte directe en la dispersibilitat, citotoxicitat i biodistribució d’aquest sistemes.
L’ús de proteïnes per functionalitzar nanopartícules és encoratjador ja que forma la corona de proteïna a la seva superfície. Tals conjugats mostren una elevada capacitat de carregar ADN i permeten la regulació de la seva alliberació mitjançant la manipulació de la composició de la corona.La terapia génica se está convirtiendo en una técnica innovadora para curar enfermedades mediante la inserción de genes dentro de las células y órganos de un individuo. El reto recae en la liberación eficiente y segura de un acido nucleico terapéutico a los órganos objectivo. De todos los vectores sintéticos desarrollados recientemente, los nanotubos de carbono son una elección interesante que ya ha demostrado prometer considerablemente como sistema de liberación gracias a su proporción anchura-altura y su capacidad de traspasar la membrana celular. El problema que surge es su limitada solubilización i la agregación espontanea in vivo. Con el objetivo de desarrollar nuevos diseños basados en nanotubos de carbono para la formación de complejos capaces te transfectar ADN a las células, con un buen registro de biocompatibilidad y viabilidad celular, se han desarrollado diferentes estrategias. En primer lugar, se ha optimizado la funcionalización covalente de los nanotubos por medio de técnicas de plasma. Este tipo de modificación permite conseguir tanto superficies altamente reactivas capaces de unir ADN a traves de una molécula enlazante, como cargadas positivamente que permiten el envoltorio del acido nucleico por interacción electrostática. En segundo lugar, se han evaluado la dispersión de nanotubos de medidas diferentes por mediado de un agente estabilizante que incluye un surfactante un polímero amfifílico y proteínas. Esta naturaleza química de la superficie del nanotubo, junto con otras propiedades físicas como su longitud o diámetro, tiene un efecto directo en la dispersibilidad, citotoxicidad y biodistribución de estos sitemas. El uso de proteínas para funcionalizar nanopartículas es alentador ya que forma la corona de proteínas en su superficie. Dichos compuestos muestran una elevada capacidad de cargar ADN y permiten la regulación de su liberación mediante la manipulación de la composición de la corona.
Gene therapy has become an increasing innovative technique to treat disease by the insertion of genes into individual’s cells and tissues. The challenge is to efficiently and safely deliver the therapeutic nucleic acid into the target cells and organs. Among the synthetic vectors recently developed, carbon nanotubes are an interesting choice as they have already demonstrated considerable promise as delivery systems due to their high aspect ratio and their capacity to translocate the cell membrane. The problem that arises is their limited solubilization and spontaneous aggregation in vivo. Aiming to engineer new carbon nanotube-based designs for the formation of complexes able to transfect DNA/RNA to cells with a good track of biocompatibility and cell viability, different strategies have been developed. Firstly, the covalent functionalization of carbon nanotubes by plasma techniques has been optimized. This type of modification allows to either achieving highly reactive surfaces able to covalently bind DNA towards a chemical linker or a positively charged nanotube surface enabling the wrapping of the nucleic acid by electrostatic interaction. Secondly, the dispersion of the differently-sized carbon nanotubes by means of a stabilizing agent including a surfactant, an amphiphilic polymer and proteins has been assessed. The chemical nature of the modifying moieties on the carbon nanotube, alongside to other physical properties such as length or diameter, has a direct effect on the dispersibility, cytotoxicity and biodistribution of these systems. The use of proteins in the nanoparticle functionalization is encouraging due to the formation of the protein corona on its surface. Such complex exhibits high DNA load capacities and allows a tunable payload release by manipulating the corona composition
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Uthe, Peter Benjamin Ashby Valerie. "The development of polycationic materials for gene delivery applications." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2010. http://dc.lib.unc.edu/u?/etd,2917.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2010.Title from electronic title page (viewed Jun. 23, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.
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TURCHIANO, GIANDOMENICO. "Defining an innovative and safe non-viral gene delivery system: perspective analysis for gene therapy applications." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/43579.
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The Sleeping Beauty (SB) transposon-based integration system is a valuable tool for functional genomics in several model organisms and represent a promising vector for gene therapy in humans. The SB transposase was found to bind the inverted repeats of regenerated salmonid transposons in a substrate-specific manner, and to mediate precise cut-and-paste transposition in fish as well as in mouse and human cells. However, a major bottleneck of any transposon-based application is the efficiency of transpositional activity. Therefore, considerable effort has been made to improve the transposition efficiency of SB by modifying its IRs and systematically mutating the transposase gene. The combined effect of these modifications resulted in the generation of the hyperactive SB100X transposase and of the high-capacity “sandwich” transposon (SA). This new system shows an almost 100-fold enhancement of transposition as compared to the first-generation transposon system. I address my work to the molecular characterization of “sandwich” SB-mediated integrants in epithelial cell lines and in primary keratinocytes. The interest in keratinocytes is related to a SB-mediated gene therapy application for the dystrophic form of epidermolysis bullosa (DEB) caused by mutations in the type-VII collagen gene (COL7A1). Delivering the >9 kb COL7A1 cDNA by a retroviral or lentiviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration. The Sleeping Beauty transposon-based integration system can potentially overcome these issues.
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LaManna, Caroline Marie. "Synthesis, characterization, and evaluation of photo-active amphiphiles for gene delivery applications." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12803.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Gene therapy has the potential to alter the landscape of medical therapeutic techniques by offering a means of introducing or knocking out genes to treat a number of diseases. Both viral and nonviral vectors are currently being utilized in gene therapy clinical trials. To overcome the obstacles in the cellular uptake and transfection pathways which impede nonviral gene delivery, novel lipids, polymers, and dendrimers are being engineered. Cationic lipid vectors have been widely characterized as gene delivery tools as they electrostatically interact with the anionic nucleic acid backbone to form a supramolecular structure (lipoplex). This complex allows the nucleic acid to be protected from enzymatic degradation during transport and interacts with the cell membrane to facilitate internalization by endocytosis. A limitation of current systems is a lack of mechanism for release of the nucleic acid, which is an integral step toward transcription and translation. The use of a charge-reversal or charge-switching amphiphile has been previously described by which the amphiphile initially has a net positive charge and is rendered negatively charged upon enzymatic removal of a terminal ester group. In order to further improve the transfection efficacy of cationic lipids and to impart an externally controlled release mechanism, we have developed a library of novel photo-active chargereversal lipids which can be triggered by ultraviolet (UV) light. In this work, we describe the synthesis and characterization of photo-active lipids for binding and releasing deoxyribonucleic acid (DNA) and evaluate the cellular uptake kinetics and transfection efficiency in vitro. The binding, release, and cellular uptake behaviors of lipoplexes were found to be dependent on lipid composition and resulting supramolecular structures. The transfection efficiency of the photo-active lipoplexes was further affected by variables associated with cellular incubation and UV exposure. Continued development of controlled release gene delivery vectors, including photoactive lipids, will enhance the understanding and utility of gene therapy by providing spatiotemporal control of the process.
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Narayanasamy, Kaarjel Kauslya. "Preparation and evaluation of polymer coated magnetic nanoparticles for applications in gene delivery." Thesis, Keele University, 2018. http://eprints.keele.ac.uk/5002/.
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With the advent of powerful gene editing tools such as CRISPR/Cas9, advances in gene therapy have gained a second wind. Despite this, disease therapy still has not progressed beyond clinical trials due to limitations in current delivery methods. The work presented in this thesis studies the development of a non-viral gene delivery method which is the nanomagnetic transfection method, which is the delivery of genes to cells using magnetic nanoparticles (MNPs) with a cationic surface charge and an external magnetic _eld. The advantage of nanomagnetic transfection over other non-viral chemical methods is the low dosage required to transfect cells coupled with a short transfection time. The presence of an external magnet provides targeting functionality, whereby the MNPs carrying the gene of interest are pulled towards the cells, thus increasing the e_ciency of cell to MNP contact. The research looks at the synthesis of MNPs using thermal decomposition to obtain particles with a narrow size distribution and exhibiting a combination of Brownian and N_eel relaxation. The MNPs were coated with polyethyleneimine (PEI), which binds and condenses DNA to deliver into cells for protein expression. PEI is known to be toxic to cells at high concentrations, hence PEI not bound to MNPs were removed using dialysis. A unique study observing the gradual loading of PEI coating on MNPs using AC susceptometry (ACS) is described. ACS provided information on the MNP coating and aggregation process that was not accessible through dynamic light scattering (DLS) due to the additional presence of non-magnetic polymer particulates in the suspensions. In combination with complementary structural characterization techniques, a simple method was derived to obtain dense, uniform PEI coatings a_ording high-stability suspensions without excessive quantities of unbound PEI to reduce cytotoxic e_ects. This method can be used for improving coating and functionalization therefore advancing MNP-drug/gene delivery studies. The PEI-coated MNPs were subsequently studied for their transfection capabilities in HeLa cells and compared to commercial MNP transfection agents. It was found that nanomagnetic transfection had higher GFP reporter expression compared to Lipofectamine and PEI. The parameters a_ecting transfection activity were determined in order to improve transfection rates of synthesized MNPs. A trade-o_ between transfection e_ciency and cytotoxicity was observed, where the presence of unbound PEI improved transfection but a_ected cell viability. To overcome this, polymers and block-copolymers with a lower charge density should be developed. The proton-sponge e_ect, which is the mechanism of MNP-PEI escape from the endolysosome was studied by measuring the AC susceptibility of MNP-PEI in live cells. However, the low transfection e_ciency of MNP-PEI and low sensitivity of the AC susceptometer made it di_cult to obtain conclusive evidence. A novel study using Raman spectroscopy to obtain _ngerprint spectra of the MNP-PEI complexes and to determine their localization in cells is reported. Individual spectra of MNP and PEI were obtained, as well as the area map of the cell, however the localization of MNPs within the cell was not possible due to the limited sensitivity of the Raman spectrometer. Finally, the e_ect of the MNP-PEI transfection agents on cells were identi_ed. It was observed that MG-63 and HeLa cells expressed increased cell stress with the formation of actin stress _bres and increased cell adhesion. Between the two transfection components, PEI antagonized the cell adhesion e_ect compared to MNPs. In addition, the genes associated with actin _bres and cell adhesion were identi_ed, which were ACTA2, ACTN1, MVCL, VCL, P4HA2, PCDHB12, SVIL, and TGFBI, which showed increased expression to MNP-PEI treatment. Collectively, the study conducted reports the development of an MNP transfection agent, from synthesis to application.
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Nelson, Ashley M. "Design of Functional Polyesters for Electronic and Biological Applications." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/74914.
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Melt polymerization and novel monomers enabled the synthesis of polyesters for electronic and biological applications. Inspiration from nature and a passion for environmental preservation instigated an emphasis on the incorporation of renewable resources into polymeric materials. Critical analysis of current research surrounding bisphenol-A replacements and ioncontaining segmented polyurethanes aided in identifying benchmark polymers, including limitations, challenges, and future needs. Structure-property-morphology relationships were investigated to evaluate the polymers for success in the proposed applications as well as to improve understanding of polyester compositions to further design and develop sophisticated polymers for emerging applications.
