My thesis is divided into five chapters, starting with a general introduction in first chapter and sample preparation and protein-NMR assignment techniques in second chapter. The remaining three chapters focus on three different areas/projects that I have worked on. Chapter 1: Introduction to nanomaterials and all the experimental techniques This chapter reviews different kinds of nanomaterials and their application utilized for protein-nanomaterial interaction in our study, along with the introduction to different spectroscopy and microscopy techniques used for the interaction studies. Starting with introduction of nanomaterials and all the experimental techniques, which constitute the arsenal for structural studies of the protein-nanomaterial interaction, different steps enroute to structural and dynamic interaction are outlined in detail. Chapter 2: Preparation and Characterization of Proteins used for nanomaterial interaction studies Proteins are generally of three kinds- globular (structured), intrinsically disordered and membrane bound. These proteins have different functions in living organisms and play a major role to maintain metabolism and other important factors. To probe protein-nanomaterial interactions, we have chosen different protein/peptides. This chapter describes the protocol/procedure used for purifying the proteins. For studying a globular protein, ubiquitin was chosen. Nanomaterial-IDP interaction was investigated using the intrinsically disordered central linker domain of human insulin like growth factor binding protein-2 (L-hIGFBP2). The hydrophobic membrane interacting part of the prion protein was chosen as a representative membrane protein. The characterization of the proteins by NMR spectroscopy is also described. Chapter 3: A nanomaterial based novel macromolecular crowding agent Carbon quantum dots (CQD) are nanomaterials with size less than 10 nm, first obtained in 2004 during purification of single-walled carbon-nanotubes. Since then CQDs have been used in a wide range of applications due to their low cost of preparation and favorable properties such as chemical inertness, biocompatibility, non-toxicity and solubility in aqueous medium. One of the applications of CQDs has been their use for imaging and tracking proteins inside cells, based on their intrinsic fluorescence. Further, quantum dots exhibit concentration dependent aggregation while retaining their solubility. Fluorescent carbon quantum dots (CQD) induce macromolecular crowding making them suitable for probing the structure, function and dynamics of both hydrophilic and hydrophobic peptides/ proteins under near in-cell conditions. We have prepared hydrophilic and hydrophobic quantum dots to see the crowding effect. After characterization of CQD, we tested the property of proteins with CQD and found that CQD behaves as a macromolecular crowding agent by mimicking near in-cell conditions. In our study, we have chosen a globular protein, an intrinsically disordered protein (IDP) and one hydrophobic membrane peptide. We have also compared the crowding property of CQD with ficoll which is widely used commercial crowding agent. The overall study tells that the CQD acts like crowding agent and can be used for the study of macromolecular crowding effect. This makes them suitable for structural and functional studies of proteins in near in-cell conditions. Chapter 4: Ubiquitin-Graphene oxide interactions Described here is the interaction of human ubiquitin with GO using NMR spectroscopy and other techniques such as Fluorescence spectroscopy, isothermal titration calorimetry (ITC), UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM). The globular protein ubiquitin interacts with GO and undergoes a dynamic and reversible association-dissociation in a fast exchange regimen as revealed by NMR spectroscopy. The conformation of the protein is not affected and the primary interaction is seen to be electrostatic in nature due to the polar functional groups present on the protein and GO sheet surface. For the first time we have shown that the interaction between ubiquitin and GO is dynamic in nature with fast and reversible adsorption/desorption of protein from the surface of GO. This insight will help in understanding the mechanistic aspects of interaction of GO with cellular proteins and will help in designing appropriate functionalized graphene oxide for its biological application. Chapter 5: Section A: Interaction of an intrinsically disordered protein (L-HIGFBP2) with graphene oxide The interaction between intrinsically disordered linker domain of human insulin-like growth factor binding protein-2 (L-hIGFBP2) with GO was studied using NMR spectroscopy and other techniques such as isothermal titration calorimetry (ITC), dynamic light scattering (DLS), zeta-potential measurements. The study revealed that the disordered protein L-hIGFBP2 interacts with GO through electrostatic interaction and undergoes a dynamic and reversible association-dissociation in a fast exchange regime. The conformation of the protein is not affected. Section B: Stability of an Intrinsically disordered protein through weak interaction with Silver nanoparticles Using NMR spectroscopy and other techniques we probed the mechanism of L-hIGFBP2–AgNP interactions which render the IDP stable. The study reveals a mechanism which involves a relatively fast and reversible association–dissociation of L-hIGFBP2 (dynamic exchange) from the surface of AgNP. The AgNP–L-hIGFBP2 complex remains stable for more than a month. The techniques employed in addition to NMR include UV-Visible spectroscopy, dynamic light scattering (DLS), zeta potential measurements and transmission electron microscopy (TEM) to probe the protein-AgNP interaction here in this section.

