<p dir="ltr">In the past decade, magnetic microrobots have gain lots of attention because of their potentials in biomedical applications, such as cell/tissue manipulation, biopsy, and drug delivery. Recent development on materials and microfabrication techniques also provide more opportunities for microrobots. Especially, the emergence of smart polymers that are responsive to environments like hydrogels has given microrobots an additional degree-of-freedom. In the meantime, the two-photon polymerization (TPP) microscale 3D printing technique has enable fabrication process that cannot be achieved easily by traditional microfabrication techniques. In general, the goal of the research presented in this dissertation is to use both hydrogels and TPP to realize novel microrobots with multiple advanced functionalities, including adaptive locomotion and micromanipulation, and modular microrobots capable of changing end-effectors for different modes of micromanipulation to facilitate the development of the field. </p><p dir="ltr">This dissertation can be divided into four main parts: (i) a proof-of-concept study on adaptive helical microrobots with finite element analysis (FEA) and dynamic calculation, (ii) material calibrations and property testing, (iii) a helical adaptive multi-material microrobot (HAMMR), and (iv) a modular microrobot achieved by a responsive mating component. A environment-responsive hydrogel is adopted here to realize the adaptive locomotion for helical microrobot and the responsive mating component for the modular microrobot. All microrobots fabricated in this dissertation are achieved by the combination of TPP and traditional photolithography techniques. </p><p dir="ltr">In part (i), FEA is applied with classic parameters for a proof-of-concept study of helical microrobot made of the classic hydrogel upon the stimulation of temperature. At different temperature, the hydrogel is going to deform and therefore the microrobot. Based on the geometrical parameters predicted by FEA before and after stimulations, dynamic calculations are then applied to predict the change of swimming performance accordingly. In part (ii), material calibrations have been done in order to realize a homogeneous material for testing (for oil-immersion mode). However, due to the limitation of the custom-built testing system, a different approach (dip-in mode) is adopted and the material properties are successfully obtained. In part (iii), two generations of HAMMRs are investigated. The first generation of HAMMR is prepared by the oil-immersion mode which shows a record-breaking swimmering velocity with the capability of adaptive locomotion. The second generation is obtained by the dip-in mode which provides the opportunities for combining FEA, dynamic calculation, and experiment to realize a comprehensive studied for such microrobot. Moveover, advances have been made to the microrobot with a functional end-effector for micromanipulation tasks. In part (iv), a modular microrobot is proposed and realized by the introduction of a responsive mating component. The responsive mating component provides a locking mechanism between different modules of the microrobot. The microrobot is able to change its end-effector to perform different types of tasks. </p><p dir="ltr">By using TPP to pattern microscale hydrogel structures, microrobots are able to be implemented with advanced functional structures. The helical microrobots capable of adaptive locomotion and micromanipulation, and the modular microrobot that can switch end-effectors for different applications are advances toward the next generation of microrobots. Moreover, a standardized method is proposed for adaptive helical microrobots towards future biomedical applications. Both the proposed helical microrobot and the modular microrobot show great potential for future application and we believe the development of these microrobots will facilitate the development of the field of microrobot.</p>

This work proposes minimally invasive solutions for spinal cord regeneration after trauma. In particular, injectable biomaterials can be precisely positioned in the lesion site, and eventually repetitively injected until the complete regeneration of the tissue. For this application, a silk fibroin functionalized with collagen type IV and laminin-derived peptides, called bio-inspired multifunctionalized silk fibroin (BMS), possessing piezoelectric properties, has been synthesized. Another approach that avoids damages to the spinal cord is proposed in the thesis as a multilayer hydrogel with piezoelectric properties that acts as a bridge between the healthy parts surrounding the injury. The multilayer hydrogel consists of i) a thin-layer of gelatin and fish collagen functionalized with VEGF for blood vessels formation, which helps the survival of the cells integrating with the pia mater of the spinal cord; ii) a BMS layer, which helps the adhesion, migration of neural stem cells and induces the sprouting of the axons thanks to the presence of Netrin (a chemoattractive protein); and iii) an adhesive layer of polydopamine (PDA) to fix the patch on the injured site. The adhesive patch exhibits a potential larger than an injectable hydrogel that could guarantee a long-term cell survival and help the axons to move towards a direction. The adhesive patch will be located on the surface of the spinal cord and the chemoattractive protein will induce the sprouting of the ascendant or descendant axons in the spinal cord to reach the axons present in the patch, restoring a signal connection. Even if not final, the results indicate that the above strategy could be explored further for the regeneration of the spinal cord.

