Browsing by Subject "Biomaterials"
- Results Per Page
- Sort Options
Item Open Access A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies(2015) Brown Peters, Erica ChoThe ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.
Item Embargo Adenosine Delivery to Mitigate Bone Disorders(2023) Newman, HunterBone is a dynamic tissue which continuously undergoes remodeling primarily through osteoblast-mediated bone formation and osteoclast-mediated bone resorption. This balance is vital in maintaining both bone homeostasis and bone regeneration. With the increase in the global elderly population, the two most prominent bone disorders of fracture and osteoporosis pose a tremendous burden to the healthcare system. While these bone disorders are increasing in prevalence, treatment options remain stagnant, demonstrating the unmet need for new clinical solutions. Strategies that induce innate repair yet eliminate the need for expensive cellular or recombinant protein-based therapies are appealing. Adenosine, a naturally occurring nucleoside, has emerged as a part of key metabolic pathway that regulates bone tissue formation, function, and homeostasis. In this dissertation, I investigate the therapeutic potential of adenosine delivery to mitigate bone disorders. Despite the regenerative capacity of bone, age-associated changes result in injuries that suffer delayed healing. Therapeutic interventions that circumvent the age-associated impairments in bone tissue and promote healing are attractive options for geriatric fracture repair. Herein, I examined the changes in extracellular adenosine signaling with aging and the potential of local delivery of adenosine to promote fracture healing in aged mice. My results showed a concomitant reduction of CD73 expression in the bone and marrow of aged mice. Local delivery of adenosine using injectable microgel building blocks and drug carriers yielded a pro-regenerative environment and promoted fracture healing in aged mice. This study provides new understandings of age-related physiological changes in adenosine levels and demonstrates the therapeutic potential of local delivery of adenosine at the fracture site to circumvent the impaired healing capacity of aged fractures. Given the multi-functionality of adenosine signaling, it is possible that extracellular adenosine delivery influences various phases of bone healing. Towards this, I examined the potential immunomodulatory effect of adenosine delivery on both the local and systemic immune system for fracture repair. My results indicated that the immune cell populations of neutrophils and macrophages did not change with adenosine treatment in the fractured callus at either 3-, 7-, or 14-days post fracture. Additionally in the peripheral blood, CD8+ and CD4+ T cell populations did not change at any of the timepoints following adenosine treatment. This study provides potential insight into the role of exogenous adenosine in the inflammatory stage of fracture healing in young animals. Aging not only poses a risk for delayed fracture healing, but also for the development of osteoporosis. Osteoporosis results in bone fragility and subsequently a higher risk for fracture incidence. This disease is characterized by an imbalance in the coupled bone remodeling process with enhanced osteoclastic activity can lead to excessive bone resorption, resulting in bone thinning. Once activated, osteoclasts bind to the bone surface and acidify the local niche. This acidic environment could serve as a potential trigger for the delivery of therapeutic agents into the osteoporotic bone tissue. To this end, I developed a pH-responsive nanocarrier-based drug delivery system that binds to the bone tissue and delivers the osteoanabolic molecule, adenosine. Adenosine is incorporated into a hyaluronic acid (HA)-based nanocarrier through a pH-sensitive ketal group. The HA-nanocarrier was further functionalized with alendronate moieties to improve binding to the bone tissues. Systemic administration of the nanocarrier containing adenosine attenuated bone loss in an ovariectomized mice model of osteoporosis and showed comparable bone qualities to that of healthy mice. Delivery of osteoanabolic small molecules, such as adenosine, that can contribute to bone formation and inhibit excessive osteoclast activity by leveraging the tissue-specific milieu could serve as viable therapeutics for osteoporosis. Overall, this dissertation offers novel findings regarding adenosine as a therapeutic to treat both fractures and osteoporosis. These findings, along with the biomaterial delivery systems developed, further advance the potential of using adenosine as a therapeutic molecule to treat bone disorders.
Item Open Access Affinity-Modulation Drug Delivery Using Thermosensitive Elastin-Like Polypeptide Block Copolymers(2010) Simnick, Andrew JosephAntivascular targeting is a promising strategy for tumor therapy. This strategy overcomes many of the transport barriers and has shown efficacy in many preclinical models, but targeting epitopes on tumor vasculature can also promote accumulation in healthy tissues. We used Elastin-like Polypeptide (ELP) to form block copolymers (BCs) consisting of two separate ELP blocks seamlessly fused at the genetic level. ELPBCs self-assemble into spherical micelles at a critical micelle temperature (CMT), allowing external control over monovalent unimer and multivalent micelle forms. We hypothesized that thermal self-assembly could trigger specific binding of ligand-ELPBC to target receptors via the multivalency effect as a method to spatially restrict high-avidity interactions. We termed this approach Dynamic Affinity Modulation (DAM). The objectives of this study were to design, identify, and evaluate protein-based drug carriers that specifically bind to target receptors through static or dynamic multivalent ligand presentation.
ELPBCs were modified to include a low-affinity GRGDS or GNGRG ligand and a unique conjugation site for hydrophobic compounds. This addition did not disrupt micelle self-assembly and facilitated thermally-controlled multivalency. The ability of ligand-ELPBC to specifically interact with isolated AvB3 or CD13 was tested using an in vitro binding assay incorporating an engineered cell line. RGD-ELPBC promoted specific receptor binding in response to multivalent presentation but NGR-ELPBC did not. Enhanced binding with multivalent presentation was also observed only with constructs exhibiting CMT < body temperature. This study establishes proof-of-principle of DAM, but ELPBC requires thermal optimization for use with applied hyperthermia. Static affinity targeting of fluorescent ligand-ELPBC was then analyzed in vivo using intravital microscopy (IM), immunohistochemistry (IHC), and custom image processing algorithms. IM showed increased accumulation of NGR-ELPBC in tumor tissue relative to normal tissue while RGD-ELPBC and non-ligand ELPBC did not, and IHC verified these observations. This study shows (1) multivalent NGR presentation is suitable for static multivalent targeting of tumors and tumor vasculature, (2) multivalent RGD presentation may be suitable for DAM with thermal optimization, and (3) ELPBC micelles may selectively target proteins at the tumor margin.