Aiming to utilize the reported [2 + 2] cycloaddition of the known mesogen 4,4’-dimethyltrans-stilbene dicarboxylate (SDE) to overcome ultraviolet (UV) induced degradation issues in electronic encasings, the synthesis of copolyesters containing SDE ensued. 1,6-Hexanediol (HD) and 1,4-butanediol comonomers in varying weight ratios readily copolymerized with SDE under melt transesterification conditions to afford a systematic series of copolyesters. Differential scanning calorimetry revealed all copolyesters exhibited liquid crystalline transitions and melting temperatures ranged from 196 °C – 317 °C. Additionally, melt rheology displayed shear thinning to facilitate melt processing. Compression molded films exhibited high storage moduli, a glassy plateau until the onset of flow, and tensile testing revealed a Young’s iii modulus of ~900 MPa for poly(SDE-HD). These properties enable a wide range of working temperatures and environments for electronic applications.
Adding complexity to linear liquid crystalline copolyesters, copolymerization with oligomeric hydroxyl-functionalized polyethers afforded segmented liquid crystalline copolyesters. 4,4’-Biphenyl dicarboxylate (BDE), commercially available diols containing 4, 5, 6, 8, or 10 methylene units, and introducing poly(tetramethylene oxide) or a Pluronic® triblock oligoethers in varying weight % were used to synthesize multiple series of segmented copolyesters. Comparing melting transitions as a function of methylene spacer length elucidated the expected even-odd effect and melting temperatures ranged from 150 °C to 300 °C. Furthermore, incorporating the flexible soft segment did not prevent formation of a liquid crystalline morphology. Complementary findings between differential scanning calorimetry and small-angle X-ray scattering confirmed a microphase-separated morphology. Thermomechanical analysis revealed tunable plateau moduli and temperature windows based on both soft segment content and methylene spacer length, and tensile testing showed the strain at break doubled from 75 weight % to 50 weight % hard segment content. The same compositions Young’s moduli decreased from 107 ± 12 MPa at 75 weight % hard segment to 19 ± 1 MPa with 50 weight % hard segment, demonstrating the mechanical trade-off and range of properties possible with small compositional changes. These segmented copolyesters could find use in high-performance applications including electronic and aerospace industries.
A two-step synthesis transformed caffeine into a novel caffeine-containing methacrylate (CMA). Conventional free radical copolymerization with a comonomer known to provide a low glass transition temperature (Tg), 2-ethylhexyl methacrylate (EHMA), allowed the investigation of the effect of small amounts of pendant caffeine on polymer properties. Thermal and iv thermomechanical testing indicated CMA incorporation dramatically increased the storage modulus, however, a microphase-separated morphology was not attained. Association of the pendant caffeine groups through non-covalent π-π stacking could present opportunities for novel thermoplastics and it is proposed that placing the pendant group further from the backbone, and potentially increasing the concentration, could aid in promoting microphase-separation.
Alkenes are reactive sites for placing functional groups, particularly those required for polyester synthesis. Methyl 9-decenoate (9-DAME), a plant-based fatty acid, provided a platform for novel biodegradable, renewable, polyesters. A formic acid hydration reaction generated an isomeric mixture of AB hydroxyester or AB hydroxyacid monomers for melt polymerization. Thermal analysis elucidated the plant-based polyesters exhibited a single transition, a Tg of about -60 °C. Aliphatic polyesters commonly crystallize, thus the isomeric mixture of secondary alcohols seemed to introduce enough irregularity to prevent crystallization. These polyesters offer an amorphous, biodegradable, sustainable replacement for applications currently using semi-crystalline poly(ε-caprolactone), which is not obtained from renewable monomers and also exhibits a -60 °C Tg. Additional applications requiring low-Tg polymers such as pressure sensitive adhesives or thermoplastic elastomers could also benefit from these novel polyesters. 9-DAME also was transformed into an ABB’ monomer after an epoxidation and subsequent hydrolysis. Successful gelation under melt transesterification conditions provided evidence that the multifunctional monomer could perform as a renewable, biodegradable, branching and/or crosslinking agent.
Novel copolyesters comprised of a bromomethyl imidazolium diol and adipic acid demonstrated potential as non-viral gene delivery vectors. Melt polycondensation produced water dispersible polyesters which bound deoxyribonucleic acid at low N/P ratios. The v polyplexes showed stability in water over 24 h and no cytotoxic effect on human cervical cancer cells (HeLa). A luciferase transfection assay revealed the copolyesters successfully underwent endocytosis and released the nucleic acid better than controls. The copolyesters with pendant imidazolium functionality also provided tunable Tgs, -41 °C to 40 °C, and the ability to electrospin into fibers upon blending with poly(ethylene oxide). These additional properties furthered potential applications to include pressure sensitive adhesives and biocompatible antibacterial bandages.Ph. D.
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Allen, Michael H. Jr. "Imidazole-Containing Polymerized Ionic Liquids for Emerging Applications: From Gene Delivery to Thermoplastic Elastomers." Diss., Virginia Tech, 2013. http://hdl.handle.net/10919/49593.
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Novel imidazole-containing polyelectrolytes based on poly(1-vinylimidazole) (poly(1VIM)) were functionalized with various hydroxyalkyl-substituents to investigate the influence of charge density and hydrogen bonding on nonviral DNA delivery. Copolymers with higher charge densities exhibited increased cytotoxicity, whereas increased hydroxyl concentrations remained nontoxic. DNA binding affinity increased with increased charge densities and increased hydroxyl content. Dynamic light scattering determined the copolymers which delivered DNA most effectively maintained an intermediate binding affinity between copolymer and DNA. Copolymers containing higher charge densities or hydroxyl concentrations bound DNA too tightly, preventing its release inside the cell. Copolymers with lower charge densities failed to protect the DNA from enzymatic degradation. Tuning hydrogen bonding concentration allowed for a less toxic and more effective alternative to conventional, highly charged polymers for the development of nonviral DNA delivery vehicles. The synthesis of amine-containing imidazolium copolymers functionalized with low concentrations of folic acid enabled the investigation of additional polymer modifications on nonviral gene delivery.
Functionalization of 1VIM with various hydroxyalkyl and alkyl groups and subsequent conventional free radical polymerization afforded a series of imidazolium-containing polyelectrolytes. Hydroxyl-containing homopolymers exhibited higher thermal stabilities and lower Tg's compared to the respective alkyl-analog. X-ray scattering demonstrated the polarity of the hydroxyl group facilitated solvation of the electrostatic interactions disrupting the
nanophase-separated morphology observed in the alkylated systems. Impedance spectroscopy determined hydroxyl-containing imidazolium homopolymers displayed higher ionic conductivities compared to the alkyl-containing analogs which was attributed to increased solvation of electrostatic interactions in the hydroxyl analogs.
Beyond functionalizing 1VIM monomers and homopolymers to tailor various properties, the synthesis of novel architectures in a controlled fashion remains difficult due to the radically unstable N-vinyl propagating radical. The regioisomer 4-vinylimidazole (4VIM) contains two resonance structures affording increased radical stability of the propagating radical. Nitroxide-mediated polymerization (NMP) and atom transfer radical polymerization (ATRP) failed to control 4VIM homopolymerizations; however, reversible addition-fragmentation chain transfer (RAFT) demonstrated unprecedented control. Linear pseudo-first order kinetics were observed and successful chain extension with additional 4VIM suggested preservation of the trithiocarbonate functionality.
Effectively controlling the polymerization of 4VIM enabled the design of amphoteric block copolymers for emerging applications. The design of ABA triblock copolymers with 4VIM as a high Tg supporting outer block and di(ethylene glycol) methyl ether methacrylate (DEGMEMA) as a low Tg, inner block, required the development of a new difunctional RAFT chain transfer agent (CTA). The difunctional CTA successfully mediated the synthesis of the ABA triblock copolymer, poly(4VIM-b-DEGMEMA-b-4VIM), which exhibited microphase separated morphologies. The amphoteric nature of the imidazole ring required substantially lower concentrations of outer block incorporation compared to traditional triblock copolymers to achieve similar mechanical properties and microphase separated morphologies.Ph. D.
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Perouzel, Eric. "Synthesis formulation and applications of new stabilisation agents for liposome based gene delivery system." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271482.
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AllenJr, Michael Harry. "Imidazole-Containing Polymerized Ionic Liquids for Emerging Applications: From Gene Delivery to Thermoplastic Elastomers." Diss., Virginia Tech, 2001. http://hdl.handle.net/10919/49593.
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Novel imidazole-containing polyelectrolytes based on poly(1-vinylimidazole) (poly(1VIM)) were functionalized with various hydroxyalkyl-substituents to investigate the influence of charge density and hydrogen bonding on nonviral DNA delivery. Copolymers with higher charge densities exhibited increased cytotoxicity, whereas increased hydroxyl concentrations remained nontoxic. DNA binding affinity increased with increased charge densities and increased hydroxyl content. Dynamic light scattering determined the copolymers which delivered DNA most effectively maintained an intermediate binding affinity between copolymer and DNA. Copolymers containing higher charge densities or hydroxyl concentrations bound DNA too tightly, preventing its release inside the cell. Copolymers with lower charge densities failed to protect the DNA from enzymatic degradation. Tuning hydrogen bonding concentration allowed for a less toxic and more effective alternative to conventional, highly charged polymers for the development of nonviral DNA delivery vehicles. The synthesis of amine-containing imidazolium copolymers functionalized with low concentrations of folic acid enabled the investigation of additional polymer modifications on nonviral gene delivery. Functionalization of 1VIM with various hydroxyalkyl and alkyl groups and subsequent conventional free radical polymerization afforded a series of imidazolium-containing polyelectrolytes. Hydroxyl-containing homopolymers exhibited higher thermal stabilities and lower Tg\'s compared to the respective alkyl-analog. X-ray scattering demonstrated the polarity of the hydroxyl group facilitated solvation of the electrostatic interactions disrupting the
nanophase-separated morphology observed in the alkylated systems. Impedance spectroscopy determined hydroxyl-containing imidazolium homopolymers displayed higher ionic conductivities compared to the alkyl-containing analogs which was attributed to increased solvation of electrostatic interactions in the hydroxyl analogs.
Beyond functionalizing 1VIM monomers and homopolymers to tailor various properties, the synthesis of novel architectures in a controlled fashion remains difficult due to the radically unstable N-vinyl propagating radical. The regioisomer 4-vinylimidazole (4VIM) contains two resonance structures affording increased radical stability of the propagating radical. Nitroxide-mediated polymerization (NMP) and atom transfer radical polymerization (ATRP) failed to control 4VIM homopolymerizations; however, reversible addition-fragmentation chain transfer (RAFT) demonstrated unprecedented control. Linear pseudo-first order kinetics were observed and successful chain extension with additional 4VIM suggested preservation of the trithiocarbonate functionality.
