Tailoring Cell-Material Interactions via Poly(acrylic acid) Brushes to Enhance Nonviral Substrate-Mediated Gene Delivery

Nonviral gene delivery modifies gene expression by transferring exogenous genetic material into cells and tissues, typically through a bolus of complexes formed by electrostatic interactions between cationic lipid or polymer vectors with negatively charged nucleic acids (e.g. DNA). Although nonviral gene delivery is safer, more cost-effective, and more flexible compared to viral systems, nonviral transfection suffers from low efficiency due to extracellular and intracellular barriers. Much research has focused on tuning physiochemical properties of the complexing vectors to improve transfection, yet the cell-material interface may prove a better platform to immobilize DNA complexes for substrate-mediated delivery (SMD) and modulate the cellular response to improve transfection to overcome transfection barriers, especially in ex vivo or site-specific applications (e.g. biomedical implants). Natural and synthetic substrate modifications have both been investigated to improve transfection via SMD, but synthetic polymer films are often considered more reproducible and tunable compared to natural substrate modifications. While synthetic polymers films have been shown improve the efficacy of SMD (e.g. self-assembled monolayers or polyelectrolytes multilayers), these films have issues with degradation and impeded release of the DNA cargo and, moreover, are not typically studied in the context of clinically relevant metals (i.e. titanium (Ti)). In this dissertation, polymer films formed with pH-responsive poly(acrylic acid) (PAA) brushes were investigated to resolve these issues by grafting to a Ti substrate, immobilizing DNA complexes through electrostatic interactions with the PAA brushes, and modulating cellular response via conjugated adhesion moieties (i.e. RGD) and adsorbed free PEI. We showed our PAA-RGD platform increased transfection in cells cultured on PEI-DNA complexes immobilized to PAA-RGD compared to PAA alone. Investigations into further tuning the PEI vector and the RGD ligand showed that reduced cytotoxicity and increased proliferation, focal adhesion formation, and endocytic pathway activation may have improved our transfection success, suggesting that PAA-RGD brushes have the potential to immobilization of therapeutic DNA complexes for applications such as Ti biomedical devices, implantable sensors, and diagnostics tools. Advisor: Angela K. Pannie

Tailoring Cell-Material Interactions via Poly(acrylic acid) Brushes to Enhance Nonviral Substrate-Mediated Gene Delivery

Nonviral gene delivery modifies gene expression by transferring exogenous genetic material into cells and tissues, typically through a bolus of complexes formed by electrostatic interactions between cationic lipid or polymer vectors with negatively charged nucleic acids (e.g. DNA). Although nonviral gene delivery is safer, more cost-effective, and more flexible compared to viral systems, nonviral transfection suffers from low efficiency due to extracellular and intracellular barriers. Much research has focused on tuning physiochemical properties of the complexing vectors to improve transfection, yet the cell-material interface may prove a better platform to immobilize DNA complexes for substrate-mediated delivery (SMD) and modulate the cellular response to improve transfection to overcome transfection barriers, especially in ex vivo or site-specific applications (e.g. biomedical implants). Natural and synthetic substrate modifications have both been investigated to improve transfection via SMD, but synthetic polymer films are often considered more reproducible and tunable compared to natural substrate modifications. While synthetic polymers films have been shown improve the efficacy of SMD (e.g. self-assembled monolayers or polyelectrolytes multilayers), these films have issues with degradation and impeded release of the DNA cargo and, moreover, are not typically studied in the context of clinically relevant metals (i.e. titanium (Ti)). In this dissertation, polymer films formed with pH-responsive poly(acrylic acid) (PAA) brushes were investigated to resolve these issues by grafting to a Ti substrate, immobilizing DNA complexes through electrostatic interactions with the PAA brushes, and modulating cellular response via conjugated adhesion moieties (i.e. RGD) and adsorbed free PEI. We showed our PAA-RGD platform increased transfection in cells cultured on PEI-DNA complexes immobilized to PAA-RGD compared to PAA alone. Investigations into further tuning the PEI vector and the RGD ligand showed that reduced cytotoxicity and increased proliferation, focal adhesion formation, and endocytic pathway activation may have improved our transfection success, suggesting that PAA-RGD brushes have the potential to immobilization of therapeutic DNA complexes for applications such as Ti biomedical devices, implantable sensors, and diagnostics tools. Advisor: Angela K. Pannie

Biomaterial substrate modifications that influence cell-material interactions to prime cellular responses to nonviral gene delivery

