The 2020 Fall Schoenborn Graduate Research Symposium Was a Success!

Background: Fibroblasts migrate directionally in response to numerous cues. For instance, in chemotaxis, cells migrate in response to gradients of soluble chemoattractants, while in haptotaxis, cells migrate in response to gradients of surface-bound extracellular matrix (ECM). The directed migration of fibroblasts supports critical biological events such as wound healing and tissue development, while errant migration exacerbates cancer metastasis. The current literature exploring these processes relies heavily on population based, endpoint assays, such as the Boyden Chamber. However, Boyden Chamber assays suffer from transient gradients, confound numerous modes of invasion, and do not provide taxis information at the individual cell level. Furthermore, fibroblast haptotaxis may be understudied given that chemotaxis publications outnumber haptotaxis publications approximately 30:1.

Results: We tackle these issues using a novel combination of microfluidic gradient generating devices and evaporative paper pumps [1, 2] in an assay that is compatible with total internal reflection fluorescence microscopy (TIRF-M). Our microfluidic design generates stable, bubble-free flow with soluble gradients of various steepnesses for over ten hours. Additionally, each experiment produces hundreds of cell trajectories, which is an order of magnitude increase over competing technologies. We analyze the high throughput data using in-house automated cell motility and cell signaling tracking software. Our results reveal that NIH/3T3 fibroblasts chemotax in a preferred platelet derived growth factor (PDGF) gradient regime that aligns with a previous model of NIH/3T3 PI3K signaling responses [3, 4]. In addition to soluble chemotactic cues, we produce surface bound haptotactic cues using a similar microfluidic approach. Our preliminary haptotaxis data shows that the IA32 fibroblast cell line haptotaxes robustly, while simultaneously disrupting and consuming fibronectin as it migrates.

Conclusions: We have developed a workflow that can produce response maps of fibroblast chemotaxis and haptotaxis over a comprehensive gradient landscape. Key to our workflow is a bubble-mitigating microfluidic device enabled by a passive pumping system that maintains stable gradients. Our high-throughput chemotactic data leads us to suggest that chemotaxing fibroblasts may be particularly adept at converting PI3K signaling into forward motility specifically in a preferred gradient regime. Furthermore, our preliminary haptotactic data leads us to hypothesize a potentially novel directed migration mechanism that we call phagotaxis, whereby cells self-generate local, steepened haptotactic gradients via endocytosis of ECM.

References:

1. Cummins, Brian M., et al. Technology (2017)
2. Shay, Tim, et al. NCSU Thesis (2017)
3. Schneider, I. C. & Haugh, J. M. J. Cell Biol. (2005)
4. Welf, E, J. Cell Biol. (2012)

Background: Among all types of Li-ion battery (LIB) separators, fibrous mats have the advantage of low cost, low mass, and high porosity. Fibrous Poly(Vinylidene difluoride) (PVDF) shows promising results because of its stability and affinity for electrolytes commonly employed in Li-ion cells. Despite numerous studies published on fibrous LIB separators, none reports structure-property relationships for the identification of an ideal structure. We investigated the properties of a melt-blowable PVDF and produced meltblown PVDF mats in scale-up equipment with the objective of elucidating its performance as a LIB separator. We also present a new class of LIB separators, PVDF-based highly branched, colloidal polymer particulates called soft dendritic colloids that are produced by shear-driven polymer precipitation within a highly turbulent nonsolvent flow, followed by filtration. We show that the morphology of the resulting PVDF particulates can be modulated from fibrous soft dendritic colloids (SDC) to thin and highly porous sheet-like particles.