Effectively controlling the polymerization of 4VIM enabled the design of amphoteric block copolymers for emerging applications. The design of ABA triblock copolymers with 4VIM as a high Tg supporting outer block and di(ethylene glycol) methyl ether methacrylate (DEGMEMA) as a low Tg, inner block, required the development of a new difunctional RAFT chain transfer agent (CTA). The difunctional CTA successfully mediated the synthesis of the ABA triblock copolymer, poly(4VIM-b-DEGMEMA-b-4VIM), which exhibited microphase separated morphologies. The amphoteric nature of the imidazole ring required substantially lower concentrations of outer block incorporation compared to traditional triblock copolymers to achieve similar mechanical properties and microphase separated morphologies.
Ph. D.
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Abbas, Aiman Omar Mahmoud. "Chitosan for biomedical applications." Diss., University of Iowa, 2010. https://ir.uiowa.edu/etd/771.
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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.
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Holmes, Christina. "A polyelectrolyte multilayer thin film system for cell adhesion, gene delivery and inductive tissue engineering applications." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=116904.
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In order for the vast therapeutic potential of tissue engineering to be realized, inductive three-dimensional (3D) scaffolds that can direct cell behaviour and tissue architecture must be developed. Although a variety of strategies which enable scaffold-based biofactor release are under investigation, the processing conditions typically employed restrict the system architectures and mechanical properties that can be produced.The main objective of this thesis work was to develop an inductive tissue engineering system for in situ 3D gene delivery, utilizing the layer-by-layer deposition technique and a model scaffold. Towards that end, a novel polyelectrolyte multilayer film system composed of glycol-modified chitosan (Glyc-CHI) and hyaluronic acid (HA) was developed and analyzed. This biocompatible Glyc-CHI/HA multilayer system was then modified to incorporate gene delivery lipoplexes, composed of plasmid DNA complexed with Lipofectamine2000TM, in order to facilitate in vitro delivery of a marker gene encoding enhanced green fluorescent protein (EGFP). The system, with and without incoporated gene delivery lipoplexes, was then adapted to be used as a LbL coating on a 3D model porous scaffold system microfabricated from poly(lactic-co-glycolic acid) (PLGA). The polyelectrolyte multilayer film system developed in this study exhibited a number of novel and useful features. Glyc-CHI/HA films composed of 5 or more bilayers were displayed significantly increased in vitro cellular adhesion, growth and viability compared to corresponding films consisting of the well characterized unmodified chitosan/HA system, while maintaining many similar physical properties. Meanwhile, multilayers incorporating gene delivery lipoplexes achieved in vitro transfection efficiencies of up to 20% in NIH3T3 and HEK293 cells, and were able to maintain transfection for at least 7 days. PLGA scaffolds LbL-coated with these Glyc-CHI/HA films supported in vitro MC3T3 cell growth and viability for a period of at least 2 weeks at levels similar to, or better than, those achieved in uncoated control scaffolds. A novel imaging technique known as optical coherence phase microscopy (OCPM) was demonstrated to enable in situ, non-invasive, label-free imaging of tissue structure and viability within our 3D tissue engineering scaffolds. Finally, coated PLGA scaffolds incorporating gene delivery lipoplexes were found to support scaffold-based in vitro transfection of HEK293 cells at levels significantly higher than uncoated scaffolds with surface adsorbed lipoplexes. Overall, this thesis work thus serves as an important first step towards using Glyc-CHI/HA multilayer films for controlled delivery of various therapeutic genes in 2D and 3D inductive tissue engineering applications.Afin de voir l'important potentiel thérapeutique que représente l'ingénierie tissulaire se réaliser, il faut d'abord voir la réalisation de matrices inductrices tridimensionnelles pouvant régir le comportement des cellules de même que l'architecture des tissus. Bien que différentes stratégies permettant la libération de molécules bioactives en matrice soient à l'étude, les conditions de traitement les plus souvent utilisées restreignent l'architecture du système et les propriétés mécaniques qui pourraient être produites.Le principal objectif de cette thèse était de développer un système inducteur d'ingénierie tissulaire pour la libération tridimensionnelle in situ de gènes à l'aide d'une technique de dépôt couche par couche et d'un modèle matriciel. Vers la fin, un nouveau système de pellicule de polyélectrolytes à couches multiples composé de chitosane modifié au glycol (Glyc-CHI) et d'acide hyaluronique avait été conçu et analysé. Ce système biocompatible à couches multiples de Glyc-CHI/HA a ensuite été modifié pour incorporer des lipoplexes issus de la libération de gènes et composés d'ADN plasmidique complexé avec du Lipofectamine2000MC, et ce, afin de faciliter la libération in vitro d'un gène marqueur qui encoderait des protéines à fluorescence verte (GFP). Avec ou sans l'incorporation des lipoplexes issus de la libération des gènes, le système a par la suite été adapté pour servir d'enveloppe couche par couche sur un système de modèle matriciel tridimensionnel et poreux microfabriqué à partir de polyacide lactique coglycolique (PLGA). Le système de pellicule de polyélectrolytes à couches multiples qui a été conçu au cours de cette étude démontrait un certain nombre de nouvelles caractéristiques utiles. Les pellicules de Glyc-CHI/HA composées de 5 bicouches présentaient une adhésion, croissance et viabilité accrues des cellules in vitro comparativement à des pellicules similaires formées du système bien connu de chitosan/HA non modifié, tout en conservant de nombreuses propriétés physiques semblables. On a pu observer que des couches multiples incorporant des lipoplexes issus de la libération de gènes parvenaient à atteindre in vitro un rendement de transfection de 20 % dans les cellules NIH3T3 et HEK293 et étaient en mesure de maintenir cette transfection pendant au moins 7 jours. Il a également été démontré que les matrices de PGLA enrobées de ces pellicules de Glyc-CHI/HA pouvaient appuyer la croissance et la viabilité in vitro de cellules MC3T3 pour une durée minimale de deux semaines, et ce, à des niveaux semblables ou supérieurs à ceux atteints par les matrices témoin non enrobées. Une nouvelle technique d'imagerie nommée microscopie de phase par cohérence optique a permis d'obtenir des images in situ non invasives et dépourvues d'étiquettes de la structure et de la viabilité des tissus à l'intérieur de nos matrices tridimensionnelles d'ingénierie tissulaire. Enfin, on a pu observer que les matrices de PLGA enrobées incorporant des lipoplexes issus de la libération de gènes appuient la transfection in vitro de cellules HEK293 dans la matrice à des niveaux nettement supérieurs à ceux des matrices non enrobées dont la surface a adsorbé des lipoplexes. En général, la présente thèse est un important premier pas vers l'utilisation de pellicules de Glyc-CHI/HA à couches multiples pour la libération contrôlée de divers gènes thérapeutiques dans les applications inductives bi- et tridimensionnelles de l'ingénierie tissulaire.
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Majewski, Alexander [Verfasser], and Axel [Akademischer Betreuer] Müller. "Dual-Responsive Polymer and Hybrid Systems: Applications for Gene Delivery and Hydrogels / Alexander Majewski. Betreuer: Axel Müller." Bayreuth : Universität Bayreuth, 2013. http://d-nb.info/1059352680/34.
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Lilley, Caroline Elizabeth. "Herpes simplex virus vectors for gene delivery to the CNS : applications in the study of Alzheimer's disease." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326175.
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Mushtaq, Yasmin. "An investigation into potential applications of spray-dried microparticles for use in the field of gene delivery." Thesis, University of Bath, 2000. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288237.
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Breitenkamp, Rebecca Boudreaux. "Oligopeptide-functionalized Graft Copolymers: Synthesis and Applications in Nucleic Acid Delivery." Amherst, Mass. : University of Massachusetts Amherst, 2009. http://scholarworks.umass.edu/open_access_dissertations/5/.
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Green, Matthew Dale. "Tailoring Structure and Function of Imidazole-Containing Block Copolymers for Emerging Applications from Gene Delivery to Electromechanical Devices." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/40352.
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The imidazole ring offers great potential for a variety of applications including gene delivery vectors, ionic liquids, electromechanical actuators, and novel monomers and polymers. The imidazole ring provides a unique building block for these applications due to its thermal stability, aromatic nature, ability to form ionic salts, and ease of functionalization. Free radical polymerization of 1-vinylimidazole (1-VIm) and free radical copolymerizations with methyl methacrylate (MMA) and n-butyl acrylate (nBA) afforded homopolymers and copolymers with tunable solution and thermal properties. Aqueous SEC provided reproducible and reliable molecular weights for poly(1-VIm) in the absence of polymer aggregates. Analysis of the thermal properties revealed ideal random copolymers with MMA and non-ideal copolymers with nBA. Small angle X-ray scattering determined that the spacing between ionic groups remained constant with increased nonionic comonomer incorporation while the spacing between adjacent polymer backbones increased.
Functionalization of 1-VIm with varying length alkyl halides and polymerization prepared a series of imidazolium homopolymers. Anion exchange reactions controlled the thermal and solution properties, and the bromide counteranion quantitatively exchanged to tetrafluoroborate (BF4), trifluoromethanesulfonate (TfO), and bis(trifluoromethanesulfonyl)imide (Tf2N). Thermogravimetric analysis revealed that thermal stability increased with decreased alkyl substituent length and larger counteranion size, and differential scanning calorimetry determined that glass transition temperature (Tg) decreased with increased alkyl substituent length and larger counteranion size. Electrochemical impedance spectroscopy determined the ionic conductivities of the imidazolium homopolymers, and analysis using the Vogel-Fulcher-Tammann equation revealed that the activation energy of ion conduction increased as alkyl substituent length increased. Polymer morphology determined using X-ray scattering also influenced the ionic conductivity. As the alkyl substituent length increased, the spacing between adjacent polymer backbones increased, which decreased the ionic conductivity due to the ion-hopping mechanism of ion conduction.
Unsuccessful attempts to control the radical polymerization of 1-VIm led to the investigation of 1-(4-vinylbenzyl)imidazole (VBIm), which is a styrenic-based monomer with excellent propagating radical stability. Triblock copolymers incorporating VBIm monomer into a soft random copolymer center block and reinforcing, hard segment outer blocks provided a template for tuning the properties of the ionomer membranes for electroactive devices. Analysis of the morphology and mechanical properties using small angle X-ray scattering and dynamic mechanical analysis determined microphase separation and optimal mechanical properties for electromechanical transducer fabrication. Testing electromechanical transducers revealed superior performance relative to the benchmark Nafion®. Optimization of triblock copolymer design criteria through varying the comonomer ratio of VBIm and nBA in the soft center block, quaternization reactions, and ionic liquid introduction influenced mechanical properties and ionic conductivity. Higher percentages of VBIm and quaternization of VBIm in the random central block increased Tg and ionic conductivity. IL selectively incorporated into the imidazole-containing phases with no leakage observed for ionic systems, reduced the center block Tg, and increased ionic conductivity.
Controlling charge density along poly(1-VIm) through well-defined alkylation reactions with 1-bromobutane provided a potential vector for nonviral gene delivery and polyanion binding. Analysis of DNA and heparin binding using gel electrophoresis revealed a decrease in N/P ratio with increased alkylation percentage. Dynamic light scattering indicated an increase in zeta potential with increasing alkylation percentages, and relatively uniform polyplex sizes in aqueous media. The MTT assay developed cytotoxicity profiles with little toxicity prior to 83% alkylation. Finally, the luciferase expression assay revealed inefficient nucleic acid delivery to multiple cell types. Synthesis of poly(1-VIm) vectors with glutathione conjugates provided an avenue for simultaneous therapeutic gene and anti-oxidant delivery in vitro. Cytotoxicity assays of cells pretreated with glutathione-conjugated poly(1-VIm) prior to oxidative stress showed that higher glutathione conjugation levels improved cell viability.Ph. D.