Gene delivery is the transfer of exogenous genetic material into somatic cells to modify their gene expression, with applications including tissue engineering, regenerative medicine, sensors and diagnostics, and gene therapy. Viral vectors are considered the most effective system to deliver nucleic acids, yet safety concerns and many other disadvantages have resulted in investigations into an alternative option, i.e. nonviral gene delivery. Chemical nonviral gene delivery is typically accomplished by electrostatically complexing cationic lipids or polymers with negatively charged nucleic acids. Unfortunately, nonviral gene delivery suffers from low efficiency due to barriers that impede transfection success, including intracellular processes such as internalization, endosomal escape, cytosolic trafficking, and nuclear entry. Efforts to improve nonviral gene delivery have focused on modifying nonviral vectors, yet a novel solution that may prove more effective than vector modifications is stimulating or “priming” cells before transfection to modulate and mitigate the cellular response to nonviral gene delivery. In applications where a cell-material interface exists, cell priming can come from cues from the substrate, through chemical modifications such as the addition of natural coatings, ligands, or functional side groups, and/or physical modifications such as topography or stiffness, to mimic extracellular matrix cues and modulate cellular behaviors that influence transfection efficiency. This review summarizes how biomaterial substrate modifications can prime the cellular response to nonviral gene delivery (e.g. integrin binding and focal adhesion formation, cytoskeletal remodeling, endocytic mechanisms, intracellular trafficking) to aid in improving gene delivery for future therapeutic applications

Free polyethylenimine enhances substrate-mediated gene delivery on titanium substrates modified with RGD-functionalized poly(acrylic acid) brushes

Substrate mediated gene delivery (SMD) is a method of immobilizing DNA complexes to a substrate via covalent attachment or nonspecific adsorption, which allows for increased transgene expression with less DNA compared to traditional bolus delivery. It may also increase cells receptivity to transfection via cell-material interactions. Substrate modifications with poly(acrylic) acid (PAA) brushes may improve SMD by enhancing substrate interactions with DNA complexes via tailored surface chemistry and increasing cellular adhesion via moieties covalently bound to the brushes. Previously, we described a simple method to graft PAA brushes to Ti and further demonstrated conjugation of cell adhesion peptides (i.e., RGD) to the PAA brushes to improve biocompatibility. The objective of this work was to investigate the ability of Ti substrates modified with PAA-RGD brushes (PAA-RGD) to immobilize complexes composed of branched polyethyleneimine and DNA plasmids (bPEI-DNA) and support SMD in NIH/3T3 fibroblasts. Transfection in NIH/3T3 cells cultured on bPEI-DNA complexes immobilized onto PAA-RGD substrates was measured and compared to transfection in cells cultured on control surfaces with immobilized complexes including Flat Ti, PAA brushes modified with a control peptide (RGE), and unmodified PAA. Transfection was two-fold higher in cells cultured on PAA-RGD compared to those cultured on all control substrates. While DNA immobilization measured with radiolabeled DNA indicated that all substrates (PAA-RGD, unmodified PAA, Flat Ti) contained nearly equivalent amounts of loaded DNA, ellipsometric measurements showed that more total mass (i.e., DNA and bPEI, both complexed and free) was immobilized to PAA and PAA-RGD compared to Flat Ti. The increase in adsorbed mass may be attributed to free bPEI, which has been shown to improve transfection. Further transfection investigations showed that removing free bPEI from the immobilized complexes decreased SMD transfection and negated any differences in transfection success between cells cultured on PAA-RGD and on control substrates, suggesting that free bPEI may be beneficial for SMD in cells cultured on bPEI-DNA complexes immobilized on PAA-RGD grafted to Ti. This work demonstrates that substrate modification with PAA-RGD is a feasible method to enhance SMD outcomes on Ti and may be used for future applications such as tissue engineering, gene therapy, and diagnostics. © 2019 Mantz, Rosenthal, Farris, Kozisek, Bittrich, Nazari, Schubert, Schubert, Stamm, Uhlmann and Pannier

Free Polyethylenimine Enhances Substrate-Mediated Gene Delivery on Titanium Substrates Modified With RGD-Functionalized Poly(acrylic acid) Brushes