Waveguide architectures were inscribed within two different stimuli-responsive hydrogels capable of actuation. An electroactive hydrogel, which deforms when placed within an electric field, is demonstrated as a method for remote actuation and steering of light outputs. Lattices of waveguide with diameters on the microscale were embedded within hydrogel prisms, achieved through a nonlinear light propagation process known as self-trapping. This process is a result of balance between the natural divergence of light and self-focusing effects caused by an irreversible positive refractive index change during photopolymerization. Waveguiding structures are inscribed in the material because of this process. Square (2D) and near-cubic (3D) lattices were inscribed in hydrogel prisms, demonstrating the ability to remotely steer one or two light outputs simultaneously using an electric field. The overall optical effect is reminiscent of camouflaging techniques observed in marine creatures (ie. cephalopods). Additionally, a novel volumetric 3D printing technique previously demonstrated by the Saravanamuttu group was implemented to fabricate hydrogel cylinders capable of photothermal actuation. Coupling a thermoresponsive hydrogel material with a photoabsorber, areas irradiated by a light source are observed to contract. These contractions lead to the deflection of waveguiding cylinders towards the light source, reminiscent of the phototropic behaviours observed in particular plants (ie. sunflowers). The results of these studies provide insight for the fabrication of functional materials through nonlinear light propagation. Understanding these systems could provide knowledge for the fabrication of other stimuli-responsive materials with light-guiding properties. / Thesis / Master of Science (MSc)

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Polymeric biomaterials have created significant advances in the field of biomedical engineering, however, very few polymeric drug delivery devices have achieved clinical and commercial success. Thus, the motivation for this thesis was to encourage long-term success of materials through expanding the fundamental understanding of polymer properties. Poly(ethylene glycol) was specifically chosen for study as its polyether backbone provides it with many unique properties that are still not fully understood, and are not seen with other similar polymers. PEG has been shown to exhibit amphiphilic character, due to its high conformational freedom, and the ability to hydrogen-bond 2-3 water molecules for each ethylene oxide subunit, creating a very structured water shell and large hydrodynamic radius. Together, the properties formed the hypothesis for the possibility for PEG to control drug release and its environment, expanding its potential in biomedical applications. This hypothesis was investigated with PEG in three states – free PEG, conjugated and blended. Free PEG was determined to inhibit melanoma cell viability by activating apoptosis via PEG effects on the osmolality of the cell medium (Chapter 3). Novel silicone hydrogels incorporating methacrylated PEG as the sole hydrophilic component showed advantageous properties for biomedical applications across a range of formulations (such as low contact angle and protein deposition), as well as altering the release of highly hydrophilic antibiotics from the materials, presumably via PEG-drug hydrogen bonding (Chapter 4). Novel siloxane-PEG blended materials were shown to have the ability to influence drug release of hydrophilic, hydrophobic and drug salts through the structure of PEG (Chapter 5). Overall, the work within this thesis expanded understanding of the abilities and limitations of PEG based on its distinct structure, and expanded the potential for PEG in biomedical applications to more than being used as simply a hydrophilic additive. / Thesis / Doctor of Philosophy (PhD) / Polymeric biomaterials have created significant advances in the field of biomedical engineering, however, very few polymeric drug delivery devices have achieved clinical and commercial success. Thus, the motivation for this thesis was to encourage long-term success of materials through expanding the fundamental understanding of polymer properties. Poly(ethylene glycol) was specifically chosen for study due to its unique exhibition of amphiphilic character and the ability to hydrogen-bond multiple water molecules, that together suggest the possibility for PEG to control drug release and its environment. Through strategic experimental designs, greater understanding of the abilities and limitations of PEG was established and shown to be the result of the distinct structure of PEG. Specifically, two novel drug delivery systems were developed with demonstrated understanding of the structure-function relationship between polymers and drugs, and the activity of PEG as a melanoma cell viability inhibitor was discovered and found correlated to the PEG structure. Overall the work within this thesis expanded the potential for PEG in biomedical applications to more than being used as simply a hydrophilic additive.