Item Open Access Bioengineered Approaches to Prevent Hypertrophic Scar Contraction(2016) Lorden, Elizabeth RBurn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.
In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.
In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.
In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.
In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.
Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.
Item Open Access Bioengineering Microporous Annealed Particle Scaffolds to Recruit Neural Progenitor Stem Cells and Promote Angiogenesis in the Stroke Core(2022) Wilson, KatrinaThere remains a significant gap in the need for regenerative therapies for stroke compared to what is currently available. An ideal therapy would be one that stimulates the formation of new tissue with the ability to regain any function previously lost due to stroke. Therefore, methods exploiting the plasticity of the brain and modulating endogenous cellular responses to promote repair in the stroke core after ischemia have become highly attractive. However, this process of neural regeneration is complex and requires a series of controlled biological events, such as recruitment and differentiation of neuron progenitor cells (NPC’s), angiogenesis, and axonogenesis. Biomaterials are now commonly used to research tissue regeneration and cellular mechanisms, both in vitro and in vivo. We have designed a biocompatible biomaterial from macroporous annealed particles (MAP) hydrogels for injection into the stroke core five days after a photothrombotic stroke. Our hyaluronic acid-based material has been modified to dictate NPC fate in vitro through maintained stemness and the formation of neurospheres or towards Tuj1 positive NPCs, as well as enhance angiogenesis and the recruitment of endogenous NPCs after stroke. We have observed the first case of significant angiogenesis throughout the entire stroke core within only 10 days after injection, or 15 days post stroke. As well as significant increase in Tuj1+ and Nestin+ cells.
Item Open Access Biomimetic Composite Scaffolds for the Functional Tissue Engineering of Articular Cartilage(2009) Moutos, Franklin ThomasArticular cartilage is the connective tissue that lines the ends of long bones in diarthrodial joints, providing a low-friction load-bearing surface that can withstand a lifetime of loading cycles under normal conditions. Despite these unique and advantageous properties, the tissue possesses a limited capacity for self-repair due to its lack of vasculature and innervation. Total joint replacement is a well-established treatment for degenerative joint disease; however, the materials used in these procedures have a limited lifespan in vivo and will likely fail over time, requiring additional - and increasingly complicated - revision surgeries. For younger or more active patients, this risk is unacceptable. Unfortunately, alternative surgical options are not currently available, leaving pain management as the only viable treatment. In seeking to discover a new therapeutic strategy, the goal of this dissertation was to develop a functional tissue-engineered cartilage construct that may be used to resurface an entire diseased or damaged joint.
A three-dimensional (3-D) woven textile structure, produced on a custom-built miniature weaving loom, was utilized as the basis for producing novel composite scaffolds and cartilage tissue constructs that exhibited initial properties similar to those of native articular cartilage. Using polyglycolic acid (PGA) fibers combined with chondrocyte-loaded agarose or fibrin hydrogels, scaffolds were engineered with anisotropic, inhomogeneous, viscoelastic, and nonlinear characteristics prior to cultivation. However, PGA-based constructs showed a rapid loss of mechanical functionality over a 28 day culture period suggesting that the inclusion of other, less degradable, biomaterial fibers could provide more stable properties.
Retaining the original 3-D architecture and fiber/hydrogel composite construction, poly (epsilon-caprolactone) (PCL)-based scaffolds demonstrated initial biomechanical properties similar to those of PGA-based scaffolds. Long-term culture of 3-D PCL/fibrin scaffolds seeded with human adipose-derived stem cells (ASCs) showed that scaffolds maintained their baseline properties as new, collagen-rich tissue accumulated within the constructs.
In an attempt to improve the bioactivity of the PCL scaffold and further induce chondrogenic differentiation of seeded ASCs, we produced a hybrid scaffold system by embedding the 3-D woven structure within a porous matrix derived from native cartilage. We then demonstrated how this multifunctional scaffold could be molded, seeded, and cultured in order to produce an anatomically accurate tissue construct with potential for resurfacing the femoral head of a hip.
In summary, these findings provide valuable insight into a new approach for the functional tissue engineering of articular cartilage. The results of this work will hopefully lead to the discovery of new strategies for the long-term treatment of cartilage pathology.
Item Open Access Design of Biomaterials toward Endogenous Skeletal Tissue Repair(2020) Zeng, YuzeRepair of skeletal tissues remains a significant challenge in patient care as there is high incidence of impaired fracture healing as well as irreversible cartilage degeneration following joint injury. To improve the repair outcome, recent advancements have been made in regenerative medicine involving administration of tissue-specific growth factors and transplantation of stem cells. While they have achieved some success, their broad clinical application is hindered by various challenges, notably high costs and safety concerns. Alternatively, strategies that enable innate repair mechanisms without cell or protein products may hold great potential for tissue repair. In this dissertation, I explore biomaterials that are low-risk, cost-effective, and capable of leveraging endogenous healing mechanisms to promote skeletal tissue health.
Adenosine, a nucleoside ubiquitously present in the human body, is a potent pro-healing small molecule. A surge in adenosine secretion ensuing from injury is integral to the natural repair mechanisms. There is growing evidence that harnessing adenosine signaling can be a powerful therapeutic strategy. However, the needed abundance of adenosine often does not persist throughout the healing process due to the fast clearance or imbalanced bone homeostasis. Herein, I describe a synthetic biomaterial containing boronate molecules that sequesters adenosine reversibly and sustains the pro-regenerative signaling locally at the injury site. I demonstrate that implantation of the biomaterial post-fracture establishes an in-situ stockpile of adenosine, resulting in accelerated healing by promoting both osteoblastogenesis and angiogenesis. This biomaterial-assisted approach can leverage the transient increase in extracellular adenosine following injury to present adenosine to cells in a temporal manner. In addition to sequestering endogenous adenosine, the biomaterial is able to deliver exogenous adenosine to the site of injury, offering a versatile solution to utilizing adenosine as a potential therapeutic for tissue repair. Given the wide distribution of adenosine in the body, this biomaterial system can have a significant impact on a wide range of diseases by modulating local adenosine signaling, thus advancing its clinical applications beyond bone health.