Substrate mediated gene delivery (SMD) is a method of immobilizing DNA complexes to a substrate via covalent attachment or nonspecific adsorption, which allows for increased transgene expression with less DNA compared to traditional bolus delivery. It may also increase cells receptivity to transfection via cell-material interactions. Substrate modifications with poly(acrylic) acid (PAA) brushes may improve SMD by enhancing substrate interactions with DNA complexes via tailored surface chemistry and increasing cellular adhesion via moieties covalently bound to the brushes. Previously, we described a simple method to graft PAA brushes to Ti and further demonstrated conjugation of cell adhesion peptides (i.e., RGD) to the PAA brushes to improve biocompatibility. The objective of this work was to investigate the ability of Ti substrates modified with PAA-RGD brushes (PAA-RGD) to immobilize complexes composed of branched polyethyleneimine and DNA plasmids (bPEI-DNA) and support SMD in NIH/3T3 fibroblasts. Transfection in NIH/3T3 cells cultured on bPEI-DNA complexes immobilized onto PAA-RGD substrates was measured and compared to transfection in cells cultured on control surfaces with immobilized complexes including Flat Ti, PAA brushes modified with a control peptide (RGE), and unmodified PAA. Transfection was two-fold higher in cells cultured on PAA-RGD compared to those cultured on all control substrates. While DNA immobilization measured with radiolabeled DNA indicated that all substrates (PAA-RGD, unmodified PAA, Flat Ti) contained nearly equivalent amounts of loaded DNA, ellipsometric measurements showed that more total mass (i.e., DNA and bPEI, both complexed and free) was immobilized to PAA and PAA-RGD compared to Flat Ti. The increase in adsorbed mass may be attributed to free bPEI, which has been shown to improve transfection. Further transfection investigations showed that removing free bPEI from the immobilized complexes decreased SMD transfection and negated any differences in transfection success between cells cultured on PAA-RGD and on control substrates, suggesting that free bPEI may be beneficial for SMD in cells cultured on bPEI-DNA complexes immobilized on PAA-RGD grafted to Ti. This work demonstrates that substrate modification with PAA-RGD is a feasible method to enhance SMD outcomes on Ti and may be used for future applications such as tissue engineering, gene therapy, and diagnostics

CMB-S4: Forecasting Constraints on Primordial Gravitational Waves

CMB-S4---the next-generation ground-based cosmic microwave background (CMB) experiment---is set to significantly advance the sensitivity of CMB measurements and enhance our understanding of the origin and evolution of the Universe, from the highest energies at the dawn of time through the growth of structure to the present day. Among the science cases pursued with CMB-S4, the quest for detecting primordial gravitational waves is a central driver of the experimental design. This work details the development of a forecasting framework that includes a power-spectrum-based semi-analytic projection tool, targeted explicitly towards optimizing constraints on the tensor-to-scalar ratio, $r$, in the presence of Galactic foregrounds and gravitational lensing of the CMB. This framework is unique in its direct use of information from the achieved performance of current Stage 2--3 CMB experiments to robustly forecast the science reach of upcoming CMB-polarization endeavors. The methodology allows for rapid iteration over experimental configurations and offers a flexible way to optimize the design of future experiments given a desired scientific goal. To form a closed-loop process, we couple this semi-analytic tool with map-based validation studies, which allow for the injection of additional complexity and verification of our forecasts with several independent analysis methods. We document multiple rounds of forecasts for CMB-S4 using this process and the resulting establishment of the current reference design of the primordial gravitational-wave component of the Stage-4 experiment, optimized to achieve our science goals of detecting primordial gravitational waves for $r > 0.003$ at greater than $5\sigma$, or, in the absence of a detection, of reaching an upper limit of $r < 0.001$ at $95\%$ CL.Comment: 24 pages, 8 figures, 9 tables, submitted to ApJ. arXiv admin note: text overlap with arXiv:1907.0447

CMB-S4

We describe the stage 4 cosmic microwave background ground-based experiment CMB-S4

CMB-S4: Forecasting Constraints on Primordial Gravitational Waves

Abstract: CMB-S4—the next-generation ground-based cosmic microwave background (CMB) experiment—is set to significantly advance the sensitivity of CMB measurements and enhance our understanding of the origin and evolution of the universe. Among the science cases pursued with CMB-S4, the quest for detecting primordial gravitational waves is a central driver of the experimental design. This work details the development of a forecasting framework that includes a power-spectrum-based semianalytic projection tool, targeted explicitly toward optimizing constraints on the tensor-to-scalar ratio, r, in the presence of Galactic foregrounds and gravitational lensing of the CMB. This framework is unique in its direct use of information from the achieved performance of current Stage 2–3 CMB experiments to robustly forecast the science reach of upcoming CMB-polarization endeavors. The methodology allows for rapid iteration over experimental configurations and offers a flexible way to optimize the design of future experiments, given a desired scientific goal. To form a closed-loop process, we couple this semianalytic tool with map-based validation studies, which allow for the injection of additional complexity and verification of our forecasts with several independent analysis methods. We document multiple rounds of forecasts for CMB-S4 using this process and the resulting establishment of the current reference design of the primordial gravitational-wave component of the Stage-4 experiment, optimized to achieve our science goals of detecting primordial gravitational waves for r > 0.003 at greater than 5σ, or in the absence of a detection, of reaching an upper limit of r < 0.001 at 95% CL