Elastin like peptides (ELPs)—derived from the protein elastin—are widely used as thermoresponsive components in biomaterials due to their LCST (lower critical solution temperature) behavior at a characteristic transition temperature (Tt). While linear ELPs have been well investigated, few reports focused on branched ELPs. Using lysine (Lys, with an additional side-chain amine) as branching units, ELP dendrimers were synthesized by solid-phase peptide synthesis (SPPS) with up to 155 amino acid residues. A secondary structure change with decreasing ratio of random coil and increasing ratio of β-turn upon heating, which is typical of linear ELPs, was confirmed by circular dichroism spectroscopy for all peptides. Conformational change did not show evident dependence on topology, while a higher Tt was observed for dendritic peptides than for their linear control peptides with the same number of GLPGL repeats. Variable-temperature small-angle X-ray scattering (SAXS) measurements showed a size increase and fractal dimension upon heating, even below the Tt. These results were further confirmed by cryogenic transmission electron microscopy (cryo-TEM), and micro differential scanning calorimetry (micro-DSC), revealing the presence of aggregates below the Tt. These results indicated the presence of a pre-coacervation step in the LCST phase transition of the ELP dendrimers. We further prepared hydrogels by crosslinking hyaluronic acid (HA) with ELP dendrimers. We invesigated their physical properties with scanning electron microscopy (SEM), swelling tests, SAXS, and model drug loading/release experiments. Most of the HA_denELP hydrogels retained transparent upon gelation, but after lyophilization and reswelling remained opaque for days. This reswelling process was carefully investigated with time-course SAXS studies, and was attributed to forming pre-coacervates in the gelation step, which slowly reswelled during rehydration. We then prepared hydrogels with H2S-releasing aroylthiooxime (SATO) groups and showed human neutrophil elastase-responsive H2S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. Furthermore, we prepared a series of wedge-shaped triblock polyethylene glycol (PEG)-ELP dendrimer-C16 (palmitic acid) conjugate amphiphiles with adjustable Tts. Various techniques were used to investigate their hierarchical structures. The triblock PEG-peptide-C16 conjugate amphiphiles were thermoresponsive and showed a morphology change from small micelles to large aggregates. However, the hydrophilic shell and strong tendency for micelle formation limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. The secondary structure was twisted from conventional β-sheet, and the thermoresponsive trend observed in typical ELP systems was not observed, either. Variable temperature NMR showed evidence for coherent dehydration of the PEG and ELP segments, probably due to the relatively short blocks. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release in hyperthermia. / Doctor of Philosophy / Elastin like peptides (ELPs) are similar to the protein elastin in terms of amino acid sequence. They are used widely as thermoresponsive (change in properties at different temperatures) components in biomaterials due to their abnormally lower solubility at higher temperatures. While linear ELPs have been thoroughly investigated, few investigations in ELP dendrimers have been studied. Dendrimers are molecules that branch in a controlled way to achieve sphere-like structures with rich surface functionalities. We synthesized the ELP dendrimers by using lysine amino acids as branching units. A protein secondary structure change, typical of ELPs, was observed for all peptide dendrimers. Secondary structure transitions showed no dependence on the molecular branching/linear structures, but a higher transition temperature (T_t) was observed for dendritic peptides than for their linear control peptides with the same number of amino acids. Various techniques confirmed the existence of aggregates below the T_ts, which was never reported before. We further fabricated hydrogels that mimic the native extracellular matrix, by connecting hyaluronic acid (HA) with ELP dendrimers. Interestingly, most of the hydrogels studied retained transparent upon gelation, but after freeze-drying and addition of water remained opaque for days. This phenomenon was attributed to forming of small aggregates in the gelation step, which resulted in slow reswelling. We then prepared hydrogels with H₂S-releasing groups, which showed human neutrophil elastase-responsive H₂S-releasing properties with potential applications in treating chronic diseases with recurring inflammation. We then prepared a series of wedge-shaped triblock poly (ethylene glycol) (PEG)- ELP dendrimer-alkyl chain molecules. The triblock molecules were thermoresponsive and showed a change from small spheres to large aggregates. However, the hydrophilic shell limited the thermoresponsive assembly, leading to slow turbidity change in the LCST transition. We found evidence of coherent assembly of the PEG and ELP parts, probably due to the relatively short polymer chains. Utilizing the micelles with hydrophobic cavity, we were able to encapsulate hydrophobic drugs, with promising applications for localized drug release for cancer treatment.

Osteoarthritis (OA) is a chronic joint disease characterized by irreversible cartilage degradation. MMP (matrix metalloproteinase) inhibitors represent a new approach to slowing OA progression by addressing cartilage degradation mechanisms. However, the success of preclinical studies failed to be translated into clinical application. One of the possible reasons is that the disease models in preclinical study can't reflect the biological complexity of human disease. Hydrogel-based cartilage constructs as in vitro models have shown promise as preclinical testing platforms due to their enhanced physiological relevance, improved prediction to human response, high-throughput drug screening, and ease of use. Metalloproteinase-13 (MMP-13) is thought to be a major contributor to the degradation of articular cartilage in OA by aggressively breaking down type II collagen. This study focused on testing MMP-13 inhibitors using a GelMA-alginate hydrogel-based OA model induced by cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). The results demonstrated a significant inhibition of type II collagen breakdown by measuring C2C concentration using ELISA after treatment with MMP-13 inhibitors. Therefore, the study highlights the GelMA-alginate hydrogel-based OA model as an alternative to human-sourced cartilage explants for in vitro drug screening, which can improve the predictability and relevance of preclinical evaluations of MMP-13 inhibitors for osteoarthritis, thereby complementing existing 2D culture, cartilage explant, and animal model studies and addressing the translational gap observed in clinical trials.