Hyaluronic acid is a key component in synovial fluid that protects cartilage and facilitates painless motion. Loss of hyaluronic acid after joint trauma disrupts the native protection mechanism and contributes to the deterioration of cartilage and subsequent osteoarthritis. Although replenishing native hyaluronic acid with viscosupplementation is commonly used in clinics, its therapeutic efficacy is largely inconsistent at least in part due to the short joint retention. To enhance the longevity and chondroprotective function of hyaluronic acid supplementation, I report a design of self-healing supramolecular biomaterial by incorporating dynamic physical crosslinking into hyaluronic acid. Consequently, the supramolecular biomaterial exhibits unique shear-thinning by reshuffling the crosslinking in response to mechanical force, resulting in improved injectability and lubrication. Furthermore, the supramolecular biomaterial is rapidly reconstructed in the absence of force, forming a stable, crosslinked network. Using a murine model of anterior cruciate ligament injury, I confirm that the supramolecular biomaterial minimizes cartilage damage with an extended joint residence in comparison with the unmodified hyaluronic acid. Therefore, the introduction of physical crosslinking to create such a self-healing biomaterial can serve as an effective solution to chondroprotection.
Together, this dissertation offers two novel biomaterial systems to support bone and cartilage health. They are developed to capture the potential of endogenous healing mechanisms, highlighting a new paradigm of biomaterial engineering for regenerative medicine.
Item Open Access Design of Biomaterials Towards Endogenous Bone Regeneration(2020) Liu, MengqianBone grafting is one of the most commonly used surgical methods to augment bone regeneration in orthopedic procedure. While using natural bones, such as autograft and allograft are considered as the gold standard techniques, they suffer from numerous drawbacks including scarcity, donor site complications, and potential disease transmission. To overcome these limitations, mineralized poly (ethylene glycol) diacrylate-co-N-acryloyl 6-aminocaproic acid (PEGDA-co-A6ACA) composed of an organic phase and an inorganic, biomineralized phase that recapitulates certain aspects of dynamic mineral environment of native has been developed. The real-world application this biomineralized material in treating bone defects in vivo depends upon a myriad of parameters including scaffold structural parameters (e.g. pore size), mechanical properties (e.g. strength and toughness), and host environments (e.g. age of the recipient). In this dissertation, I explored these biomaterial and biological parameters for biomaterials mediated bone regeneration through leveraging endogenous healing mechanism. First of all, I evaluated the potential of mineralized biomaterials to induce bone repair of a critical-sized cranial defect in the absence of exogenous cells and growth factors. I demonstrated that the mineralized biomaterial alone can support complete bone formation within critical-sized bone defects through recruitment of endogenous cells and neo-bone tissue formation in mice. By providing a bone-specific mineral environment, these biomaterials induce osteogenic commitment of recruited host progenitor cells and support the maintenance of cells relevant for the formation and function of bone tissues, including vascularization of the implant during repair. Based on these findings, I further investigated the effect of the scaffold pore size on in vivo ectopic bone formation. Biomineralized PEGDA-co-A6ACA hydrogels were made to have an interconnected macroporous network with different pore size ranges (45-53 μm, 90-106 μm, 160-180 μm, 212-250 μm or 300-355 μm) and similar overall porosity between 65% to 70%. Using these scaffolds, I evaluated their abilities to promote ectopic bone formation upon subcutaneous implantation in wild-type mice as a function of time. I found that scaffolds with pore sizes larger than 100 μm showed similar bone formation abilities, whereas in scaffolds with pore sizes 45-53 μm, cell infiltration only happened at the peripheral region of the scaffolds. Results from this study revealed that pore size of the scaffolds had a prominent influence on the extent of cell infiltration and bone ingrowth. While such biomaterial-mediated in situ tissue engineering is highly attractive, success of this approach relies largely on the regenerative potential of the recruited endogenous cells, which is anticipated to vary with age of the host. To this end, I investigated the effect of the age of the host on mineralized biomaterial-mediated bone tissue repair using critical-sized cranial defects as a model system. Mice of varying ages, 1-month-old (juvenile), 2-month-old (young-adult), 6-month-old (middle-aged), and 14-month-old (elderly), were used as recipients. I showed that the biomineralized scaffolds support bone tissue formation by recruiting endogenous cells for all groups albeit with differences in an age-related manner. The age of the recipient mice had a significant influence on the quantity and quality of the neo-bone tissues characterized in terms of bone mineral deposition and bone tissue-specific markers, where delayed bone formation and decreased quantity of neo-bone tissue formation were observed in older mice. The real-world applications of the biomineralized materials for aiding bone tissue regeneration are greatly limited by the lack of mechanical strength and toughness of the materials. To enhance the mechanical property of the biomineralized scaffold, I further proposed a double network (DN) hydrogel system with an asymmetric network structure, where the first network is tightly cross-linked by A6ACA with crosslinker N, N'-Methylenebisacrylamide (bisacrylamide), and the second network is loosely crosslinked PEGDA. The effects of bisacrylamide crosslinker concentration (2 mol.%, 4 mol.% and 6 mol.%), and molecular weight (Mn: 3.4 kDa, 6 kDa, 10 kDa, and 20 kDa) of 20 w/v % PEGDA on mechanical properties of the resultant DN-hydrogels were investigated and compared to those of single network (SN) hydrogels of the same composition. Findings from this study showed that increase in crosslinker concentration of the first network was correlated with lower ultimate compressive strain, higher compressive strength, toughness and elastic modulus. Furthermore, DN-hydrogels prepared in this work displayed swelling ratios ranging from 569 ± 20% to 1948 ± 12%. Among all compositions, DN-hydrogel with 6 mol.% bisacrylamide and PEGDA 10 kDa demonstrated the highest compressive strength (3.47 ± 0.35 MPa), highest toughness (0.60 ± 0.03 MJ/m3), and elastic modulus (1.04 ± 0.09 MPa). Using this composition, porous DN-hydrogels with interconnected pore architecture were fabricated through polymethylmethacrylate (PMMA) bead leaching method. Resultant porous hydrogels demonstrated potent biomineralization capabilities, and the matrix-bounded CaP minerals were able to undergo dissolutions. Given the high strength and biomineralization capacity, DN-hydrogels reported here could be useful for developments of tissue engineering scaffolds for bone tissue regenerations. Overall, this dissertation explores different biomaterial designs and biological factors in biomaterial-mediated in vivo bone tissue repair, providing materials insights that are useful to researchers and engineers in designs of biomaterials to leverage endogenous healing mechanism for tissue regeneration and repair.