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Liu, Jie. "Development of multifunctional siRNA delivery systems and their applications in modulating gene expression in a cardiac ischemia-reperfusion model." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/53391.
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RNA interference (RNAi) is a conservative post-transcriptional gene silencing mechanism that can be mediated by small interfering RNAs (siRNAs). Given the effectiveness and specificity of RNAi, the administration of siRNA molecules is a promising approach to cure diseases caused by abnormal gene expression. However, as siRNA is susceptible to degradation by nucleases and it can hardly penetrate cell membranes due to its polyanionic nature, a successful translation of the RNAi mechanism for therapeutic purposes is contingent on the development of safe and efficient delivery systems. This dissertation described the development of novel siRNA delivery systems on the basis of polymeric and dendrimeric materials and also demonstrated the application of one optimized delivery system to deliver therapeutic siRNAs in a cardiovascular disease model in vivo. We studied a linear peptide polymer made from cell penetrating peptide monomers and investigated the contribution of the polymeric structure, degradability, and ligand conjugation to the siRNA loading capacity, biocompatibility, and transfection efficiency of polymeric materials. With the obtained knowledge and experience, we invented a neutral crosslinked delivery system aiming to solve the inherent drawbacks of traditional cationic delivery systems that are based on electrostatic interactions. The new concept utilized buffering amines to temporarily bind siRNA and a crosslinking reaction to immobilize the formed particles, and targeting ligands modified on the neutral dendrimer surface further enhanced the interactions between the delivery vehicles and target cells. The obtained delivery system allowed stability, safety, controllability, and targeting ability for siRNA delivery, and the method developed here could be transformed to other polymeric or dendrimeric cationic materials to make them safer and more efficient. To exploit the therapeutic potential of siRNA delivery, we developed a tadpole-shaped dendrimeric material to deliver siRNA against an Angiotensin II receptor in a rat ischemia-reperfusion model. Our results showed that the nonaarginine-conjugated tadpole dendrimer was capable of delivering siRNA effectively to cardiac cells both in vitro and in vivo, and the successful down-regulation of the Angiotensin II receptor preserved the cardiac functions and reduced the infarct size post-myocardial infarction. This dissertation paves a way for transforming multifunctional non-viral siRNA delivery systems into potent therapeutic strategies for the management of cardiovascular diseases.
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Boehm, Michael. "EXPERIMENTAL INVESTIGATION OF TWO-PHASE PENETRATING FLOW OF NEWTONIAN AND NON-NEWTONIAN POLYMERIC FLUIDS AND DEVELOPMENT OF PRACTICAL APPLICATIONS IN DRUG/GENE DELIVERY." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1253548237.
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Wagner, Darcy E. "Calcium Phosphate Nanoparticle Synthesis and Manufacture using Microwave Processing for Biomedical Applications." University of Toledo / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1310179406.
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Montellano, Lopez Alejandro. "Polyamidoamine dendritic fullerene derivatives for biological and material applications." Doctoral thesis, Università degli studi di Trieste, 2012. http://hdl.handle.net/10077/7392.
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2010/2011Fullerene C60 science can been broadly divided into the study of three areas: (i) its reactivitythat permits the obtention of modified fullerene derivatives, which can typically found (ii) biological and (iii) material applications. The main goal of this thesis is the synthesis and characterization of a library of monoadducts, bisadducts and hexakisadducts of fullerene C60 containing different generations of PAMAM dendron. On this purpose we have firstly studied the functionalization of fullerene C60 by means of 1,3-dipolar cycloaddition. On this purpose, we moved from the classic conditions by employing MW irradiation as the heating source, combined with ionic liquids as the solvent phase in order to obtain remarkable differences in the reactivity and the polyaddition selectivity. In a second stage we have been dealing with the separate synthesis of four different generations of PAMAM dendron and different fulleropyrrolidine moieties including: monoadduct and five bisadducts isomers to finally attach them via amidation. We have complete this library with the employment of the Bingel-Hirsch reaction to attach twelve units of a PAMAM first generation dendron to the carbon cage to obtain a Th symmetric hexakisadduct. As a result, a variety of fullerene derivatives with an enhanced water solubility was obtained, opening the door to their utilization for biological applications. Thus, we can distinguished between those containing terminal, positive-charged amines that can be used to efficiently complex oligonucleotides and those that contains one or more terminal-free carboxylic acid that can be used as anchor points for further functionalization. Concerning to those potentially used for transfection, the broad range of examples described in this thesis will permit to examine the role of the dendron moiety, the fullerene, and the distribution of the positive charges around the fullerene sphere, as key points into the complexation and transfection processes. Furthermore, complexation studies of some of these derivatives has been performed, all of them exhibiting a high affinity towards DNA complexation, demonstrating the great potential of these derivatives for transfection. In the last part of this thesis, we have focused on the synthesis of two porphyrin-dendrofullerene dyads with or without an amide linker. Since the water solubility usually goes hand in hand with a good number of charges we have incorporated an asymmetric tryspyridilporphyrin conferring three more positive charges to the final structure, that provides an additional solubility to the final molecule. This system is of the most interest since electron transfer processes could be studied in polar media. In addition, electrostatic interactions could be further exploited with negative charged systems to build up high complex systems.
XXIV Ciclo
1985
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Bertucci, Alessandro. "Hybrid organic-inorganic interfaces for biomedical applications." Thesis, Strasbourg, 2015. http://www.theses.fr/2015STRAF008/document.
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Le travail de recherche de cette thèse consiste en le développement de nouveaux matériaux hybrides organiques-inorganiques pour des applications en nanotechnologie, nanomédicine et diagnostic. Dans ce contexte, des cristaux poreux de zéolite-L ont été utilisé comme nano-vecteur pour faire de la transfection d’ADN et d’ANP, en combinaison avec le relargage de molécules hôtes placées dans les pores. Des nanoparticules de silice mesoporeuses multifonctionnelles ont été utilisées pour traiter le glioblastome, en combinant la thérapie génique avec l’administration durable d’un principe actif. Des nano-coquilles hybrides biodégradables ont été encore développés pour encapsuler des protéines et les relâcher dans les cellules vivantes. Dans le domaine de la détection d’acides nucléiques, des fibres optiques à cristal photonique, fonctionnalisées avec des sondes d’ANP, ont été exploitées comme plateformes optiques pour faire de la détection ultra-sensible d’oligonucléotides ou d’ADN génomique. Enfin, la squelette de l’ANP a été modifié à créer des sondes fluorescentes pour reconnaître et détecter la présence des séquences cibles spécifiquesThe research work presented throughout this thesis focuses on the development of novel organic-inorganichybrid materials for applications in nanotechnology, nanomedicine and diagnostics. In such a context, porous zeolite-L crystals have been used as nanocarriers to deliver either DNA or PNA in live cells, in combination with the release of guest molecules placed into the pores. Multifunctional mesoporous silica nanoparticles have been designed to treat glioblastoma, combining gene therapy with the sustained delivery of a chemotherapy agent. Biodegradable hybrid nano-shells have been furthermore created to encapsulate proteins and release them in living cells upon degradation of the outer structure in reductive environment. In the field of nucleic acid detection, photonic crystal fibers, functionalized with specific PNA probes, have been exploited as optical sensing devices to perform ultra-sensitive detection of DNA oligonucleotides or genomic DNA. Eventually, the PNA backbone has served as scaffold to synthesize fluorescent switching probes able to recognize and to detect the presence of specific target sequences
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Mindemark, Jonas. "Functional Cyclic Carbonate Monomers and Polycarbonates : Synthesis and Biomaterials Applications." Doctoral thesis, Uppsala universitet, Polymerkemi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-169677.
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The present work describes a selection of strategies for the synthesis of functional aliphatic polycarbonates. Using an end-group functionalization strategy, a series of DNA-binding cationic poly(trimethylene carbonate)s was synthesized for application as vectors for non-viral gene delivery. As the end-group functionality was identical in all polymers, the differences observed in DNA binding and in vitro transfection studies were directly related to the length of the hydrophobic poly(trimethylene carbonate) backbone and the number of functional end-groups. This enabled the use of this polymer system to explore the effects of structural elements on the gene delivery ability of cationic polymers, revealing striking differences between different materials, related to functionality and cationic charge density. In an effort to achieve more flexibility in the synthesis of functional polymers, polycarbonates were synthesized in which the functionalities were distributed along the polymer backbone. Through polymerization of a series of alkyl halide-functional six-membered cyclic carbonates, semicrystalline chloro- and bromo-functional homopolycarbonates were obtained. The tendency of the materials to form crystallites was related to the presence of alkyl as well as halide functionalities and ranged from polymers that crystallized from the melt to materials that only crystallized on precipitation from a solution. Semicrystallinity was also observed for random 1:1 copolymers of some of the monomers with trimethylene carbonate, suggesting a remarkable ability of repeating units originating from these monomers to form crystallites. For the further synthesis of functional monomers and polymers, azide-functional cyclic carbonates were synthesized from the bromo-functional monomers. These were used as starting materials for the click synthesis of triazole-functional cyclic carbonate monomers through Cu(I)-catalyzed azide–alkyne cycloaddition. The click chemistry strategy proved to be a viable route to obtain structurally diverse monomers starting from a few azide-functional precursors. This paves the way for facile synthesis of a wide range of novel functional cyclic carbonate monomers and polycarbonates, limited only by the availability of suitable functional alkynes.
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El, jundi Ayman. "DEGRADABLE DOUBLE HYDROPHILIC BLOCK COPOLYMERS FOR HEALTH APPLICATIONS." Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTS141.
