Students will investigate surface modifications to controllably tailor biopolymer adhesion. In order to prevent the destructive growth of biofilms, we must understand the important parameters (e.g., chemical, physical, environmental) that contribute to biofilm adhesion. Students will learn a novel laser-induced stress wave technique for loading thin films, materials characterization techniques, and biofilm culture. The adhesion of model biopolymer films on surfaces with prescribed chemistries (self-assembled monolayers) and topography (surface roughness, surface patterning using nano fabrication techniques) will be measured by the laser-induced stress wave technique. Once surface design parameters are explored, the adhesion of cultured bacterial films will be characterized with the potential to expand to other cell types.
Incorporation of membrane proteins in functional biomaterials has been demonstrated for specific systems, but is difficult to consistently isolate and immobilize membrane proteins in their stabile and active state (1-4). We will develop a novel system to address this issue by first embedding the protein in small lipoprotein assemblies named nanodiscs, and then immobilizing the nanodiscs to silica particles via physisorption and covalent attachment with lipid-functional silanes (5,6). Diacylglycerol kinase (DGK) will be used as the model membrane protein during the development of the system (7). For Theme 1, the REU student will learn basic biochemical and molecular biology techniques to assemble the nanodisc and conduct activity assay in Dr. Wei’s lab. For Theme 2, the student will investigate the immobilization of the protein-incorporated nanodiscs to micron-scale mesoporous silica particles and characterize the location and activity of the immobilized protein in the labs of Dr. Rankin and Knutson.
1. Wagner ML, Tamm LK (2000) Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker. Biophys J 79: 1400–1414.
2. Sackmann E (1996) Supported membranes: scientific and practical applications. Science 271: 43–48.
3. Richter RP, Berat R, Brisson AR (2006) Formation of solid-supported lipid bilayers: an integrated view. Langmuir 22: 3497–3505.
4. Kusters I, Mukherjee N, de Jong MR, Tans S, Koçer A, et al. (2011) Taming Membranes: Functional Immobilization of Biological Membranes in Hydrogels. PLoS ONE 6(5): e20435. doi:10.1371/journal.pone.0020435
5. Schuler, M. A., Denisov, I. G., Sligar, S. G. (2013) “Nanodiscs as a new tool to examine lipid-protein interactions.” Methods Mol Biol. 974, 415-433.
6. Sloan, C. D., Marty, M. T., Sligar, S. G., Bailey, R. C. (2013) “Interfacing lipid bilayer nanodiscs and silicon photonic sensor arrays for multiplexed protein-lipid and protein-membrane protein interaction screening.” Anal. Chem., 85(5), 2970-2976.
7. Li, DF, Lyons, JA, Pye, VE, Vogeley, L, Aragao, D, Kenyon, CP, Shah, STA, Doherty, C, Aherne, M, Caffrey, M, Crystal structure of the integral membrane diacylglycerol kinase, Nature, 497, 7450, 2013, 521-524
Many promising therapeutics are fundamentally limited by a combination of poor solubility, stability, and temporal biodistribution. Nanoparticle drug carriers are a simple approach for the solubilization and stabilization of therapeutics. This exciting new field of therapeutics is limited by the transport of the therapy in vivo. Most intravenously delivered nanoparticles do not have sufficient time to interact with the intended site, owing to their rapid clearance from the bloodstream. Because of this rapid clearance, some drugs may need to be administered several times a week. We seek to find a way of getting these nanoparticles to stay in the bloodstream longer, so we can inject them less frequently.
In the human body, cells often function as nature’s delivery-vehicle. One specific example is how red blood cells deliver oxygen throughout the body. Interestingly, these cells circulate in the bloodstream 10-100x longer than most nanoparticles. In this project, we seek to attach nanoparticles to red blood cells, where they can “hitch hike” around the body for weeks. This summer, we will explore the concept of drug release from a small patch of nanoparticle-loaded polymer on red blood cells. In the Berron Lab, the student will learn to make these polymer coatings on glass, study the release of a cancer drug from these patches, and make the patches on cells. In the Shin lab, the student will study if these patches change the cell’s ability to travel through the circulatory system.
Objective: To develop patches for the outside of red blood cells, to allow long delivery times for a drug.
Major project outcomes:
- Design drug loaded polymer coatings on glass.
- Quantify the release of a cancer drug from these patches.
- Determine how patch design influences the release of drugs from the patch.
- Put patches on cells.
- Evaluate how the patches influence red blood cell function.
Potential methods the student will use: polymer chemistry, photochemistry, cell culture, flow cytometry, cell analysis, fluorescent microscopy techniques.