Item Open Access Designing Multi-Epitope Supramolecular Nanofiber Vaccines for Autoimmunity and Infectious Disease(2021) Shores, Lucas ScottWhile promoting a highly targeted immune response, linear peptide epitopes generally have reduced immunogenicity and a lack of conformational specificity which has hampered the development of peptide-based vaccines. In contrast, supramolecular assembly is capable of considerably enhancing immunogenicity and promoting long-term immune responses to peptide epitopes. Immune responses generated with different vaccine platforms but targeted to the same peptide epitope, however, can vary greatly in magnitude and phenotype depending on the physicochemical cues, adjuvant, and density with which that immune epitope is displayed, influencing vaccine outcomes. Greater understanding of how design criteria such as adjuvant choice, epitope density, and multi-epitope immunization adjust the magnitude and phenotype of immune responses—in addition to how these changes in magnitude and phenotype affect therapeutic outcomes—would greatly benefit the development of peptide-based vaccines for treatment and prevention of disease. In this effort, the Collier lab has previously established that the density of T-cell epitope content within molecular self-assemblies can alter the cellular phenotype of antigen-specific T-cells in mice. Further modifications to nanofiber physical properties such as size and surface charge have also been recently shown to modulate the immunogenicity of peptide nanofiber constructs. It remains unclear, however, the extent to which combinations of B- and T-cell epitope content within nanofibers affects the antibody and T-cell responses. Further, the magnitude of antibody and T-cell responses can be magnified by the presence of adjuvants, but with adjuvant specific alterations to antibody subclass and T-cell phenotype. This work capitalizes on these advances in supramolecular nanofiber vaccines and extends them further in three separate contexts. Using peptide epitopes from inflammatory cytokines (Chapter 3), HIV protein mimics (Chapter 4), and influenza A virus (Chapter 5), we demonstrate how titrating epitope content controls adaptive immune responses and how these responses are influenced by the presence of different adjuvants. While previous work with adjuvants has identified general principles of phenotype, typically characterized as Th1 vs Th2 responses, this work seeks to understand and expand context-specific effects, e.g. optimized phenotypes for anti-cytokines immunizations compared to infectious disease. In Chapter 3, epitopes were predicted, tested, and optimized in the Q11 system to determine an immunogenic IL-17A epitope for the treatment of psoriasis. The epitopes were identified through a literature search and Kolaskar-Tongaonkar antigenicity prediction. High-scoring epitopes were tested in mice for immunogenicity and one was selected based on antibody titer. The formulation space combining the selected epitope with different combinations of T-cell epitope was also characterized, and combinations of B- and T-cell epitope content were determined to be predictive of antibody titer in this system. Responses were then enhanced with either a Th1 or a Th2 biasing adjuvant and tested for efficacy in a mouse model of psoriasis. Reduced disease symptoms were correlated with specific antibody subclasses in a first demonstration of the importance of antibody subclass for active immunotherapy. To further understand the quantitative importance of B cell signaling induced by supramolecular nanofibers, we developed a system of peptide-based activation of human B cells in vitro. In Chapter 4, we describe the first use, to our knowledge, of a peptide epitope for activation of the VRC01 Ramos B cell line. This cell line was described in 2017 and contains a B-cell Receptor (BCR) that is identical to the anti-HIV broadly-neutralizing monoclonal antibody VRC01. Other groups had previously published epitope mimicking peptide epitopes, termed mimotopes, which bind the VRC01 monoclonal antibody despite little to no sequence similarity to the native protein. We tested the highest performing mimotopes with the Q11 system and found an epitope-density-dependent effect for one of the mimotopes that was independent of nanofiber concentration. We then tested this epitope at a variety of epitope densities, with or without adjuvant. This study provides evidence that epitope density on supramolecular nanofibers has the potential to directly influence B-cell signaling, and how adjuvants can modulate this effect. Finally, we applied a Design-of-Experiments (DOE) approach to a multi-epitope system and combined it with the novel adjuvant KEYA20Q11. As attempts to make a universal influenza vaccine have become more refined, the specificity of peptide-based vaccines has had increased interest for their ability to avoid immunodominant epitopes in the native protein. Combining T-cell and B-cell responses has further shown promise in the field, but there remains a significant deficit in methods to enhance the T-cell response to a given epitope. In Chapter 5, we first optimize peptide nanofiber formulations in the context of adjuvant-free formulations. The addition of adjuvants does not alter the optimal response profile for antibody and T cell responses in this context, and we first use traditional adjuvants to enhance these responses before challenging with influenza virus. Unfortunately, this did not result in protection from viral challenge and a novel adjuvant based on the therapeutic polypeptide Glatiramer Acetate was used. This adjuvant provides universal T-cell help to enhance B-cell responses and increases the magnitude of co-assembled CD4+ and CD8+ responses. The protective efficacy of the peptide nanofiber formulation was enhanced by a combination of this novel adjuvant with the more traditional adjuvant of cyclic-di-AMP. While formulations with and without KEYA20Q11 both provided equal protection from influenza-induced weight loss, the enhanced T cell responses from the KEYA20Q11 group correlated with reduced viral titers. This work demonstrates how quantitative optimization in conjunction with adjuvant choice can lead to the enhancement of universal B- and T-cell responses for protection from influenza. Taken as a whole, this work represents a significant contribution to the understanding of how peptide epitope content and phenotype of adaptive immune responses influences their efficacy in the contexts of autoimmune and infectious disease. While not all materials-based vaccine platforms may follow the precise rules laid out through the use of the supramolecular peptide systems, the quantitative approach and analysis of immune cell phenotype should be broadly applicable. This work relies heavily on responses from in vivo models or from human cell responses which broadens the translatability of the approach. The completion of this work should facilitate the next generation of supramolecular nanofiber vaccines for autoimmunity and infectious disease.