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Les copolymères amphiphiles dégradables à base de poly (éthylène glycol) PEG et de polyesters aliphatiques (poly(ε-caprolactone) (PCL), poly(lactide) (PLA), poly(glycolide) (PGA)) sont largement utilisés dans les applications médicales en raison de leur innocuité et leur acceptation par les autorités de santé. Cependant leur capacité à répondre aux enjeux auxquels sont confrontés les nanomédicaments (ciblage, réponse programmée etc…) est limitée du fait de l’absence de groupes fonctionnels. Pour pallier cette limitation, ce travail de thèse s’intéresse aux voies de modification post-polymérisation de copolymères amphiphiles PEG-b-PCL donnant un accès simple à des familles de copolymères à blocs double hydrophiles (DHBC) dégradables. Nous nous intéressons en particulier à la synthèse de DHBC en trois étapes incluant une étape de photoaddition thiol-yne qui permet à partir d’un même précurseur macromoléculaire la synthèse de familles de DHBC à bloc PEG et blocs PCL fonctionnalisés en chaîne latérale à caractère neutre, cationique ou anionique. Le potentiel de ces DHBC pour la formulation d’actifs au sein de nanosystèmes de libérations pH-répondants est tout d’abord évalué à l’aide d’un anti-cancéreux à large spectre d’activité antitumorale. Dans un second temps, la formulation de micelles de complexes polyioniques tripartites de siRNA pour application en thérapie génique à partir de DHBC anioniques est étudiée. Enfin, la préparation de nanocomplexes DHBC/gadolinium pour imagerie médicale par résonance magnétique nucléaire (IRM) est abordéeBiodegradable amphiphilic copolymers based on poly(ethylene glycol) PEG and aliphatic polyesters (poly(ε-caprolactone) (PCL), poly(lactide) (PLA), poly(glycolide) (PGA)) are widely used in medical applications due to their safety and their acceptance by health authorities. However, their ability to address the challenges faced by the nanomedicines (targeting, programmed response etc…) is limited due to the absence of functional groups. To overcome this limitation, this work focuses on the post-polymerization modification strategies of amphiphilic PEG-b-PCL giving easy access to families of degradable double hydrophilic block copolymers (DHBC). We are particularly interested in the three-step synthesis of DHBC including a thiol-yne photoaddition step which allows, starting from the same macromolecular precursor, the synthesis of DHBC families composed of PEG blocks and side chain functionalized PCL blocks with a neutral, cationic or anionic character. The potential of these DHBC for the formulation of active pharmaceutical ingredients within pH-responsive drug delivery nanosystems is first evaluated using an anti-cancer agent with a broad spectrum of antitumor activity. In another part, we study the formulation of tripartite polyionic complex micelles with an anionic DHBC and siRNA for applications in gene therapy. Finally, the preparation of DHBC/gadolinium nanocomplexes for medical imaging by nuclear magnetic resonance (MRI) is discussed
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Barua, Neil U. "Convection-enhanced drug delivery and its application to Alzheimer's disease." Thesis, University of Bristol, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617593.
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Bremner, K. Helen. "Application of nuclear localization sequences to non-viral gene delivery systems." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273725.
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Zhang, Dongwei. "Application of microneedles to enhance delivery of micro-particles from gene guns." Thesis, Loughborough University, 2013. https://dspace.lboro.ac.uk/2134/13744.
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Gene gun assisted micro-particle delivery system is an excellent method for the delivery of DNA into target tissue so as to carry out gene transfection in the target cells. The gene gun is primarily a particle accelerator which accelerates DNA-coated micro-particles to sufficient velocities to breach the target layer enabling the micro-particles to penetrate to a desired depth and target the cells of interest to achieve gene transfer. However, an inevitable problem in this process is the tissue/cell damage due to the impaction of the pressurized gas and micro-particles on the target. The purpose of this research is developing a new conceptual system which improves the penetration depth of micro-particles at less imposed pressure and particle injection velocity. This is achieved by applying a microneedle array and ground slide in the gene gun system, thus a study involving microneedle assisted micro-particle delivery is conducted in this work. Microneedle array is used to create holes in the target which allows a number of micro-particles to penetrate through the skin which enhances the penetration depth inside target. The ground slide is used to load a pellet of the micro-particles and prevent the pressurized gas to avoid the impaction on the target. The operation principle is that the pellet is attached to ground slide which is accelerated to a sufficient velocity by the pressurized gas. The pellet is released from the ground slide which separates into individual micro-particles by a mesh and penetrates to a desired depth inside the target. An experimental rig to study various aspects of microneedle assisted micro-particle delivery is designed in this PhD research. The passage percentage of the micro-particles and size of the separated micro-particles are analysed in relation to the operating pressure, mesh pore size and Polyvinylpyrrolidone (PVP) concentration to verify the applicability of this system for the micro-particle delivery. The results have shown that the passage percentage increases from an increase in the mesh pore size and operating pressure and a decrease in PVP concentration. A mesh pore size of 178 μm and pellet PVP concentration of 40 mg/ml were used for the bulk of the experiments in this study as these seem to provide higher passage percentage and the narrow size distribution of the separated micro-particles. In addition, the velocity of the ground slide is detected by the photoelectric sensor and shown that it increases from an increase in operating pressure and reaches 148 m/s at 6 bar pressure, A further analysis in the penetration depths of the micro-particles to determine whether they achieve enhanced penetration depths inside the target after using microneedles is carried out. A skin mimicked agarose gel is obtained from comparing the viscoelastic properties of various concentration of agarose gel in comparison with the porcine skin, which is assumed to mimic the human skin. These experiments are used to relate the micro-particle penetration depth with the operating pressure, microneedle length and particle size. In addition, a theoretical model is developed based on the experimental data to simulate the microneedle assisted micro-particle delivery which provide further understanding of the microneedle assisted micro-particle delivery. The developed model was used to analyse the penetration depth of micro-particles in relation to the operation pressure, target properties, microneedle length and particle size and density. The modelling results were compared with the experimental results to verify the feasibility of the microneedle assisted micro-particle delivery for micro-particles delivery. As expected, both experimental and theoretical results show that the micro-particles achieve an enhanced penetration depth inside target. The maximum penetration depth of micro-particles is increased from an increase in operating pressure, microneedle length, particle size and density.
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Jeng, Lily. "Application of endostatin using nonviral gene delivery toward the regeneration of articular cartilage." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/67204.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Cataloged from PDF version of thesis.
Includes bibliographical references (p. 191-207).
Articular cartilage is avascular, and defects have limited capacity for spontaneous healing. Angiogenesis may interfere with maturation of naturally avascular tissues. Our rationale is that the use of endostatin, a potent angiogenesis inhibitor, will facilitate the formation of hyaline cartilage during regeneration. The objective of this thesis was to develop a system with a novel approach for treating cartilage defects, namely endostatin-producing cartilaginous constructs. The constructs were engineered using nonviral gene therapy, through evaluation of select variables, including regulators (culture media, endostatin plasmid load, method of pEndo lipoplex incorporation, and oxygen tension), scaffold formulation, and cell type. We also investigated select aspects of the in vivo cartilage defect model in which the construct can be implanted, including the post-surgical rehabilitation protocol and the use of osteogenic protein (OP)- 1. The principal achievement was the engineering of endostatin-expressing cartilaginous constructs in vitro using chondrocytes and mesenchymal stem cells, collagen sponge-like scaffolds and hydrogels, and chondrogenic medium. Peaks in endostatin protein were observed during the first few days of culture, followed by decreases. The endostatin levels were comparable to therapeutic levels in vitro and physiological levels in vivo. Most of the endostatin protein was released into the expended medium; little retention was observed, including in scaffolds supplemented with heparan sulfate, chondroitin sulfate, and heparin. In vivo work examining chondral defects in the goat knee demonstrated that long-term post-operative immobilization, even with periodic passive motion exercise, resulted in significant joint degeneration. Cell-seeded scaffolds were observed in the defect 2 months following implantation and short-term immobilization, and yielded results at least as good as historical data obtained using other treatment techniques, including autologous chondrocyte implantation and microfracture, suggesting that a cell-seeded scaffold is a viable option for cartilage repair. There was no significant benefit of multiple treatments of OP-I on chondral defects. Neovascularization was observed in the largely fibrous reparative tissue filling the chondral defects, providing further rationale for the use of endostatin. A notable finding was the observation of laminin and type IV collagen, 2 common basement membrane molecules, in both in vitro engineered cartilaginous constructs and in vivo cartilage repair samples.
by Lily Jeng.
Ph.D.
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Sun, Qian. "Application of Polyelectrolyte Layer-By-Layer Self-Assembly on Polymer-Based Gene Delivery System." Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519429.
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Carlisle, Robert. "The application of adenovirus transduction mechanisms to enhance the activity of synthetic gene delivery systems." Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273726.
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Watanabe, Satoshi. "Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders." Kyoto University, 2015. http://hdl.handle.net/2433/199213.
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Millar, Benjamin Mark Glassell. "Studies of membrane fusion by influenza haemagglutinins and their application to liposome delivery systems in gene therapy." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267530.
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Shu, Yi. "Assembly of Phi29 pRNA Nanoparticles for Gene or Drug Delivery and for Application in Nanotechnology and Nanomedicine." University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1336683831.
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Mott, Landon Alexander. "TOWARDS THE RATIONAL DESIGN AND APPLICATION OF POLYMERS FOR GENE THERAPY: INTERNALIZATION AND INTRACELLULAR FATE." UKnowledge, 2019. https://uknowledge.uky.edu/cme_etds/99.
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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.
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Zheng, Yu. "Synthesis and conformational study of trans-2-aminocyclohexanol-based pH-triggered molecular switches and their application in gene delivery." Scholarly Commons, 2013. https://scholarlycommons.pacific.edu/uop_etds/153.
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Trans-2-Aminocyclohexanol (TACH) is a promising model for pH-triggerable molecular switches with a variety of potential applications. In particular, such a switch, when incorporated into cationic liposomes, provides a novel design of the pH-sensitive helper lipids for gene delivery. Protonation of TACH molecules results in a strong intramolecular hydrogen bond between the amino and its neighboring hydroxyl groups, which triggers a conformational flip, and forces changes of the relative position of other substituents on the ring. In this work, a library of TACH-lipids has been designed and built based on structural modifications of both hydrophilic headgroups and hydrophobic tails, and their conformational behavior has been studied by 1 H NMR. NMR-titration has been done to quantitatively monitor the conformational switch for TACH derivatives. It was discovered that conformational behavior of TACH-lipids is independent from the length or shape of their hydrophobic tails. Therefore, a simplified model was suggested based on TACH with diethyl groups instead of hydrocarbon tails. Conformational study of these models has demonstrated that the position of equilibrium shift A [special characters omitted] BH + can be effectively changed by altering structure of NR 2 R 3 group. Furthermore, the pH-induced conformational flip occurs in a certain pH range that mostly depends on the basicity of group NR 2 R 3 , allowing a broad tuning of the pH-sensitivity of TACH-based conformational switches in a wide range of acidity. The hydrophilic OH group was also modified to influence the conformational equilibrium. External stimuli including addition of acid, change of solvent and of the solution ionic strength also showed impact on conformation equilibrium to different extents. To explore the potential to serve as pH-sensitive helper lipids in gene delivery, a variety of TACH-lipids were incorporated into lipoplexes together with the cationic lipid DOTAP to mediate DNA transfection in Bl6F1 and HeLa cancer cell lines. The lipoplex comprising TACH-lipid 3o (R 1 = C 19 H 37 ; R 2 R 3 = CF 3 CH 2 NH) exhibited one to two orders of magnitude better transfection efficiency than the one with the conventional helper lipid DOPE while only inducing slight higher cytotoxicity. Thus, the lipid can be suggested as a novel helper lipid for efficient gene transfection with low cytotoxicity.
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Urnauer, Sarah [Verfasser], and Ernst [Akademischer Betreuer] Wagner. "Improved synthetic gene delivery vehicles for advanced bioimaging-guided tumor-targeted application of the sodium iodide symporter (NIS) as theranostic gene / Sarah Urnauer ; Betreuer: Ernst Wagner." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1148941177/34.