Results: Through a scale-up system, we obtained high-quality meltblown PVDF with high homogeneity, low number of defects, an average fiber diameter of 1.4 μm, and pore size as low as 0.9 μm. Small fiber diameter provides high-surface area and high electrolyte uptake. We show interactions of the meltblown PVDF with the electrolyte lead to a morphology change in the fibers. The highest ionic conductivity was ~ 9.6 mS/cm, and the first-cycle capacity was 140 mAh/g (Li/LiCoO2). After melt-pressing, the thickness and pore size decrease, but the mats electrolyte absorbency and conductivity decrease commensurately. PVDF SDC separators show high porosity (up to 80%) and high particle surface area, which results in high conductivity (1.2 mS/cm), high-electrolyte uptake (325%), and high-cell capacity (112 mAh/g in Li/LiCoO2 cell) with <10% loss after 50 cycles. Both processes yield separators with low thermal shrinkage (<5% at 90 oC) and high tensile strength (<0.5% offset at 1000 psi), with the highest-performing separator possessing low average fiber diameter with a wide diameter distribution.

Conclusions: Both meltblowing and shear-driven precipitation are facile and versatile processes for high volume fabrication of LIB separators with one single polymer without necessarily requiring post-processing and with characteristics similar to commercially available battery separators. Fibrous LIB should be fabricated with a low fiber diameter (<1 μm) but also with a wide diameter distribution. When the strength and openness of the microfiber support is coupled with the highly permeable nanofibers for a low average pore size, an ideal fibrous Li ion battery separator is obtained.

References:

1. Luiso, S., Henry, J. J., Pourdeyhimi, B. & Fedkiw, P. S. (2020). Fabrication and Characterization of Meltblown Poly(vinylidene difluoride) Membranes. ACS Appl. Polym. Mater., 2, 2849 2857
2. S. Roh, A. H. Williams, R. S. Bang, S. D. Stoyanov, and O. D. Velev (2019). Soft dendritic microparticles with unusual adhesion and structuring properties. Nature Materials, vol. 18, no. 12. pp. 1315–1320.

Background: Stimuli responsive materials have been a topic of intensive scientific interest due to their perspective applications in engineered materials response to stimuli such as optical, mechanical, and magnetic fields. This ability to tune the material properties could be very valuable in numerous fields such as biomedicine, soft robotics, coatings, catalysts, filtration membranes, and 3D printed materials [1]. Magnetic-field-directed assembly of superparamagnetic nanoparticles, for example, allows for the remote control of the particles’ organization as well as the generation of anisotropic nanostructured materials [2]. [3,4].

Results: Magnetic interactions can be used to form smart gel systems. We present here a system composed of soft micromagnets of polydimethylsiloxane (PDMS) beads that have internally embedded magnetic nanoparticles (MNPs). The organization of the internalized magnetic nanoparticles can be controlled via magnetic-field directed assembly, allowing for the generation of permanent, anisotropic, chained nanoparticle structures in PDMS beads. Once magnetized, the beads’ magnetic interactions result in the formation of linear and branched networks. The chain-containing microspheres could have a permanently embedded dipole moment and hence, residual polar magnetization. We also present two types of magneto-capillary gels that differ in the location of the MNPs. The capillary bridges are composed of a fatty acid that wets and brings together the PDMS beads, thus forming the gels. The first magnetically responsive capillary gel (MRCG1) has the MNPs in the PDMS beads and MRCG2 has the MNPs in the capillary bridges. We evaluate the differences in rheological properties of the gels and compare their magnetic properties. We also discuss their self-repairing abilities.

Conclusions: We proved the tunability of the new chained soft bead systems by conducting magnetization, demagnetization, and re-magnetization experiments that show evidence of the successful reformation of percolating networks. We are currently using magnetic interaction templating to study and manipulate the magnetic response of our systems. The direct structure templating could potentially improve the precision of rheological measurements by anchoring the gels to 3D printed patterns We will discuss how the control of the capillary forces and magnetic interactions can be used to form new smart multiphasic gel systems. Overall, we have constructed and are developing further a rich toolbox of structures and interactions for making novel magneto-responsive gel networks.

References:

1. Schneider, Monica, Koos, Erin, and Willenbacher, Norbert (2016). Highly conductive, printable pastes from capillary suspensions. Sci. Rep., 6:1-10.
2. Fameau, Anne-Laure, Lam, Stephanie, and Velev, Orlin D. (2013). Multi-stimuli responsive foams combining particles and self-assembling fatty acids. Chem. Sci., 4: 3874-3881.