Item Open Access Development and Characterization of Mechanically Robust, 3D-Printable Photopolymers(2017) Sycks, Dalton3D printing has seen an explosion of interest and growth in recent years, especially within the biomedical space. Prized for its efficiency, ability to produce complex geometries, and facile material processing, additive manufacturing is rapidly being used to create medical devices ranging from orthopedic implants to tissue scaffolds. However, 3D printing is currently limited to a select few material choices, especially when one considers soft tissue replacement or augmentation. To this end, my research focuses on developing material systems that are simultaneously 1) 3D printable, 2) biocompatible, and 3) mechanically robust with properties appropriate for soft-tissue replacement or augmentation applications. Two systems were developed toward this goal: an interpenetrating network (IPN) hydrogel consisting of covalently crosslinked poly (ethylene glycol) diacrylate (PEGDA) and ionically crosslinked brown sodium alginate, and semi-crystalline thiol-ene photopolymers containing spiroacetal molecules in the polymer main-chain backbone. In addition to successfully being incorporated into existing 3D printing systems (extrusion-deposition for the PEGDA-alginate hydrogel and digital light processing for the thiol-ene polymers) both systems exhibited biocompatibility and superior thermomechanical properties such as tensile modulus, failure strain, and toughness. This work offers two fully-developed, novel polymer platforms with outstanding performance; further, structure-property relationships are highlighted and discussed on a molecular and morphological level to provide material insights that are useful to researchers and engineers in the design of highly tuned and mechanically robust polymers.
Item Open Access Development of Genetically Encoded Zwitterionic Polypeptides for Drug Delivery(2019) Banskota, SamagyaThe clinical utility of many peptide, protein and small molecule drugs is limited by their short in-vivo ¬half-life. To address this limitation, we report a new class of biomaterials that have a long plasma circulation time. In particular, taking inspiration and cues from natural proteins and synthetic polymers, we have worked to create polypeptide-based drug carriers that are biocompatible and biodegradable. These peptide polymers or polypeptides can be attached to therapeutics with molecular precision as they are designed from the gene level.
In the first part of this thesis (Chapter 3-4), we report on the development of a new class of biomaterials called zwitterionic polypeptides (ZIPPs) that exhibit “stealth” behavior, and when fused to therapeutics, improve their pharmacological efficacy. To identify an optimal polypeptide design, we first synthesized a library of ZIPPs by incorporating various oppositely charged amino acids within an intrinsically disordered polypeptide motif, (VPX1X2G)n, where X1 and X2 are cationic and anionic amino acids, respectively, and n is the number of repeats. The (VPX1X2G)n motif is derived from the disordered region of human tropo-elastin. By systematically varying the identity of the charged amino acids and the chain length of the polypeptide, we determined the optimal polypeptide sequence that maximizes the pharmacokinetics for intravenous and subcutaneous routes of administration. We show that a combination of lysine and glutamic acid in the ZIPP confer superior pharmacokinetics, for both intravenous and subcutaneous administration, compared to uncharged control polypeptides. We report detailed physicochemical characterization of this new class of polypeptide-based drug carriers and show its clinical utility for drug delivery by using it to deliver a peptide drug. The peptide drug used is Glucagon like peptide 1 (GLP1) – a therapeutic peptide that is approved for treatment of type 2 diabetes but has seen limited clinical utility because of its short two-minutes half-life. We find that the GLP1-ZIPP conjugate reduced blood glucose level for up to 3 days in a diet induced obesity model of type-2 diabetes in mice after a single s.c. injection. This is a 70-fold improvement over the injection of the unmodified drug and a 1.5-fold improvement over an uncharged polypeptide control.
To further demonstrate the clinical utility of ZIPPs, in the second part of this thesis (Chapter 5), we used ZIPPs to create a nanoparticle system that can package and deliver hydrophobic chemotherapeutic drugs to the tumor with higher efficacy and lower toxicity. Such nanoparticle drug carriers are attractive for systemic delivery of chemotherapeutics because they improve the half-life of the drug, protect the drug from early degradation, and increase selective accumulation of drugs in tumors via the enhanced permeation and retention effect (EPR). The EPR effect is a consequence of the leaky vasculature and poorly developed lymphatic drainage system present in the tumors. These attributes of nanoparticles are significant and desirable because drug delivery systems that can improve circulation time and tumor accumulation of chemotherapeutics have the ability to improve the patient prognosis and survival by controlling the tumors at their local sites. To that end, we conjugated paclitaxel a chemotherapy drug that is used to treat different types of cancer to ZIPPs and showed that it imparts sufficient amphiphilicity to the polypeptide chain to drive its self-assembly into sub-100 nm nanoparticles. We report that ZIPPs can increase the systemic exposure of paclitaxel by 17-fold compared to the free drug and 1.6-fold compared to uncharged recombinant control. Treatment of mice bearing highly aggressive prostate cancer or colon cancer with a single dose of ZIPP-Paclitaxel nanoparticles leads to a near complete-eradication of the tumors (5 out of 7 cures in prostate cancer) and (2 out of 7 cures in colon cancer) and it outperforms Abraxane, which is an FDA approved taxane nanoformulation and current gold standard for paclitaxel delivery.
