Browsing by Subject "Polyplex"

Now showing 1 - 5 of 5
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    Complexation Between DNA and Hydrophilic-Cationic Diblock Copolymers
    (2017-08) Jung, Seyoung
    DNA-polycation complexes (polyplexes) are gene delivery vehicles that offer a number of advantages over viral vehicles: high genetic payload capacity, low toxicity, and low cost. Among the many polycation architectures, hydrophilic-cationic diblock copolymers are particularly promising due to their versatility. Compared to the emphasis on biological evaluation of polyplexes, however, in-depth understanding of the polyplex formation process (and the parameters involved) is rather sparse. Thus, this thesis discusses the physics of polyplex formation. First, a collection of solution-state techniques are employed to elucidate the effects of diblock composition, ionic strength, DNA morphology, and pH on DNA-diblock binding. Specifically, the binding thermodynamics and strength - measured using isothermal calorimetry and circular dichroism spectroscopy, respectively - turn out to show a strong dependence on the cationic content in the diblock, leading to wide ranges of polyplex size, dispersity, and stability. Second, the effects of hydrophilic block structure, charge density, and polyplex formation route are investigated. It is found that bulkier hydrophilic blocks translate to larger polyplexes when DNA is in excess and that hydrophilic block solubility controls polyplex stability when the diblock is in excess. With fixed material and concentration, different polyplex sizes and dispersities can be achieved by varying solution ionic strength or DNA-polycation mixing route. To this end, the analysis of polyplex formation presented in this thesis provides original insights into physical variables that can be adjusted to tune the DNA-polycation binding behavior and resulting polyplex properties, which can aid in the guided development of polymeric DNA delivery systems.
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    Elucidating Glycopolycation Structure-Function Relationships For Improved Gene Therapy
    (2017-06) Phillips, Haley
    The gene therapy field is devoted to treating disease by adding, altering, or inhibiting gene expression. This type of therapy holds great promise for the treatment and even cure of monogenic diseases such as cystic fibrosis, Duchenne muscular dystrophy, hemophilia A and B, and epidermolysis bullosa. To produce therapeutic effect, nucleic acids must be delivered and expressed in cells of interest. Deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) in many forms can be delivered using viral or non-viral vehicles. Viral vectors provide efficient DNA delivery; however, packaging limitations and occasional safety issues such as immune responses are major issues. In contrast, non-viral vectors are cheaper and easier to mass produce and can package any length of nucleic acid; however, non-viral vectors struggle to deliver genetic cargo at therapeutically beneficial levels. Polymers with the ability to condense and protect genetic material make promising non-viral vectors. They are relatively easy to produce compared to viral vehicles, can safely package various plasmid sizes, and have shown significant uptake in a wide variety of human cell lines. Cationic polymers complex with the negatively charged phosphodiester backbone of DNA or RNA, forming inter-polyelectrolyte complexes termed polyplexes. Herein, we explore using experiments in vitro, ex vivo, and in vivo to probe the structure-function relationships dictating polyplex gene delivery and other glycomaterial applications.
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    Improving polymer-mediated DNA vaccine delivery.
    (2011-06) Palumbo, Rebecca Noelle
    Vaccination using antigen-encoding plasmid DNA has great potential to generate strong immune response against delivered antigen. In order to effectively generate immune response, antigen must be delivered to antigen presenting cells, primarily dendritic cells (DCs). Using cationic polymers as a delivery vehicle can provide many advantages, including protection of DNA from degradation, ability to add targeting moieties, and easy modification of structure to optimize various properties. We have investigated the use of polyplexes as a DNA delivery vehicle in a variety of settings. We demonstrated the feasibility of using the CD40L as a DC targeting moiety, a protein capable of both binding and stimulating DC maturation, using coated nanoparticles. We have also studied the possibility of delivering antigen through transfection of bystander cells rather than direct expression by DCs using an in vitro model. We confirmed the ability of these DCs to present antigen, become mature, and stimulate T cells. Finally, we studied the interaction of cationic polymer complexes in vivo, both in respect to local tissue dispersion and interaction with specific cell types, using fluorescently labeled DNA. Through these experiments we have illuminated potential pathways for optimizing DNA vaccine efficiency using polymer complexes with slightly different structures.