Surface-plasmon resonance (SPR) sensing is a widely used optical technique for detecting and analyzing biochemical interactions. It has found applications in diverse fields such as medical diagnostics, drug discovery, and environmental monitoring. A key challenge for SPR is maximizing sensitivity to a target analyte while minimizing response to interfering species. Dr. Hastings’ group has developed several SPR sensors that use optical methods to distinguish specific and non-specific interactions. Dr. Berron’s group has developed stable, low-density self-assembled monolayers that are ideal for functionalizing the gold surfaces common to SPR sensors. The tunable density of exposed functional groups should allow optimization of the interaction with a chosen target, while suppressing non-specific protein binding. In addition, the SPR technique itself will increase our understanding of the kinetics of protein adsorption on low-density monolayers. Students working on this project will study the interaction of low density monolayers with certain proteins, the functionalization of these monolayers to target specific interactions, and/or the further development of multi-mode surface-plasmon resonance sensor systems.
The immobilization of enzymes (for biocatalysis) and specific protein channels (for selective transport) is an important area of research where both selective separations and reactivity is needed. Layer-by-layer (LbL) assembly technique, most commonly conducted by intercalation of positive and negative polyelectrolytes, is a powerful, versatile and simple method for assembling supramolecular structures and the development of nanostructured materials. Non-stoichiometric immobilization of charged polyelectrolyte assemblies within confined pore geometries leads to an enhanced volume density of ionizable groups in the membrane phase. The immobilization of enzymes for bio-catalysis (such as, Glucose oxidase, Catalase) in LBL assembly provides highly enhanced activity. The protein channel creation will require crosslinking with segments of the LbL assembly. The REU student will be working on the synthesis of functionalized materials, formation of LBL assembly in membrane pores, permeability and enzyme activity measurements, and protein channel separation selectivity. This project will advance their knowledge further in functionalization chemistry, material characterization, and applications in selective separation and biocatalytic reaction area.
Infection remains a primary complication following surgical treatment of injuries to the face, with mandibular and maxillary fractures being two of the most common. The use of prophylactic antibiotics remains controversial, with reports of increased complications and development of resistant microorganisms. Cationic antimicrobial peptides, such as human β-defensin 3 (hBD3), are naturally occurring molecules possessing broad antimicrobial activity. The overall goal of this project is to develop coatings that induce localized expression of hBD3 for preventing colonization of fracture fixation hardware and the consequent establishment of infection. First, polyplexes will be developed to enable efficient transfection and subsequent secretion of the peptide. Second, these vectors will be encapsulated into biodegradable formulations for coating on metallic plates or as an osteoconductive bone filler.
In this collaborative project between Drs. Puleo and Pack, the undergraduate student researcher will conduct experiments that fit two themes. In Theme 1, biodegradable materials will be formulated with plasmid encoding an antimicrobial peptide to kill bacteria. For Theme 2, the delivery system, the drug delivery profiles, and in vitro bioactivity of released molecules will be characterized.
Microfiltration membranes are commonly used for filtration of suspended solids, bacteria, etc. However, one can create advanced membranes by functionalization of the pores with appropriate macromolecules, nanostructured catalysts, and providing applications ranging from stimuli-responsive flux and separations at low pressure, toxic metal capture, green chemical synthesis, to toxic organics destruction. Various base membranes, such as, cellulosics, polycarbonate, alumina, poly vinylidene fluoride (PVDF), polysulfone, polyamide, etc. can be used to create responsive membranes using simple functionalization approaches. The REU students will work on creation of highly porous phase inversion membranes and subsequent functionalization of pores by stimuli responsive polymers (such as, poly-glutamic acid). Degradation kinetics of model organic pollutants will be studied. In addition, synthesis will include layered assembly for enzyme catalyzed green synthesis of chemicals. The use of SEM, TEM and other advanced instruments will further enhance student’s knowledge in the membrane material characterization area.
Metastasis is the cause of 90 % of cancer related deaths, and one protein that has been associated with metastasis is CD151. CD151 is a tetraspanin that, while present on all cells, is up regulated in cancers with poor prognoses. However, literature is conflicting about the role of CD151 in metastasis due to the fact that in vivo models have poor visualization and low control over variables, and until recently, many in vitro techniques have not been able to mimic the environment inside of a blood vessel. Therefore, it has been impossible to run studies to fully examine the effects of CD151 on metastasis. With the employment of microfluidics, it has been possible to study this phenomenon to gain greater understanding of this process. This project aims to have students utilize modern microfluidic assays for studying the effects of CD151 on metastasis. Students will utilize microscopy imaging techniques, cell culture, and microfabrication processes in order to study this phenomenon.
All living systems have a need to transport critical nutrients throughout their structures. This need is a critical challenge in the next generation of medical devices which use live cells to perform basic functions. It is also one of the primary challenges in engineering thick three-dimensional tissues. In these systems, the flow of nutrients needs to be uniform throughout the material at the micron-scale. In vivo, this is accomplished by an integrated circulatory system, but the detailed multi-scale geometry involved is particularly difficult to recreate ex vivo. In this project, we seek to use lithography-based microfabrication to generate 3D cell/hydrogel structures with embedded microfluidic channels.