In summary, this doctoral research is multidisciplinary, which integrates the field of protein engineering, molecular biology, bioconjugate chemistry, soft matter physics and cancer biology for rational design of biomaterials for drug delivery.
Item Embargo Engineering the microstructure and spatial bioactivity of granular biomaterials to guide vascular patterning(2023) Anderson, Alexa R.In tissues where the vasculature is either lacking or abnormal, biomaterial interventions can be designed to induce vessel formation and promote tissue repair. The porous architecture of biomaterials plays a key role in influencing cell infiltration and inducing vascularization by enabling the diffusion of nutrients and providing structural avenues for vessel ingrowth. Microporous annealed particle (MAP) scaffolds are a class of biomaterial that inherently possess a tunable, porous architecture. These materials are composed of small hydrogel particles, or microgels, that pack together to produce an interconnected, porous network. We first demonstrated that the particle fraction in MAP scaffolds serves as a bioactive cue for cell growth. To control this bioactive cue, we developed methods to form MAP scaffolds with user-defined particle fractions to reproducibly assess mechanical properties, macromolecular diffusion, as and cell responses. We then modulated the microstructure of the MAP scaffolds by changing microgel size as well as the spatial bioactivity using heterogeneous microgel populations to promote de novo assembly of endothelial progenitor-like cells into vessel-like structures. Through a combination of in silico and in vitro experimentation, we found that the microstructure (dimension of the void), integrin binding, and growth factor sequestration were all shown to guide vascular morphogenesis. We then demonstrated that the findings produced in a reductionist model of vasculogenesis translated to an in vivo effect on vessel formation in both dermal wounds and glioblastoma tumors.
Item Open Access Generation of Functional Hepatocyte-Like Cells (HLCs) from Human Adipose-Derived Stem Cells (ADSCs) in 2D and 3D(2019) Arinda, Beryl NgabiranoMortality and morbidity rates caused by acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and chronic liver disease continue to rise because of drug induced failure or viral hepatitis, currently with 2,000 cases annually in the United States. Liver transplantation is the only intervention that has shown the most promising patient outcomes, but this approach has major shortcomings like shortage of donor livers, lifelong immunosuppression and a risk of organ rejection after transplantation. Additionally, with rapid liver deterioration and subsequent multi-organ failure characterized by ALF and ACLF conditions, there are high mortality rates as patients await a liver transplant or wait for their livers to regenerate. As such, bioartificial liver support devices provide an alternative to improve patient survival through either offloading liver functions to allow for liver regeneration or by allowing the patient time to receive a liver transplant. These bioartificial liver support devices are designed to perform essential liver functions through incorporation of an active cellular component that performs the liver functions and their success is therefore heavily reliant on the performance of the incorporated cell lines. Because of this, limited sources of these characteristic cell lines with hepatic function is a great challenge being faced in the research and development of the devices.
Adipose-derived stem cells (ADSCs) are a great candidate as a stem cell source for differentiation of hepatocyte-like cells because they can be easily obtained in large quantities with little donor site morbidity or discomfort and have been successfully differentiated into multiple cell lineages. In this study, we investigate the possibility of differentiating human ADSCs into functional hepatocyte-like cells. Furthermore, we investigated the ability to differentiate ADSCs into hepatocyte-like cells in both 2D and 3D environments. We found that induced ADSCs can produce high levels of some hepatocyte functions, like albumin secretion. However, other functions, like urea secretion and cytochrome P450 metabolic activity, while present, are not yet at sufficient levels to be comparable to primary hepatocytes.
Item Embargo Hydrogel-Mediated Gene Delivery from Granular Scaffolds for Applications in Biologics Manufacturing and Regenerative Medicine(2023) Kurt, Evan MichaelNucleic acid delivery has applications ranging from tissue engineering to biologics development and manufacturing to vaccines and infectious disease. To improve delivery and extend viable expression over time, we turn to biomaterials as a method for sustained nucleic acid release and enhanced cell culture or tissue interaction. Here, we describe how cationic polymer and lipid condensed nucleic acids can be effectively loaded into injectable granular hydrogel scaffolds by stabilizing the condensed nucleic acid into a lyophilized powder, loading the powder into a bulk hydrogel, and then fragmenting the gel into hydrogel microparticles. The resulting microgels contain non-aggregated nucleic acid particles, can be annealed into an injectable microporous scaffold, and can effectively deliver nucleic acids to cells with a sustained rate of expression. We explore how this technology can improve the production of biologics, like antibodies and viruses, to overcome limitations of current batch processes. Our scaffolds allow for continuous biologics manufacturing, with sustained production upwards of 30 days. We also explore how our platform can improve tissue regeneration in disease models like dermal wounds by delivering nucleic acid drugs, namely DNA, mRNA, and therapeutic viruses. The loaded granular scaffolds allow cells to readily repopulate the missing tissue and drugs be locally released and taken up over time. Overall, our scaffold delivery approach is a customizable platform that can be tuned for many different applications.
Item Open Access Improving Indwelling Glucose Sensor Performance: Porous, Dexamethasone-Releasing Coatings that Modulate the Foreign Body Response(2015) VallejoHeligon, Suzana GabrielaInflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.
First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.
The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.
Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.
The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.