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    Quinine Copolymer Reporters For Enhanced Gene Editing And Raman Imaging
    (2022-01) Van Bruggen, Craig
    After decades of development, gene therapy has finally reached the forefront of medicine and has led to new cures for genetic disorders and the development of life-saving vaccines. The field has been buoyed by the development of more precise and user-friendly targeted nucleases, such as those used for clustered regularly interspersed palindromic repeats (CRISPR)-based editing. These useful gene-editing technologies, however, are still stymied by the challenge of delivering exogenous nucleic acids and proteins into the cells of interest. The emerging gene therapy industry is investing heavily in developing more efficient and safe non-viral vehicles as alternatives to costly and immunogenic viral vectors. Cationic polymers are promising non-viral vectors due to their manufacturing scalability, their chemical stability, and their synthetic tunability. Improvements in delivery efficiency are necessary, however, for widespread adoption of polymeric vehicles for gene therapy. One challenge in improving performance, however, is the difficulty and limited methodology for elucidating the intracellular mechanics of polymeric vehicles. In this thesis, I describe my research focused on the development of a novel quinine-containing polymer, called a Quinine Copolymer Reporter (QCR), that enhanced transient transfections of cultured cells with plasmids and improved gene editing of cultured cells through the simultaneous delivery of the CRISPR-associated protein Cas9 and DNA donor template. In addition, I describe collaborative research performed with colleagues in the research group of Prof. Renee Frontiera that characterized a band in quinine’s Raman spectrum that is diagnostic of its chemical environment. Using this chemical sensitivity in conjunction with Raman microscopic imaging, we help elucidated the intracellular unpackaging mechanisms of the QCR-nucleic acid complexes.
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    Synthesis and Characterization of Polycations with Various Structural Features for Nucleic Acid Delivery
    (2014-07) Wu, Yaoying
    Cationic polymers have been widely explored as non-viral nucleic acid delivery vectors for gene therapy, as they are able to complex with nucleic acids, protect genetic materials from degradation, and facilitate the internalization of transgene expression process. To understand how various structural elements impact the nucleic acid delivery, several classes of polycations were synthesized and examined for their in vitro transfection performance. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed for the synthesis of several series of diblock glycopolycations with different carbohydrate containing blocks, including poly(2-deoxy-2-methacrylamido glucopyranose) (PMAG) and poly(methacrylamidotrehalose) (PMAT). Amine containing monomers were subsequently polymerized through chain extension to yield cationic blocks for nucleic acid binding, including N-[3-(N, N-dimethylamino) propyl] methacrylamide (DMAPMA), N-(2-aminoethyl) methacrylamide (AEMA), aminoethylmethacrylate (AEMT), N-methyl aminoethylmethacrylate (MAEMT), N,N-dimethyl aminoethylmethacrylate (DMAEMT), and N,N,N-trimethylammoniumethylmethacrylate (TMAEMT). Initially, it was demonstrated that these polymers were all able to complex plasmid DNA into polyplex structures and prevent colloidal aggregation of polyplexes in physiological salt conditions. More importantly, glycopolymers with PMAT block can prevent polyplexes from aggregation, and exhibit the ability to protect polyplexes through the cycle of lyophilization and reconstitution. The role of charge type, block length, and cell type on transfection efficiency and toxicity were studied for the in vitro transfection in both HeLa (human cervix adenocarcinoma) and HepG2 (human liver hepatocellular carcinoma) cells by comparing the polyplexes formulation created with PMAG-b-PAEMA and PMAG-b-PDMAPMA. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased, polyplex toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Cell type played a major role in efficiency, these glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend signifying that the MAG block could aid in hepatocyte transfection. Lastly, a new class of lipophilic polycations were designed, poly(alkylamidoamine) (PAAA) was synthesized to understand the role of lyophilicity in pDNA transfection. PAAAs with various length of lipophilic linker (C3 to C6) were synthesized via step-growth polymerization, and examined for their in-vitro transfection efficiency and cytotoxicity in multiple cell types, including HDFa (human dermal fibroblasts, adult) cells, HeLa (human cervix adenocarcinoma) cells, HMEC (human mammary epithelial cells), and HUVEC (human umbilical vein endothelial cells). The PAAA vehicles exhibited comparable or even superior transfection efficiency to Lipofectamine 2000, a leading lipid-based transfection reagent. It was revealed that, for the PAAA polymers examined in this study, an increase in lipophilicity leads to higher cytotoxicity, but also raised the transfection efficiency in HeLa cells. Overall, we demonstrated the great potential of carbohydrate containing polymer block, which has potential to serve as a targeting moiety, stealthy coating, and even lyo-protectant for polyplex formulations. Different amine types were examined for their nucleic acid delivery ability, and we conclude that tertiary amine containing monomer, DMAPMA, exhibits high toxicity along with low transfection efficiency while primary amine block shows relatively lower toxicity and higher transfection efficiency. Finally, the lipophilic polycations, PAAAs, were shown to be important for improving transfection efficiency in multiple cell types.