Objective: To develop hydrogel-based microfluidic devices that mimic in vivo blood flow
Major project outcomes:
- Develop a technique for creating multi-layered PEG-diacrylate and collagen microfluidic devices.
- Measure the accuracy of the patterning and adhesion strength between layers.
- Measure the distribution of materials flowing through the micropatterned device.
- Incorporate cells in the devices and measure long-term viability.
Potential methods the student will use: microfluidic device manufacturing, cell culture, polymer chemistry, photochemistry, microscopy techniques.
Iron oxide nanoparticles have the ability to enhance the production of reactive oxygen species (ROS) within cells through catalyzing the Haber-Weiss reaction (Fenton chemistry) which produces the hydroxyl radical. Cancer cells are more susceptible to oxidative insults compared to normal cells due to fast cell proliferation and metabolism so additional ROS stress induced by exogenous agents can overwhelm the relatively low antioxidant capacity and disrupt the redox homeostasis inside cancer cells leading to selective tumor cell toxicity. Iron oxide nanoparticles have been previously studied due to their multitude of biological applications, inherent biocompatibility, magnetic properties, and lack of protein adsorption after proper coating. Therefore, iron oxide nanoparticles coated with dextran will be used to improve the efficacy of radiation by enhancing the intracellular ROS production.
Due to the half-life of the hydroxyl radical being on the order of a nanosecond, the location of the production of the hydroxyl radical production is very important. If ROS can be generated at or within the nuclear envelope, the probability of interacting with the DNA and resulting in a double strand break increases. Therefore, the iron oxide nanoparticles will be conjugated with a Nuclear Localizing Signal (NLS) peptide. This signal has been shown to interact with cytosolic factors forming stable complexes that are docked at the nuclear pore complex (NPC) in the nuclear membrane. In order to target the nuclear envelope, it is important for the nanoparticle to escape the endosome after receptor-mediated or non-specific endocytosis. The NLS conjugated to the iron oxide nanoparticle surface must be able to interact with the cytoplasm in order to facilitate exiting the endosome.
As an undergraduate scholar, your contribution to this project with focus on developing an iron oxide nanoparticle conjugated with a NLS for localization of the nanoparticle at the nuclear envelope. The iron oxide nanoparticle can then catalyze the Haber-Weiss reaction through Fenton chemistry to enhance intracellular ROS generation with the expectation that this oxidative stress combined with radiation treatment will lead to synergistic treatment of lung cancer. This project will involve cell culture, confocal microscopy and nanoparticle characterization techniques.
Surface-plasmon resonance (SPR) sensing has become a mainstay of drug discovery, and there are many emerging applications in food safety, environmental monitoring, and medical diagnostics [1, 2]. However, interfering effects such as non-specific binding and changes in solution refractive index limit SPR sensing in complex media. Drs. Hastings and Wei are developing surface plasmon resonance sensors that compensate for these effects by exciting multiple surface-plasmon modes on thin metal films or metallic nanostructures [3-14]. Thanks to NSF support, they recently demonstrated that U-shaped gold nanostructures with three localized resonances can compensate for both non-specific protein binding and index changes in liquid volumes less than a cubic micron . This project offers an excellent platform for an REU experience because the instrumentation and methodologies are in place, and the REU student can immediately focus novel systems and translational applications. In Theme 1, the students will evaluate sensing architectures involving new metal nanostructure geometries and their functionalization for protein recognition. In Theme 2, the students will focus on evaluating these sensors’ performance for detection and bio-interaction analysis in the presence of interfering effects.
 A. P. F. Turner, “Biosensors: sense and sensibility,” Chemical Society Reviews, vol. 42, pp. 3184-3196, April 2013.
 H. Šípová and J. Homola, “Surface plasmon resonance sensing of nucleic acids: A review,” Analytica Chimica Acta, vol. 773, pp. 9-23, April 2013.
 N. Nehru, E. U. Donev, G. M. Huda, L. L. Yu, Y. N. Wei, and J. T. Hastings, “Differentiating surface and bulk interactions using localized surface plasmon resonances of gold nanorods,” Optics Express, vol. 20, pp. 6905-6914, Mar 2012.
 N. Nehru, L. Yu, Y. Wei, and J. T. Hastings, “Reference Compensated Localized Surface Plasmon Resonance based Sensor,” in IEEE Nano: 12th International Conference on Nanotechnology, Birmingham, UK, 2012.
 P. D. Keathley and J. T. Hastings, “Nano-gap-Enhanced Surface Plasmon Resonance Sensors,” Plasmonics, vol. 7, pp. 59-69, Mar 2012.