Item Open Access LOcal Void Analysis of MAP scaffolds (LOVAMAP)(2022) Riley, LindsayOur lab designs hydrogel microparticles (HMPs) that are interlinked to form microporous annealed particle (MAP) scaffolds for wound healing applications. The therapeutic effects of MAP are attributed, in part, to the void space between particles that creates inherent micro-porosity through which cells can infiltrate and migrate unhindered. Cell behavior is influenced by local geometry, and our goal is to design scaffolds that influence cells toward pro-healing behaviors. To accomplish this, we need a methodology for quantitatively characterizing the void space of MAP scaffolds in order to study the relationships between internal microarchitecture and therapeutic outcomes. The work presented here is a visually-rich dissertation that covers our approach for analyzing the void space of packed particles. We use techniques from computational geometry and graph theory to develop a robust methodology for segmenting the void space into natural pockets of open space and outputting a set of descriptors that characterize the space. Our methods are developed using simulated MAP scaffolds covering a range of particle compositions, including mixed particle sizes, stiffnesses, and shapes. Our software, called LOcal Void Analysis of MAP scaffolds (LOVAMAP), has allowed us to study many aspects of void space, including global descriptors like void volume fraction, local ‘pore’ measurements of size and shape, and additional features like ligand availability, paths, isotropy/anisotropy, and available regions for unhindered migration based on size. LOVAMAP is an enabling technology that can be used for analyzing real scaffolds or studying simulated scaffolds to inform material design. It serves as a platform for void space analysis that can easily be built upon to encompass ever-growing innovations in scaffold characterization.
Item Open Access Microencapsulation of Octylcyanoacrylate for Applications as a Healing Agent in a Self-healing Bone Cement(2011) Brochu, AliceTotal joint replacement surgeries are performed on thousands of patients every year, yet these implants are subject to failure following prolonged exposure to the harsh environment of the body as well as the complex loading patterns seen in biological joints. The generation of wear debris from both the articulating surfaces and the poly(methyl methacrylate) (PMMA) bone cement used to anchor the replacements in place serves to accelerate wear and subsequent failure of the device. Self-healing approaches that employ an encapsulated healing agent embedded in a catalyst-containing matrix have been developed to restore mechanical function to materials that undergo crack damage; following capsule rupture, and healing agent release and polymerization serves to halt microcrack propagation. However, existing encapsulated systems do not adhere to biomaterials constraints. In this work, interfacial polymerization of polyurethane (PUR) in an oil-in-water emulsion was used to achieve encapsulation of octylcyanoacrylate (OCA), a medical grade adhesive used in sutureless surgeries. The optimized encapsulation procedure was determined by studying the effects of solvent, surfactant, and temperature on the final product. The average size and size distribution, capsule shell thickness, percent fill and reactivity of encapsulated agent, and shelf life of these capsules were studied and are now suitable for incorporation into PMMA and assessment as potential healing agent systems.
Item Unknown Molecular and Macroscale Engineering of Sublingual Nanofiber Vaccines(2020) Kelly, Sean HShort peptides are poorly immunogenic when delivered sublingually – under the tongue. This challenge has prevented widespread investigation into sublingual peptide vaccines, leaving their considerable potential untapped. Sublingual immunization is logistically favorable due to its ease of administration and can raise both strong systemic immune responses and mucosal responses in tissues throughout the body. Peptide epitopes are highly specific, allowing for the generation of immune responses directed solely against precisely selected targets, without accompanying off-target antibody or T-cell production. To enable sublingual peptide immunization, we designed a strategy based on molecular self-assembly of epitopes within nanofibers. We then extract and expound several key implications from this finding, including insights into the mechanism of action, development of a translatable administration modality, and application to a currently unmet clinical need.
The design of our sublingual peptide immunization strategy is described in the first part of this thesis (Chapter 3). We sought to utilize nanomaterial delivery of peptides to enhance sublingual immunogenicity, a strategy that has proved successful in several other immunization routes. However, the salivary mucus layer is a significant barrier to nanomaterial delivery, particularly for supramolecular materials, due to its ability to entrap and clear these materials rapidly. We designed β-sheet nanofibers conjugated at a high-density to the mucus-inert polymer polyethylene glycol (PEG) to shield them from the mucus layer. Strikingly, sublingually delivered PEGylated peptide nanofibers (PEG-Q11) raised extremely durable antibody and T-cell responses against peptide epitopes when mixed with a mucosal adjuvant. We showed that PEG decreases nanofiber interactions with mucin in vitro, and extends the residence time of nanofibers at the sublingual space in vivo. Further, we showed that we could achieve similar results by adapting the use of PASylation (modification with peptide sequences rich in Pro, Ala, and Ser) to mucosal delivery.
In the second part of this thesis (Chapter 4) we designed a supramolecular strategy for enhancing sublingual nanofiber immunization. Mucosal adjuvants, such as cyclic-di-nucleotides (CDNs), can promote sublingual immune responses but must be co-delivered with the antigen to the epithelium for maximum effect. We designed peptide-polymer nanofibers displaying nona-arginine (R9) at a high density to promote complexation with CDNs via bidentate hydrogen-bonding with arginine side chains. We co-assembled PEG-Q11 and PEG-Q11R9 peptides to titrate the concentration of R9 within nanofibers. In vitro, PEG-Q11R9 fibers and cyclic-di-GMP or cyclic-di-AMP adjuvants had a synergistic effect on enhancing dendritic cell activation that was STING-dependent and increased monotonically with increasing R9 concentration. However, intermediate levels of R9 within sublingually-administered PEG-Q11 fibers were optimal for sublingual immunization, suggesting a balance between polyarginine’s ability to sequester CDNs along the nanofiber and its potentially detrimental mucoadhesive interactions. These findings reveal important design considerations for the continuing development of sublingual peptide nanofiber vaccines.
We sought to enhance the translational capacity of our immunization strategy by designing a highly accessible vaccine method (described in Chapter 5). Significant barriers exist to improving vaccine coverage in lower- and middle-income countries, including the costly requirements for cold-chain distribution and trained medical personnel to administer the vaccines. To address these barriers, we built upon our sublingual nanofiber platform to design a heat-stable and highly porous tablet vaccine that can be administered via simple dissolution under the tongue. We produced SIMPL (Supramolecular Immunization with Peptides SubLingually) tablet vaccines by freeze-drying a mixture of self-assembling peptide-polymer nanofibers, sugar excipients, and adjuvant. We showed that even after heating for 1 week at 45 °C, SIMPL tablets could raise antibody responses against a peptide epitope from M. tuberculosis, in contrast to a conventional carrier vaccine (KLH) which lost sublingual efficacy after heating. Our approach directly addresses the need for a heat-stable and easily deliverable vaccine to improve equity in global vaccine coverage.