Background: Sweat is an important biofluid for monitoring individuals’ health as it contains a number of essential biomarkers. However, sampling sweat for analysis still remains challenging as most of the commercially available sweat sensing devices are either invasive in nature or work only under active perspiration [1]. These devices fail to function under low-sweating conditions and are incapable of deriving sweat measurements from subjects at rest. We demonstrate a new principle for the design of flexible and wearable devices, which are capable of extracting sweat under both sedentary and actively perspiring conditions using osmotic pressure difference for pumping, and evaporation for liquid disposal [2]. The device is constructed of silicone, polyacrylamide hydrogel patch, and paper microfluidic conduit with a site of evaporation at the end (evaporation pad). The hydrogel is equilibrated with glycerin, glucose, or NaCl solution to build up the desired osmotic strength.

Results: In-vitro testing with gelatin-based model skin platform has initially revealed that both glucose and glycerin treated hydrogels facilitate high dye (model biomarker in model skin) accumulation on the evaporation pad, with glucose being the highest. Human trials with devices embedded with a glucose hydrogel have shown the potential of the prototype to extract sweat containing lactate under both resting and non-resting (during exercise) conditions within a period 2 hours. Results reveal the following two observations: (1) sweat lactate increases with exercise and (2) there is no direct correlation between the blood and sweat lactate. Integration of a “glycerogel” (hydrogel treated with pure glycerin) hosted patch with microneedles have shown the potential of this combined system to sample model biomarker from model skin. Such an arrangement is essential for long-term interstitial fluid (ISF) sampling since ISF glucose levels are similar to that in blood. A continuous sweat lactate sensing platform is also currently being established on the prototype by using enzymatic electrochemical sensors on a polyimide film, which is adhered directly onto the paper microfluidic channel. Initial testing with this integrated platform has shown that this wearable prototype can be easily tuned for continuous sweat lactate monitoring.

Conclusions: The novel simple, low cost, continuous sweat sampling platform with specific sensors can either be worn directly onto the skin or be used as a wearable, that can reveal a throve of health information. We are presently expanding our prototype to a lateral flow assay (LFA) platform and working towards utilizing it for early disease diagnosis from sweat.

References:

1. Min S, Balooch G, Kang D, Koh A, Kim J, Pielak RM, et al. (2016). A soft, wearable microfluidic device for the capture, storage, and colorimetric sensing of sweat. Sci Transl Med., 8(366), 366ra165-366ra165.
2. Shay, T., Saha, T., Dickey, M. D. & Velev, O. D. Principles of long-term fluids handling in paper-based wearables with capillary-evaporative transport (2020). Biomicrofluidics 14(034112), 1-9.

Background: Wearable electronics have attracted great attention over the past decade due to their potential applications in health care and human-machine interfaces. Next generation wearable technology demands compatible energy sources that are soft, stretchable, and sustainable. Efforts have been made to develop energy harvesters that can convert human motion/ambient mechanical energy to electrical energy.[1-4] Triboelectric and piezoelectric harvesters are prominently used to convert mechanical energy to electrical energy.[1-4] Triboelectric harvesters have gained great popularity due to their relatively straightforward fabrication. They induce electrical current based on contact electrification (rubbing two surfaces) and electrostatic induction (oscillating the distance between charged surfaces).[1,3-5] Triboelectric harvesters generate high voltages and low currents. They require moisture free environments to enable contact electrification.[1,3-5] Piezoelectric harvesters, in contrast, require extensive fabrication techniques and loss in energy conversion when converted to flexible devices, as their fundamental materials are intrinsically hard and brittle.[1-3]

Results: We report a new approach to energy harvesting that utilizes liquid metals. These devices generate around 1mW/m2 by harnessing energy from mechanical motion. We have characterized the behavior of these devices as a function of a variety of parameters including material properties and physical deformation. The devices behave as expected and the response of the devices to deformation match a physics-based model.

Conclusions:The liquid metal based soft devices generate electrical signal when deformed, which may be useful for energy harvesting as well as self-powered sensors. These devices can be used to monitor human activities thereby find many applications in wearable electronics, dynamic tactile surfaces, healthcare systems like rehabilitation and prosthetics.

