Click-Chemistry Based Molecularly Responsive Hydrogel as Biodegradable Scaffolds for 3-D Cell Culture: Design and Preparation of Building Blocks
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According to the American Cancer Society, every year over one million Americans are diagnosed with one of the recognized forms of cancer. Cancer metastasis is associated with poor patient prognosis. In order to better understand how cancer grows and metastasizes, researchers are interested in the development of platforms that could model the environment of tumor tissue and enable the study of the effect of cancer cell-secreted molecules on cell replication, extracellular matrix remodeling, and cell migration leading to metastasis. Due to their tissue-like properties, hydrogels have the ability to serve as such model systems by providing a three-dimensional scaffold in which cancerous cells could be cultured and studied. Hydrogels are formed from physically or chemically cross-linked hydrophilic polymers that form insoluble networks. Due to their high water content, hydrogels have mechanical properties that are similar to those of tissue; therefore, hydrogels could become ideal platforms for the study of cell growth and migration. The long-term goal of this work is to develop a hydrogel system that is molecularly responsive and can be synthesized at physiological conditions that could be used as a model the study of cancer cell growth. In order to achieve this goal, in this work we focused on: (1) the development of an aptamer complex to be used as crosslinker within the hydrogel network that is responsive towards the cancer cell-secreted protein vascular endothelium growth factor (VEGF), (2) the development of methods for the synthesis of hydrogels under mild conditions via copper-free click chemistry, and (3) the integration of cell adhesive properties to the hydrogels through a cyclo-Arginine-Glycine-Aspartic Acid (cRGD) peptide that mimics fibrin and collagen, some of the most abundant protein components of the extracellular matrix. The aptamer complex was designed to be able to hybridize a physiological conditions but denature in the presence of the molecular target VEGF, yielding a system that is responsive and degradable. Through the use of copper-free click chemistry, the hydrogels could be synthesized in the absence of toxins like free radicals, metals, and ultraviolet light that are typically used in polymerization reactions but that have deleterious effects on cells. The bioorthogonal reaction between azide and dibenzylcyclooctyne utilized in the copper-free click chemistry reaction between azide and dibenzylcyclooctyne utilized in the copper-free click chemistry reaction is able to rapidly link polymeric precursors, thus providing a method for immediate the encapsulation of cancer cells within the hydrogel scaffold. The encapsulated cells are then able to interact with the mimicry peptide cRGD and signal the cells to begin growing and proliferating. In doing so, a hydrogel that is molecular responsive, nontoxic, and capable of encapsulating cells is fashioned in order to serve as three-dimensional platform for their study. Chapter 1 provides a background on the various concepts on which the proposed biomaterial design is based. Chapter 2 discusses the design and in vitro evaluation of VEGF-responsive aptamer complexes to be used in later work as crosslinkers of molecularly controlled biodegradable hydrogels. Chapter 3 describes the work that was performed for the development of synthetic protocols to be used for the preparation of the hydrogels using click chemistry. Chapter 4 describes the methods used for solid-phase synthesis of the mimicry peptide cRGD. Finally, Chapter 5 summarizes the work and provides concluding remarks.