To demonstrate the clinical usefulness of our technology, we designed a sublingual vaccine against uropathogenic E. coli (UPEC), the pathogen that causes most urinary tract infections (UTIs). Sublingual peptide immunization is uniquely advantageous for immunization against UTIs, as sublingual immunization raises antibodies in the blood and urinary tract, and peptide epitopes allow for targeting UPEC without perturbing commensals. We co-assembled PEG-Q11 nanofibers containing three different B-cell epitopes from UPEC along with a helper T-cell epitope, allowing us to raise simultaneous antibody responses against three targets systemically and in the urinary tract. These antibodies were highly specific for UPEC, exhibiting no binding to a non-pathogenic strain. Further, these antibodies demonstrated clinical potential by protecting mice from an intraperitoneal UPEC challenge. Finally, we showed the ability to use SIMPL tablets to raise anti-UPEC responses in rabbits, which contain an oral cavity with key similarities to humans.
This thesis demonstrates a critical enabling technology for sublingual peptide immunization and builds upon this technology to report findings with key implications for supramolecular biomaterial design, accessible vaccination, and treatment of UPEC-mediated diseases. This interdisciplinary research synthesizes and utilizes knowledge from materials science, immunology, vaccinology, pharmaceutical sciences, and pathology to present an important original contribution to the field of immune engineering.
Item Unknown Multiphase, Multicomponent Systems: Divalent Ionic Surfactant Phases and Single-Particle Engineering of Protein and Polymer Glasses(2011) Rickard, DeborahThis thesis presents an analysis of the material properties and phase behavior of divalent ionic surfactant salts, and protein and polymer glasses. There has been extensive interest in understanding the phase behavior of divalent ionic surfactants due to the many applications of ionic surfactants in which they come into contact with divalent ions, such as detergency, oil recovery, and surfactant separation processes. One goal of determining the phase boundaries was to explore the option of incorporating a hydrophobic molecule into the solid phase through the micelle-to-crystal bilayer transition, either for drug delivery applications (with a biologically compatible surfactant) or for the purpose of studying the hydrophobic molecule itself. The liquid micellar and solid crystal phases of the alkaline earth metal dodecyl sulfates were investigated using calorimetry, visual inspection, solubilization of a fluorescent probe, and x-ray diffraction. The Krafft temperature and dissolution enthalpy were determined for each surfactant, and partial composition-temperature phase diagrams of magnesium dodecyl sulfate-water, calcium dodecyl sulfate-water, as well as sodium dodecyl sulfate with MgCl2 and CaCl2 are presented. As a proof of concept, fluorescence microscopy images showed that it is, in fact, possible to incorporate a small hydrophobic molecule, diphenylhexatriene, into the solid phase.
The second, and main, part of this thesis expands on work done previously in the lab by using the micropipette technique to study two-phase microsystems. These microsystems consist of a liquid droplet suspended in a second, immiscible liquid medium, and can serve as direct single-particle studies of drug delivery systems that are formed using solvent extraction (e.g., protein encapsulated in a biodegradable polymer), and as model systems with which to study the materials and principles that govern particle formation. The assumptions of the Epstein-Plesset model, which predicts the rate of droplet dissolution, are examined in the context of the micropipette technique. A modification to the model is presented that accounts for the effect a solute has on the dissolution rate. The modification is based on the assumption that the droplet interface is in local thermodynamic equilibrium, and that the water activity in a solution droplet can be used to determine its dissolution (or dehydration) rate. The model successfully predicts the dissolution rates of NaCl solutions into octanol and butyl acetate up to the point of NaCl crystallization. The dehydration of protein solutions (lysozyme or bovine serum albumin) results in glassified microbeads with less than a monolayer of water coverage per protein molecule, which can be controlled by the water activity of the surrounding organic medium. The kinetics of dehydration match the prediction of the activity-based model, and it is shown how the micropipette technique can be used to study the effect of dissolution rate on final particle morphology. By using a stable protein with a simple geometry (lyosyzme), this technique was be used to determine the distance dependence of protein-protein interactions in the range of 2-25 Å, providing the first calculation of the hydration pressure decay length for globular proteins. The distance-dependence of the interaction potential at distances less than 9 Å was found to have a decay length of 1.7 Å, which is consistent with the known decay length of hydration pressure between other biological materials. Biodegradable polyesters, such as poly(lactide-co-glycolide) (PLGA), are some of the most common materials used for the encapsulation of therapeutics in microspheres for long-term drug release. Since they degrade by hydrolysis, release rates depend on water uptake, which can be affected by processing parameters and the material properties of the encapsulated drug. The micropipette technique allows observations not possible on any bulk preparation method. Single-particle observations of microsphere formation (organic solvent extraction into a surrounding aqueous phase) show that as solvent leaves the microsphere and the water concentration in the polymer matrix becomes supersaturated, water phase separates and inclusions initially grow quickly. Once the concentration in the polymer matrix equilibrates with the surrounding aqueous medium, the water inclusions continue to grow due to dissolved impurities, solvent, and/or water-soluble polymer fragments resulting from hydrolysis, all of which locally lower the water activity in the inclusion. Experiments are also presented in which glassified protein microbeads were suspended in PLGA solution prior to forming the single microspheres. This technique allowed the concentration of protein in a single microbead/inclusion to be determined as a function of time.
Item Open Access Novel Enzyme-Mediated Ternary Radical Initiating System for Producing HydrogelsJoseph, Neica; West, Jennifer L; Su, Teng