 J. Guo, P. D. Keathley, and J. T. Hastings, “Dual-mode surface-plasmon-resonance sensors using angular interrogation,” Optics Letters, vol. 33, pp. 512-514, Mar 2008.
 P. D. Keathley and J. T. Hastings, “Optical properties of sputtered fluorinated ethylene propylene and its application to surface-plasmon resonance sensor fabrication,” Journal of Vacuum Science & Technology B, vol. 26, pp. 2473-2477, Nov 2008.
 J. T. Hastings, J. Guo, P. D. Keathley, P. B. Kumaresh, Y. Wei, S. Law, et al., “Optimal self-referenced sensing using long- and short- range surface plasmons,” Opt. Express, vol. 15, pp. 17661-17672, Dec 2007.
 J. T. Hastings, N. Nehru, and M. D. Bresin, “Focused electron-beam induced deposition of plasmonic nanostructures from aqueous solutions,” in Proc. SPIE 8613, Advanced Fabrication Technologies for Micro/Nano Optics and Photonics VI, San Francisco, CA, 2013, pp. 861306-861306-7.
 N. Nehru and J. T. Hastings, “Optical sensing characteristics of nanostructures supporting multiple localized surface plasmon resonances,” in Proc. SPIE 8594, Nanoscale Imaging, Sensing, and Actuation for Biomedical Applications X, San Francisco, CA, 2013, pp. 859405-859405-6.
 L. Webb and J. T. Hastings, “Silver in dual-mode Surface-Plasmon Resonance sensors,” in IEEE Southeastcon, 2009, pp. 13-17.
 J. T. Hastings, “Optimizing Surface-Plasmon Resonance Sensors for Limit of Detection Based on a Cramer Rao Bound,” IEEE Sensors Journal, vol. 8, pp. 170-175, Feb. 2008.
 M. Florea, J. T. Hastings, and C. Adeola, “Wavelength shift analysis techniques and methods for dual mode surface plasmon resonance sensors,” in IEEE Southeastcon, 2008, pp. 432-435.
 J. T. Hastings, “Spectral peak-shift estimation with wavelength dependent sources and detectors,” IEEE Transactions on Signal Processing, vol. 56, pp. 5269-5272, Oct 2008.
 N. Nehru, L. Yu, Y. Wei, and J. T. Hastings, “Using U-Shaped Localized Surface Plasmon Resonance Sensors to Compensate for Nonspecific Interactions,” Ieee Transactions on Nanotechnology, vol. 13, pp. 55-61, Jan 2014.
Drug delivery of proteins from controlled-release matrices is complicated by the poor stability of the proteins in the solvents used to prepare the matrices, as well as the need to store the protein without degradation.1 One approach has been to use crystalline proteins that do not dissolve in the solvents and can be incorporated into the matrix.2 However, this limits the number of proteins that can be used. In Theme 1, the students will prepare particles of prestabilized protein/sugar matrices that can then be incorporated into biodegradable polymeric matrices. In Theme 2, the release kinetics of the protein, including in vivo stability of the protein upon release, will be studied. In Theme 3, the students will manufacture implants that can serve as drug delivery implants. An essential element of this project is the use of solid-state NMR spectroscopy to evaluate the protein in situ within the matrices, which can then be used to predict protein stability.
(1) Wu, F.; Jin, T. “Polymer-Based Sustained-Release Dosage Forms for Protein Drugs, Challenges, and Recent Advances”, AAPS Pharmscitech 2008, 9, 1218.
(2) Pechenov, S.; Shenoy, B.; Yang, M. X.; Basu, S. K.; Margolin, A. L. “Injectable controlled release formulations incorporating protein crystals”, J. Control. Release 2004, 96, 149.
Plants offer an extraordinary variety of bioactive small molecule metabolites that are potentially valuable as pharmaceuticals, nutraceuticals and agrochemicals. Production of these metabolites in plant cell cultures (e.g., hairy root cultures), can be directed through genetic manipulation or environment, such as exposure to pathogens. This proposal aims to generate nanoparticles which bind selective components of plant microbial pathogens and which are taken up by plant cells in cultures to elicit a specific defensive secondary metabolite. Porous, high surface area silica nanoparticles will be designed as a pathogen carrier into the plant cells, where the particle size, pore size, and surface functionalization will be the properties that determine the effectiveness of the carrier (Theme 1). The viability of hairy root cultures, particle uptake, and secondary metabolite production will be used as measures of the effectiveness of the carrier/pathogen combination (Theme 2). This research takes advantage of our ability to synthesis tailored silica platforms and evidence for plant cell viability following the uptake of nanoparticles. Elicitation of selective defensive metabolites through nanoparticle delivery has application to both the production of high value added therapeutics as well as understanding the mechanism of the pathogens, which have difficulty passing into the cell directly.