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Abbasi, Sana. "Preparation and in vitro characterization of modified bio-degradable albumin-based nanoparticles for the efficient delivery of therapeutic drugs and genes in breast cancer applications." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106547.
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Breast cancer is considered the second most commonly diagnosed type of cancer across the world. The common modes of treatment are limited by severe side-effects that hinder the efficacy of the drugs, compromise the patients' quality of life and often lead to other disorders. One of the main focuses of nanobiotechnology research is to develop novel anti-cancer drug delivery systems that improve the drug efficacy, limit harmful side effects and also allow for the delivery of developing therapeutics that are rapidly degraded in circulation, such as small interfering RNA (siRNA). Nano-carriers are helpful particularly in anti-cancer drug delivery due to the Enhanced Permeability and Retention (EPR) effect. In the current research study, we developed and investigated the use of surface modified HSA nanoparticles for the delivery of anti-cancer therapeutics in breast cancer applications. Results showed formation of modified HSA nanoparticles of sizes below 150 nm and contained a positive surface charge. The cellular uptake of the nanoparticles was higher in coated particles (average: ~70%) than uncoated particles. Furthermore, the cytotoxicity assessment of modified HSA nanoparticles suggested that empty particles are biocompatible and non-toxic to cells. Therefore, the presented PEI-enhanced and TAT-coated HSA nanoparticles form an appealing delivery system for anti-cancer therapeutics with a potential for clinical application.Le cancer du sein est considéré comme le deuxième type de cancer le plus couramment diagnostiqué à travers le monde. La plupart des traitements sont characterisés par des effets secondaires nocifs qui limitent l'efficacité des médicaments, compromettent la qualité de vie des patients et conduisent souvent à d'autres troubles nocifs. L'un des principaux axes de recherche en nanobiotechnologie est de développer un nouveaux système de délivrance qui permet d'améliorer l'efficacité du médicament, de limiter les effets secondaires nocifs et aussi de permettre la livraison de molecules qui sont rapidement dégradées dans la circulation, tels que les petits ARN interférents (siRNA). Les nano-transporteurs sont utiles en particulier dans l'administration de médicaments anticancerigenes en raison de leur perméabilité accrue et de leur conservation (EPR). Dans l'étude de la recherche actuelle, nous avons développé et étudié l'utilisation de nanoparticules HSA à surface modifiée pour la livraison de médicaments anticancéreux dans les applications de cancer du sein. Les résultats ont montré la formation de nanoparticules HSA de tailles modifiées en dessous de 150 nm contenant une charge de surface positive. L'absorption cellulaire des nanoparticules est plus élevée dans les particules enrobées (moyenne: ~ 70%) que les particules non enrobée. Par ailleurs, l'évaluation de la cytotoxicité des nanoparticules HSA modifiées a suggéré que les particules vides sont biocompatibles et non toxiques pour les cellules. Par conséquent, les nanoparticules HSA revêtues de TAT et PEI-améliorée forment un système de prestation idéale pour les thérapies anti-cancereuses avec un potentiel d'application clinique.
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Reis, Fábio Pedro. "Development of amphiphilic block copolymers for gene delivery applications." Master's thesis, 2016. http://hdl.handle.net/10316/32660.
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Dissertação de Mestrado Integrado em Engenharia Biomédica apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.A terapia genética tem tido um crescente interesse devido às suas potencialidades no tratamento de doenças, como o cancro, infeções e até mesmo doenças genéticas. Para que a terapia genética seja bem sucedida, um dos fatores mais importantes é o desenvolvimento de sistemas adequados para a libertação controlada de material genético. Têm vindo a ser desenvolvidos vários métodos de transferência de genes por vetores não virais de modo a superar os problemas de segurança associados aos vetores virais. Os vetores não virais têm diversas vantagens quer na segurança, quer na prevenção da potencial imunogenicidade e toxicidade, permitindo a administração de repetidas doses, e a facilidade no estabelecimento de boas práticas de fabrico.[1] Neste sentido, têm sido amplamente utilizados copolímeros de bloco anfifílicos (ABCs) em aplicações farmacêuticas, como é o caso das tecnologias de libertação controlada em terapia genética. Os ABCs têm sido extensivamente utilizados devido à sua composição química única, a qual é caracterizada por segmentos hidrofílicos e hidrofóbicos que, em solução aquosa são capazes de se auto-agregar em diferentes morfologias.[2] As diversas características presentes nos copolímeros anfifílicos torna-os adequados veículos de entrega de material genético. Devido aos recentes avanços das técnicas de polimerização radicalar por desativação reversível (RDRP) é agora possível sintetizar copolímeros de bloco com estruturas e funcionalidades específicas. Estes permitem o desenvolvimento de sistemas de libertação controlada de fármacos mediada pelo pH, biocompatíveis e de baixa citotoxicidade. A polimerização radical por transferência de átomo (ATRP) é uma das técnicas RDRP mais eficientes, versáteis e robustas. Com o intuito de reduzir a quantidade de cobre necessária para controlar as polimerizações, foram propostas novas alterações no método de ATRP, tais como a polimerização radicalar por transferência de átomo com ativador suplementar e agente redutor (SARA ATRP).[3] Assim, o objetivo deste trabalho foi investigar novos sistemas poliméricos para entrega de material genético, compostos por copolímeros de bloco que respondem a determinados estímulos: poli(oligo(óxido de etileno) metil éter) metacrilato-bloco- poli[metacrilato de 2-(N-dimetilamino)etil] (POEOMA-b-PDMAEMA), poli(oligo(óxido de etileno) metil éter) metacrilato-bloco-poli(2-diisopropilamino metacrilato de etilo) (POEOMA-b-PDPA). Os copolímeros de bloco POEOMA-b- xii PDMAEMA, POEOMA-b-PDPA, POEOMA-b-(PDPA-co-PDMAEMA) e o homopolímero PDPA foram sintetizados com diferentes pesos moleculares, por um método desenvolvido recentemente, designado SARA ATRP, que utiliza reduzidas concentrações de catalisador (cobre). Os polímeros resultantes foram caracterizados pelas técnicas de espectroscopia de ressonância magnética nuclear (NMR) e de cromatografia de exclusão molecular (SEC) Diferentes parâmetros como pKa, tamanho e carga superficial das partículas foram avaliados a fim de estudar o potencial destes copolímeros em aplicações biomédicas. Os copolímeros mais promissores foram enviados para o Centro de Investigação de Ciências da Saúde da Universidade da Beira Interior para avaliar as suas capacidades de complexação de genes e citotoxicicdade, obtendo resultados muito promissores. Palavras-chave: RDRP, SARA, ATRP, copolímeros de bloco, copolímeros de bloco sensíveis ao pH, libertação controlada de genes
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ALAJANGI, HEMA KUMARI. "INVESTIGATION ON NUCLEIC ACID INTERACTION WITH VARIOUS CATIONIC LIGANDS FOR GENE DELIVERY APPLICATIONS." Thesis, 2017. http://dspace.dtu.ac.in:8080/jspui/handle/repository/15796.
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A new paradigm in medicine called gene therapy involves altering of genes entailed in disease by delivering therapeutic gene (nucleic acid based drugs). Delivering such therapeutic gene to the specific target site is an irresistible challenge to drug delivery scientists. However, the polyanionic nature of DNA limits the cellular membrane interactions. Hence, successful therapeutic DNA based gene delivery highly relies on the efficient extracellular and intracellular delivery of DNA molecules for empowering target interaction.(1-5) Various Carrier molecules have been designed specifically to enter cells & deposit therapeutic genes. Vectors can be viral or non-viral. Viral vectors are one of the successful gene delivery systems available, such as retrovirus, adenovirus (types 2 and 5), adeno-associated virus, herpes virus, pox virus, human foamy virus (HFV), and lentivirus. The genomes of the viral vector have been modified by deleting some areas of their genomic sequences so that their replication becomes deranged and it makes them more safe, but these viral systems has accompanied with some problems, such as their marked immunogenicity that causes induction of inflammatory system leading to degeneration of transducted tissue; and toxin production, including mortality, the insertional mutagenesis; and their limitation in transgenic capacity size. These limitations could be overcome by using synthetic non-viral gene carrier systems. (6)
Non-viral vectors are being used in improving potential therapeutic effect of DNA because they are safe and non-immunogenic as compared to viral vectors. Even though lower transfection efficiency of non-viral vectors are proven compared to viral-mediated gene transfection, the observed shortcomings could be overcome by appropriate structural designing of carriers like cationic polymers or lipids. For example, non-viral vectors can easily be targeted to a target tissue or cell by coupling of cell- or tissue-specific targeting moieties on the carrier. Additionally, by controlling
Synopsis
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the size of DNA-cationic vector complex the bio-distribution, cellular internalization and intracellular trafficking of the micro- or nanoparticle can be influenced. Most importantly, the success of the non-viral gene therapy is dependent on the various extra- and intracellular barriers that affect the efficiency of all gene delivery systems, including cellular uptake, endosomal escape, nuclear uptake and gene expression.(7-9)
Poly cationic vector such as poly (amidoamine) (PAMAM) dendrimer is one of the versatile non-viral based gene delivery systems for efficient targeted delivery of DNA amongst numerous other cationic vectors.(10-12) In particular, PAMAM dendrimer is shown as a potential vehicle especially through several routes of administration, including oral, ocular, parenteral and transdermal.(10,13,14) The uniqueness of these dendrimers are that they are monodisperse, hyperbranched and of controlled size and molecular weight, possessing number of terminal groups with the increasing generation number.(15) More interestingly, PAMAM-DNA complexes have been found to be highly soluble and stable in almost all the physiological conditions and also observed to be resistant to nuclease digestion by Bielinska et al.(16) These distinctive characteristics of PAMAM dendrimer coupled with their architecture has attracted attention of several research groups and their applications in biology and medicine have been explored.(10,17-19)Additionally, various research groups also commenced to determine dendrimer toxicity and immunogenicity and reported that cytotoxicity increases with increase in generation number i.e. smaller the generation of PAMAM dendrimer less the chances of cytotoxicity.(20,21)In spite of high cost and high cytotoxicity, there exist enormous successful targeted transfection reports using unmodified and modified dendrimers of higher generations.(11,21-23) It is pertinent to mention that even though PAMAM dendrimer-based gene transfection reagents such as Superfect and Priofect are already commercially available, these products are based on toxic high generation dendrimers and also not cost effective.
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"Modified inorganic nanostructures: cytotoxicity and biological applications in gene and drug delivery." 2014. http://repository.lib.cuhk.edu.hk/en/item/cuhk-1291830.
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Zhao, Yan. "PEGylated polyethyleneimine-entrapped gold nanoparticles for enhanced and targeted gene delivery applications." Master's thesis, 2015. http://hdl.handle.net/10400.13/1102.