Tailoring Cell-Material Interactions via Poly(acrylic) Acid Brushes to Enhance Nonviral Substrate-Mediated Gene Delivery

Nonviral gene delivery modifies gene expression by transferring exogenous genetic material into cells and tissues, typically through a bolus of complexes formed by electrostatic interactions between cationic lipid or polymer vectors with negatively charged nucleic acids (e.g. DNA). Although nonviral gene delivery is safer, more cost-effective, and more flexible compared to viral systems, nonviral transfection suffers from low efficiency due to extracellular and intracellular barriers. Much research has focused on tuning physiochemical properties of the complexing vectors to improve transfection, yet the cell-material interface may prove a better platform to immobilize DNA complexes for substrate mediated delivery (SMD) and modulate the cellular response to improve transfection to overcome transfection barriers, especially in ex vivo or site-specific applications (e.g biomedical implants). Natural and synthetic substrate modifications have both been investigated to improve transfection via SMD, but synthetic polymer films are often considered more reproducible and tunable compared to natural substrate modifications. While synthetic polymers films have been shown improve the efficacy of SMD (e.g. self-assembled monolayers or polyelectrolytes multilayers), these films have issues with degradation and impeded release of the DNA cargo and, moreover, are not typically studied in the context of clinically relevant metals (i.e. titanium (Ti)). In this dissertation, polymer films formed with pH-responsive poly(acrylic acid) (PAA) brushes were investigated to resolve these issues by grafting to a Ti substrate, immobilizing DNA complexes through electrostatic interactions with the PAA brushes, and modulating cellular response via conjugated adhesion moieties (i.e. RGD) and adsorbed free PEI. We showed our PAA-RGD platform increased transfection in cells cultured on PEI-DNA complexes immobilized to PAA-RGD compared to PAA alone. Investigations into further tuning the PEI vector and the RGD ligand showed that reduced cytotoxicity and increased proliferation, focal adhesion formation, and endocytic pathway activation may have improved our transfection success, suggesting that PAA-RGD brushes have the potential to immobilization of therapeutic DNA complexes for applications such as Ti biomedical devices, implantable sensors, and diagnostics tools

Tailoring Cell-Material Interactions via Poly(acrylic) Acid Brushes to Enhance Nonviral Substrate-Mediated Gene Delivery

Nonviral gene delivery modifies gene expression by transferring exogenous genetic material into cells and tissues, typically through a bolus of complexes formed by electrostatic interactions between cationic lipid or polymer vectors with negatively charged nucleic acids (e.g. DNA). Although nonviral gene delivery is safer, more cost-effective, and more flexible compared to viral systems, nonviral transfection suffers from low efficiency due to extracellular and intracellular barriers. Much research has focused on tuning physiochemical properties of the complexing vectors to improve transfection, yet the cell-material interface may prove a better platform to immobilize DNA complexes for substrate mediated delivery (SMD) and modulate the cellular response to improve transfection to overcome transfection barriers, especially in ex vivo or site-specific applications (e.g biomedical implants). Natural and synthetic substrate modifications have both been investigated to improve transfection via SMD, but synthetic polymer films are often considered more reproducible and tunable compared to natural substrate modifications. While synthetic polymers films have been shown improve the efficacy of SMD (e.g. self-assembled monolayers or polyelectrolytes multilayers), these films have issues with degradation and impeded release of the DNA cargo and, moreover, are not typically studied in the context of clinically relevant metals (i.e. titanium (Ti)). In this dissertation, polymer films formed with pH-responsive poly(acrylic acid) (PAA) brushes were investigated to resolve these issues by grafting to a Ti substrate, immobilizing DNA complexes through electrostatic interactions with the PAA brushes, and modulating cellular response via conjugated adhesion moieties (i.e. RGD) and adsorbed free PEI. We showed our PAA-RGD platform increased transfection in cells cultured on PEI-DNA complexes immobilized to PAA-RGD compared to PAA alone. Investigations into further tuning the PEI vector and the RGD ligand showed that reduced cytotoxicity and increased proliferation, focal adhesion formation, and endocytic pathway activation may have improved our transfection success, suggesting that PAA-RGD brushes have the potential to immobilization of therapeutic DNA complexes for applications such as Ti biomedical devices, implantable sensors, and diagnostics tools