References:

1. Zhou, M. et al. A review on heat and mechanical energy harvesting from human – Principles, prototypes and perspectives. Renewable and Sustainable Energy Reviews 82, 3582–3609 (2018).
2. Sim, H. J. et al. Flexible, stretchable and weavable piezoelectric fiber: Flexible, Stretchable and Weavable Piezoelectric. Advanced Engineering Materials 17, 1270–1275 (2015).
3. Yang, Y. et al. Liquid-Metal-Based SuperStretchable and Structure-Designable Triboelectric Nanogenerator for Wearable Electronics. ACS Nano 12, 2027–2034 (2018).
4. Gao, S. et al. Wearable high-dielectric constant polymers with core–shell liquid metal inclusions for biomechanical energy harvesting and a self-powered user interface. Journal of Materials Chemistry A 7, 7109–7117 (2019).
5. Wang, Z. L., Jiang, T. & Xu, L. Toward the blue energy dream by triboelectric nanogenerator networks. Nano Energy 39, 9–23 (2017).

Background:Clostridium difficile infection is the leading cause of diarrhea and colitis (inflammation of the colon) in North America and Europe. There is a growing concern about the failure of the first line of treatment for C. diff infection (metronidazole and vancomycin), and thus current attention is focused on the search for alternative treatment options like biologic drugs that might be safer or more efficacious [1]. A viable strategy is the development of targeted peptide-based therapeutics that prevent and treat C. diff infections by deactivating the pathogen while leaving the resident gut microbiota unharmed. During infection, the C. diff pathogen produces two large virulent toxins (Toxin A and B) that share 71% sequence homology. The glucosyltransferase domain (GTD) of these toxins acts by binding UDP (Uridine diphosphate)-glucose, hydrolyzing it into glucose and UDP, and attaching the glucose monomer to the human Rho family of GTPases. Glycosylation of the GTPases by TcdA and TcdB GTDs leads to disruption of the cytoskeleton of the host cells, apoptosis, and cell death [2]. The objective of this project is to computationally design peptide inhibitors that bind with high affinity and specificity to the glucosyltransferase domain of C. diff toxins A and B to inhibit their activity. We have developed a computational approach to discover peptide sequences that bind to a biomolecular target with high affinity. Our peptide search algorithm employs Monte Carlo methods, self-consistent mean-field theory, and the concerted rotation (CONROT) technique [3]. The algorithm requires a reference ligand, an experimentally determined peptide sequence that has a good binding affinity to the target protein, to start the search.

Results: We have discovered two peptides, NPA and NPB, that can inhibit the glucosyltransferase activity of Toxin A GTD using EGWHAHTGGG (Kd 100 nM) as the reference ligand, a peptide previously identified by the Feig lab using phage display. Simulations of NPA and NPB at the binding site of Toxin A showed that they have a high affinity for Toxin A. Calculations of the binding free energy for these peptides based on atomistic molecular dynamics simulations suggest that the computationally discovered peptides are better binders than the reference ligand. Experimental results demonstrate highly effective neutralization of TcdA toxicity for NPA in jejunum cells (human small intestine) but no efficacy when tested in human colonic epithelial cells (large intestine).

Conclusions: Current work is focused on furthering our understanding of the interaction of these peptides with the toxins, redesigning shorter peptide inhibitors for Toxin A, and discovering peptides that can bind to Toxin B.

References:

1. Ananthakrishnan, AN (2011). Clostridium difficile infection: epidemiology, risk factors and management. Nat. Rev. Gastroenterol. Hepatol., 8(1): 17-26.
2. Alvin, J.W. and Lacy, D.B (2017). Clostridium difficile toxin glucosyltransferase domains in complex with a non-hydrolyzable UDP-glucose analogue. J. Struct. Biol., 198(3): 203-209
3. Xiao, X., Agris, P.F and Hall, C.K (2017). Extended Concerted Rotation Technique Enhances the Sampling Efficiency of the Computational Peptide-Design Algorithm. J. Chem. Theory. Comput. 13, 5709-5720.