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Gene therapy, which involves the transfer of nucleic acid into target cells in patients, has become one of the most important and widely explored strategies to treat a variety of diseases, such as cancer, infectious diseases and genetic disorders. Relative to viral vectors that have high immunogenicity, toxicity and oncogenicity, non-viral vectors have gained a lot of interest in recent years. This is largely due to their ability to mimic viral vector features including the capacity to overcome extra- and intra-cellular barriers and to enhance transfection efficiency. Polyethyleneimine (PEI) has been extensively investigated as a non-viral vector. This cationic polymer, which is able to compact nucleic acid through electrostatic interactions and to transport it across the negatively charged cell membranes, has been shown to effectively transfect nucleic acid into different cell lines. Moreover, entrapment of gold nanoparticles (Au NPs) into such an amine-terminated polymer template has been shown to significantly enhance gene transfection efficiency.
In this work, a novel non-viral nucleic acid vector system for enhanced and targeted nucleic acid delivery applications was developed. The system was based on the functionalization of PEI with folic acid (FA; for targeted delivery to cancer cells overexpressing FA receptors on their surface) using polyethylene glycol (PEG) as a linker molecule. This was followed by the preparation of PEI-entrapped Au NPs (Au PENPs; for enhancement of transfection efficiency). In the synthesis process, the primary amines of PEI were first partially modified with fluorescein isothiocyanate (FI) using a molar ratio of 1:7. The formed PEI-FI conjugate was then further modified with either PEG or PEGylated FA using a molar ratio of 1:1. This process was finally followed by entrapment of Au NPs into the modified polymers. The resulting conjugates and Au PENPs were characterized by several techniques, namely Nuclear Magnetic Resonance, Dynamic Light Scattering and Ultraviolet-Visible Spectroscopy, to assess their physicochemical properties.
In the cell biology studies, the synthesized conjugates and their respective Au PENPs were shown to be non-toxic towards A2780 human ovarian carcinoma cells. The role of these materials as gene delivery agents was lastly evaluated. In the gene delivery studies, the A2780 cells were successfully transfected with plasmid DNA using the different vector systems. However, FA-modification and Au NPs entrapment were not determinant factors for improved transfection efficiency. In the gene silencing studies, on the other hand, the Au PENPs were shown to effectively deliver small interfering RNA, thereby reducing the expression of the B-cell lymphoma 2 protein. Based on these results, we can say that the systems synthesized in this work show potential for enhanced and targeted gene therapy applications.
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Radu, Daniela Rodica. "Mesoporous silica nanomaterials for applications in catalysis, sensing, drug delivery and gene transfection /." 2004.
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Sequeira, Marta Baptista. "Internship and Monograph reports entitled ”Cell Membrane-Coated Nanosystems for Gene Delivery Applications"." Master's thesis, 2020. http://hdl.handle.net/10316/93115.
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Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de FarmáciaO crescimento exponencial da terapia génica tem proporcionado o sucesso de várias estratégias laboratoriais aplicadas à entrega de ácidos nucleicos para a terapia de várias doenças inatas e adquiridas. Contudo, devido à existência de barreiras extracelulares e intracelulares, que dificultam a eficiência e a integridade da entrega de material genético, tem-se verificado limitações nas aplicações clínicas desta terapia. Inicialmente, a utilização de estratégias como, por exemplo, a modificação química de ácidos nucleicos resultou, apenas, em aplicações clínicas para administrações locais e tópicas. Posteriormente, a segurança, custo reduzido e a elevada capacidade de transfecção de genes através de vetores não virais avaliada em ensaios para a distribuição sistémica de genes in vivo, promoveu o desenvolvimento de nanopartículas com estruturas inorgânicas e orgânicas para o transporte de material genético. No entanto, a falta de complementaridade biológica na totalidade e de evasão imunológica das nanopartículas icentivou o desenvolvimento de novas estratégias inspiradas em mecanismos e componentes celulares biológicos. Ultimamente, experiências com abordagens inovadoras de nanossistemas biomiméticos têm reportado um aumento da capacidade de direcionamento, interação com mecanismos, internalização celular e farmacocinética das nanopartículas. Os contemporâneos e multifuncionais nanossistemas biomiméticos de nanopartículas revestidas por membranas celulares emergentes consistem no encapsulamento de nanopartículas sintéticas enriquecidas pelo revestimento de membranas celulares, por métodos “top-down”. Este método de revestimento de nanopartículas possui a capacidade de preservar as biofuncionalidades das membranas utilizadas incluindo a complexidade de componentes presentes na sua superfície. Existe uma grande variedade de tipos de membrana utilizados de acordo com os tipos de células disponíveis, como por exemplo, glóbulos vermelhos, células cancerosas, glóbulos brancos, plaquetas e células estaminais com capacidade de revestimento de nanopartículas diferentes. O revestimento de nanoparticulas com membranas celulares tem potenciado a estabilidade e a proteção de ácidos nucleicos, prolongado a sua circulação sanguínea, promovido a utilização de nanopartículas transportadoras de ácidos nucleicos providas das biofuncionalidades da membrana utilizada para o seu revestimento e permitido a entrega de ácidos nucleicos e fármacos sinergicamente num único sistema com várias aplicações no diagnóstico, terapia e teranóstico de várias doenças oncológicas, infeciosas e cardiovasculares. Esta monografia descreve detalhadamente as nanopartículas revestidas por membranas celulares utilizadas na entrega eficiente e inteligente de material genético, os desafios inerentes e as perspetivas futuras desta tecnologia para promover a entrega direcionada de ácidos nucleicos e ampliar as suas aplicações terapêuticas.
The exponential growth of gene therapy has propelled various successful laboratory strategies focussed on nucleic acid (NA)-based therapies applied in several innate and acquired diseases. However, hurdles in the efficient and integral delivery of the genetic material ascribed to distinct human body extracellular and intracellular barriers have been broadly hindered the successful clinical translation of NA-based therapies. Initially, attempts with NAs chemical modifications achieved clinical translations status in local and topical administrations. Posteriorly, studies with non-viral vectors for systemic NA delivery in vivo demonstrated safety, reduced cost, and high transfection capacity, induced inorganic and organic nanoparticles (NPs) improvements as gene carriers. Nevertheless, their lack of complete biological complementarity and immune evasion capacity hurdles have led to the development of innovative biointerfacing strategies. Recently, several studies have reported an emerging era of novel biomimetic nanosystems endowed with not only increased targeting and biointerfacing features, but also enhanced cellular internalization and improved pharmacokinetics. Cell membrane-coated NPs are multifunctional and innovative biomimetic nanosystems consisting of synthetic NPs cores coated with cell membranes by a top-down approach. Such cell membrane coatings are able to preserve the natural biofunctionality of parent cell membranes and inherit the vast complex surface repertoire present on the surface cells’ membranes, including a diversity of membrane types on account of the different cell types available, such as red blood cells, cancer cells, white blood cells, platelet cells, and stem cells with the ability to coat distinct nanoparticle cores. Overall, cell membrane-coated NPs for gene delivery have displayed enhanced NA protection and stability, prolonged NA blood circulation half-life, improved NA-loaded cores provided with cell membranes biofunctionalities and allowed both versatile and multivalent drug and NA delivery toward synergistic approaches, evidencing diverse applications in the diagnostic, therapeutic, and theranostic of a myriad of diseases including cancer, infectious and cardiovascular diseases. This review integrates a detailed compilation of the recent cell membrane-coated nanosystems as efficient, safe and smart NA nanocarriers, critically addressing the challenges and future perspectives toward enhanced cell-targeted NA delivery and improved NA therapeutics.
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"Surface-immobilized adeno-associated virus nanoparticles for applications in controlled gene delivery and biosensing." Thesis, 2011. http://hdl.handle.net/1911/70344.
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Adeno-associated virus (AAV) is a 25 nm replication deficient DNA virus most commonly studied for human gene therapy applications. The work detailed in this thesis investigates the controlled delivery of AAV from surfaces for both transduction and biosensing applications. First, AAV was tested for compatibility with substrate-mediated gene delivery approaches. Two reverse transduction applications were investigated: (1) spatial localization of cells and virus vectors for tissue engineering applications and (2) live cell genetic microarrays. To drive the proper differentiation and assembly of cells within tissue engineering constructs, gene expression patterns may need to be tightly regulated. To localize adhesive proteins and AAV, polydimethylsiloxane stamps and protein adhesive alkanethiols were used. By adsorbing AAV onto adhesive proteins, including human fibronectin, laminin, collagen I, elastin and poly-l-lysine, both cell adhesion and gene delivery were localized to a defined pattern. Gene delivery was efficient on all protein surfaces, with higher expression observed on laminin surfaces. AAV was also patterned using a robotic spotter to create live cell genetic microarrays, creating localized cell islands expressing GFP. This potentially high-throughput technique could be extended to study complex genetic interactions within cells, such as stem cells or induced pluripotent stem cells. Additionally, AAV was explored as a biosensor by modifying virus output functionalities. Wild-type AAV2 externalizes an N-terminus region containing a phospholipase A2 (PLA2) domain during intracellular processing, allowing the virus to escape the endosomal pathway and deliver genetic cargo. This externalization can be replicated outside of cells through heat treatment. AAV2-ΔPLA2-His was created by replacing the PLA2 domain with a nickel binding hexahistidine tag. This replacement allows heat-treated mutant virus to bind a nickel affinity column. Finally, directed evolution was used to (1) improve the ability of AAV to deliver genes into target cells or (2) alter AAV biosensor inputs. Virus libraries were created using error-prone polymerase chain reaction (EP-PCR) to introduce random amino acid modifications into the protein capsid. The error rate for these libraries was estimated to be between 5-7 errors per cap gene. Combining surface immobilization with directed evolution could allow for precise control of AAV for gene delivery and biosensing.
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Ting, Christina Lei. "A Novel Method for Studying Nucleated Pathways in Membranes: Development and Applications for Gene Delivery." Thesis, 2013. https://thesis.library.caltech.edu/7193/2/Ting_Christina_L_2013_5.pdf.
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The development of a safe, selective, and efficient gene delivery system is key to the success of human gene therapy. In polymer-based gene delivery systems, biocompatible polymers electrostatically bind and condense the genetic material into protective nanoparticles. These nanoparticles must subsequently overcome several challenges, which remain poorly understood. In particular, once internalized by the cell, the nanoparticles are trapped inside a membrane-bound compartment called the endosome. In the proton sponge hypothesis, the buffering capacity of the polymers leads to an increase in osmotic pressure that eventually ruptures the endosomal membrane and releases the trapped nanoparticles.
To obtain a mechanistic understanding of the endosomal escape, we first develop a coarse-grained model to study the equilibrium interaction between a positively charged nanoparticle and a lipid membrane. Results indicate the existence of a pore with an inserted particle, whose metastability depends on the membrane tension and particle properties (size and charge). These pores are subsequently shown to lower the critical tension necessary for membrane rupture, thus possibly enhancing the release of the trapped genetic material from the endosome.
Next, we address the actual escape pathway, which is likely a thermally nucleated process and cannot be simulated directly or studied by equilibrium methods. Hence, we develop a novel method for studying minimum free energy paths in membranes. Our results indicate that thermally nucleated rupture may be an important factor for the low rupture strains observed in lipid membranes. Under the moderate tensions found in this regime, there are multiple pathways for crossing the membrane: (1) particle-assisted membrane rupture, (2) particle insertion into a metastable pore followed by translocation and membrane resealing, and (3) particle insertion into a metastable pore followed by membrane rupture. This suggests a direct role of the nanoparticle in the endosomal escape not previously envisioned in the proton sponge hypothesis, and illustrates the importance of having an induced tension on the membrane.