In 2D cell cultures uptake/release of various metabolic analytes such as glucose, lactate or metabolic by-products like hydrogen peroxide from/to the extracellular environment results in concentration gradients. The magnitude, direction, and time scales of these gradients carries information that is essential for internal cellular processes and/or for communication with neighboring cells. This PhD research work focusses on the design, fabrication and characterization of electrochemical microelectrode arrays (MEAs) optimized to be positioned in commonly used 2D cell culture setups. Importantly, by simultaneously measuring accurate concentration transients and associated gradients/uxes near the cell surface (surface concentration) the capability of the device to quantify kinetic rates and distinguish mechanisms involved in various cellular processes is demonstrated. An in-situ transient calibration technique suitable for amperometric MEAs is developed and the technique is validated by quantitatively measuring dynamic concentration profiles with varying spatial (100-800 µm) and time (s to hrs.) scales set up from an electrically controlled diffusion reaction system. With the proposed MEA design and technique three physiological applications are demonstrated. Firstly, the position able 1D MEA was employed real time to quantitatively measure the hydrogen peroxide scavenging rates from astrocyte vs glioblastoma cell cultures. With the ability to extract to dynamic surface concentration and fluxes, the cell lines were shown to have hydrogen peroxide uptake rates dependent on local surface concentrations. Moreover, the cancerous glioblastoma cells demonstrated an upregulated linear peroxide scavenging mechanism as compared to astrocytes. For the next phase, spatial scales of 1D MEA device along the size and functionalization scheme of the electrodes in the MEA was further modified to selectively sense glucose and lactate to enable extracellular metabolic profiling of cancer vs normal cell lines. Secondly, measurement of glucose concentration profiles demonstrated an increased glucose uptake rate in glioblastoma as compared to astrocytes. Additionally, sigmoidal (allosteric) vs Michaelis - Menten glucose uptake kinetics was observed in glioblastoma vs astrocytes. Moreover, the presence of a glucose sensing mechanism was observed in glioblastoma cells due to the dependence of the glucose uptake rate on initial exposed concentration rather than surface concentration. Finally, simultaneous multi-analyte (glucose and lactate) gradient measurements were performed on genetically modified mouse pancreatic cancer cell lines. While glucose uptake rate was shown to increase with increasing extracellular glucose concentration for one of the cell lines, the lactate release rate was observed to be independent of the initial extracellular glucose dose.

<div>Delayed fracture healing is a major health issue involved with aging. Therefore, strategies to improve the pace of repair and prevent non-union are needed in order to improve patient outcomes and lower healthcare costs. In order to accelerate bone fracture healing noninvasively, we sought to develop a drug delivery system that could safely and effectively be used to deliver therapeutics to the site of a bone fracture. We elected to pursue the promising strategy of using small-molecule drug conjugates that deliver therapeutics to bone in an attempt to increase the efficacy and safety of drugs for treating bone-related diseases.</div><div>This strategy also opened the door for new methods of administering drugs. Traditionally, administering bone anabolic agents to treat bone fractures has relied entirely on local surgical application. However, because it is so invasive, this method’s use and development has been limited. By conjugating bone anabolic agents to bone-homing molecules, bone fracture treatment can be performed through minimally invasive subcutaneous administration. The exposure of raw hydroxyapatite that occurs with a bone fracture allows these high-affinity molecules to chelate the calcium component of hydroxyapatite and localize primarily to the fracture site.</div><div>Many bone-homing molecules (such as bisphosphonates and tetracycline targeting) have been developed to treat osteoporosis. However, many of these molecules have toxicity associated with them. We have found that short oligopeptides of acidic amino acids can localize to bone fractures with high selectivity and with very low toxicity compared to bisphosphonates and tetracyclines.</div><div>We have also demonstrated that these molecules can be used to target peptides of all chemical classes: hydrophobic, neutral, cationic, anionic, short, and long. This ability is particularly useful because many bone anabolics are peptidic in nature. We have found that acidic oligopeptides have better persistence at the site of the fracture than bisphosphonate-targeted therapeutics. This method allows for a systemic administration of bone anabolics to treat bone fractures, which it achieves by accumulating the bone anabolic at the fracture site. It also opens the door for a new way of treating the prevalent afflictions of broken bones and the deaths associated with them.</div><div>We further developed this technology by using it to deliver anabolic peptides derived from growth factors, angiogenic agents, neuropeptides, and extracellular matrix fragments. We found several promising therapeutics that accelerated the healing of bone fractures by improving the mineralization of the callus and improving the overall strength. We optimized the performance of these molecules by improving their stability, targeting ligands, linkers, dose, and dosing frequency.</div><div>We also found that these therapeutics could be used to accelerate bone fracture repair even in the presence of severe comorbidities (such as diabetes and osteoporosis) that typically slow the repair process. We found that, unlike the currently approved therapeutic for fracture healing (BMP2), our therapeutics improved functionality and reduced pain in addition to strengthening the bone. These optimized targeted bone anabolics were not only effective at healing bone fractures but they also demonstrated that they could be used to speed up spinal fusion. Additionally, we demonstrated that acidic oligopeptides have potential to be used to treat other bone diseases with damaged bone.</div><div>With these targeted therapeutics, we no longer have to limit bone fracture healing to casts or invasive surgeries. Rather, we can apply these promising therapeutics that can be administered non-invasively to augment existing orthopedic practices. As these therapeutics move into clinical development, we anticipate that they will be able to reduce the immobilization time that is the source of so many of the deadly complications associated with bone fracture healing, particularly in the elderly.</div>