Finally, the methodology developed in this work represents the most advanced theoretical technique for describing nucleation pathways in soft condensed matter systems that also include hard-particle degrees of freedom. We expect the method to be useful for studying a wide range of nucleation phenomena beyond membrane systems, for example, in nanoparticle polymer composites.
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Campos, Samuel Knox. "Metabolic biotinylation of the adenoviral capsid: Avidin-based applications and studies of ligand-targeted gene delivery." Thesis, 2005. http://hdl.handle.net/1911/18742.
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Adenoviral vectors have great potential for use in gene therapy and genetic immunization. The targeting of Ad vectors to the relevant tissue and cell types in vivo could greatly improve their safety and performance by lowering the effective dosage required for therapeutic levels of gene expression. Redirection of Ad vector tropism will require physical modifications of the adenoviral capsid but direct genetic modification of the Ad capsid has so far been limited to small peptides.
A novel system for the attachment of targeting ligands to the Ad capsid, based on the extremely strong avidin-biotin interaction, is described herein. The genetic insertion of a biotin acceptor peptide (BAP) into the fiber, protein IX, or hexon components of the Ad capsid has resulted in vectors that are metabolically biotinylated upon production in host cells. Avidin-dependent redirection of transduction through a variety of biotinylated ligands is greatly dependent on the nature of the biotinylated capsid protein. While targeted transduction via the fiber was efficient through a broad array of ligand-receptor interactions, redirection of binding and uptake through the more abundant protein IX and hexon resulted in poor transduction. Although the basis of these differences has not been determined, it most likely reflects functional differences between the capsomeres during the process of vector uptake and trafficking. This study represents the first direct comparison of transduction through the various capsomeres and strongly suggests that future targeting efforts should be focused on fiber modification.
In addition to the functional studies on Ad-IX-BAP, structural analysis by cryoelectron microscopy and particle reconstruction is presented. The C-terminal BAP fusion was used as a structural tag to visualize the position of IX within the capsid. Results contradict all previous reports on the location of IX and suggest the surface accessible density currently assigned as IIIa is actually attributable to protein IX. These studies highlight the need for a more thorough analysis of adenoviral structure and the complex interactions between its components.
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Ke, Jin-He, and 柯錦和. "Design, synthesis and evaluation of cell uptake-favorable polycation for gene delivery and other biomedical applications." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/78358436799944799445.
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博士國立臺灣大學
高分子科學與工程學研究所
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Therapeutic nucleic acid delivery has been considered as a powerful strategy for treating gene-related diseases. Development of safe and efficient gene delivery vector is essential for clinical use in gene therapy. Polycations are the major type of the nonviral gene vectors widely investigated for gene delivery. The purpose of this dissertation attempts to develop an efficient with minimal toxicity polycationic gene vector for gene delivery. The first part of this dissertation gives a broad discussion of the current comprehension of the biological barriers and common discussed polycations in gene delivery. In the second part of this dissertation, a series of poly (N-substituent acrylamide)s (PAms) that differ in alkylamine side chain was synthesized via free radical polymerization. The PAms were designed to examine the effects of the methylene numbers (from two to twelve) in the alkylamine side chain on cytotoxicity, plasmid DNA (pDNA) binding affinity, cellular uptake efficiency and gene expression. The cytotoxicity of PAms evaluated in HEK293 cells using the MTT assay showed a trend of decreasing toxicity with the side chain length and the IC50 values of all PAms were lower than that of polyethylenimine (PEI) control. The primary amine-based polymers were able to efficiently condense pDNA to form complexes with size ranging from 100 to 350 nm. The gene transfection ability of PAms is dominantly determined by the specific side chain length that P8Am (with octylamine side chain) reveals higher gene expression than other PAms containing the same backbone structure. Although the gene transfection efficiency of PEI was better than all of PAms, PAms were found not to be uptake-limited. This was supported by the effect of chloroquine on transfection activity, based on the protease inhibition activity of chloroquine. Especially, complexes formed from P8Am displayed high uptake level relative to PEI, which was attributed to the proper structure of P8Am to compact pDNA to form stable nanoparticles under the heparin replacement assay. This offers the understanding to polymer structure that influences the transfection ability and gives useful information to develop efficient polymeric gene vector. In the third part of this dissertation, chemical modification was performed to give P8Am multi-functionalities to overcome the gene delivery barriers encountered during transfection. Hence, a novel cationic polymer was developed by conjugating imidazole and polyethylene glycol (PEG) on poly(N-(8-aminooctyl)acrylamide) (P8Am) to exhibit high gene expression with low cytotoxicity and the resistance against erythrocyte agglutination and serum inhibition. Cytotoxicity results indicated that these P8Am derivatives in varied substitutions were more of biocompatibility than unmodified P8Am and PEI control. Moreover, the particle size and zeta potential experiment demonstrated that they were capable of complexing pDNA into sub-micro (135 ~ 625 nm) and positive charge (+10 ~ +43 mV) particles, while high degree of substitution might impede their pDNA complexation ability that formed less positive and larger polyplexes. Flow cytometry analysis demonstrated the cellular uptake efficiency was depended on the degree of substitution; low degree of substitution would mediate high uptake efficiency. The gene transfection ability was evaluated by luciferase assay that revealed low substitution P8Am-IM11 (substituted with 11 mole % of imidazole moiety) and P8Am-PG7 (substituted with 7 mole % of PEG moiety) transfected cells more efficient than unmodified P8Am, respectively. Therefore, the multi-functional P8Am derivative, P8Am-IM11-PG7 – containing both imidazole and PEG, was developed according to the optimized contents. In the presence of serum, P8Am-IM11-PG7 polyplexes significantly enhanced the gene transfection efficiency relative to unmodified P8Am polyplexes. Moreover, it exhibited minimal cytotoxicity and the erythrocyte aggregation assay showed that P8Am-IM11-PG7 polyplexes revealed good blood compatibility as compared to P8Am polyplexes and PEI polyplexes. This indicated that by the efforts of chemical modification, P8Am-IM11-PG7 could possess required abilities to overcome the difficulties encountering in gene transfection. However, the chemical strategy seems to impede the cell-uptake favorable property of P8Am. In the fourth part of this dissertation, quaternary polyplexes were prepared by sequential addition of polycations (polyethylenimine (PEI) or poly (N-(8-aminooctyl)-acrylamide) (P8Am)) for loading pDNA into the core polyplexes and poly (acrylic acid) (PAA) for reversing charges to deposit additional polycation (PEI or P8Am) layer. It was found the cytotoxicity and cellular uptake expression of PEI core polyplexes could be improved by coating a cell uptake-favorable P8Am layer. Conversely, P8Am could not facilitate endosomal release through the proposed proton sponge effect so the PEI core was required for the P8Am-coated quaternary polyplexes to ensure efficient transfection. Consequently, an efficient and safe non-viral gene vehicle was constructed by layer-by-layer deposition, using alternate polyanion and polycation with required functionalities to overcome the obstacles met in the process of transfection. Maximum transfection activity with minimal toxicity was observed when the quaternary polyplex of pDNA/PEI/PAA/P8Am was prepared at a weight ratio of 1/1.5/3/5. Conversely, the same composition in different position such as the cell-favorable P8Am core was externally deposited with the endosome lytic moiety, PEI showed high toxicity and low efficiency. This indicates the pDNA/PEI/PAA/P8Am sequence for a quaternary polyplex is as important as the functional polymer selection for designing safe and reliable gene delivery vehicles. We demonstrate here that gene delivery efficiency may be improved by increasing the uptake level and the endosomal buffering release through an additional layer of cell uptake-favorable polycations associated with the core polycations possessing endosomal release ability. In the last part of the dissertation, achievements of each chapter in this dissertation were concluded, and some suggestions and prospection were provided according to the present findings.
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Sum, Chi Hong. "Optimized Production and Purification of LCC DNA Minivectors for Applications in Gene Therapy and Vaccine Development." Thesis, 2014. http://hdl.handle.net/10012/8232.
Full textAbstract:
Linear covalently closed (LCC) DNA minivectors serve to be superior to conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors due to enhancements to both transfection efficiency and safety. Specifically, LCC DNA minivectors have a heightened safety profile as insertional mutagenesis is inhibited by covalently closed terminal ends conferring double-strand breaks that cause chromosomal disruption and cell death in the low frequency event of chromosomal integration. The development of a one-step, E. coli based in vivo LCC DNA minivector production system enables facile and efficient production of LCC DNA minivectors referred to as DNA ministrings. This novel in vivo system demonstrates high versatility, generating DNA ministrings catered to numerous potential applications in gene therapy and vaccine development.
In the present study, numerous aspects pertaining to the generation of gene therapeutics with LCC DNA ministrings have been explored with relevance to both industry and clinical settings. Through systematic assessment of induction duration, cultivation strategy, and genetic/chemical modifications, the novel in vivo system was optimized to produce high yields of DNA ministrings at ~90% production efficiency. Purification of LCC DNA ministrings using anion exchange membrane chromatography demonstrated rapid, scalable purification of DNA vectors as well as its potential in the separation of different DNA isoforms. The application of a hydrogel-based strong Q-anion exchange membrane, with manipulations to salt gradient, constituted effective separation of parental supercoiled CCC precursor pDNA and LCC DNA. The resulting DNA ministrings were employed for the generation of 16-3-16 gemini surfactant based synthetic vectors and comparative analysis, through physical characterization and in vitro transfection assays, was conducted between DNA ministring derived and CCC pDNA derived lipoplexes. Differences in DNA topology were observed to induce differences in particle size and DNA protection/encapsulation upon lipoplex formation. Lastly, the in vivo DNA minivector production system successfully generated gagV3(BCE) LCC DNA ministrings for downstream development of a HIV DNA-VLP (Virus-like particle) vaccine, thus highlighting the capacity of such system to produce DNA ministrings with numerous potential applications.
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Liu, Yao-Chung, and 劉耀中. "Preparation of Fluorescent Silica Nanotubes and their Application in Gene Delivery." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/91528758601262070204.
Full textAbstract:
碩士國立臺灣師範大學
化學研究所
92
The silica nanotubes have a number of advantages that make them potential candidate for biological applications. Firstly, nanotubes have inner voids that can be filled with species ranging in size from large proteins to small molecules. Secondly, nanotubes have distinct inner and outer surfaces that can be functionalized easily. By using template method, a large number of nanotubes were synthesized and their sizes were controlled preciously. Silica nanotubes were synthesized through a sol-gel reaction using the anodic aluminum oxide membrane (AAO) as a template. The fluorescent silica nanotubes were prepared by dopping water-soluble nanocrystal CdSe(ZnS). The fluorescent silica nanotubes were used to monitor the localization of nanotubes in living cells and found to be localized in the cytoplasm. The fluorescent silica nanotubes carrying the GFP gene were employed to investigate the potential application of the nanotubes in gene delivery. The expression of GFP demonstrates a new biological application of nanotubes as a biomolecule carrier.