Permanent URI for this collection
Browse
Recent Submissions
Publication Decoupling the Effects of Charge Density and Hydrophobicity on the Phase Behavior and Viscoelasticity of Complex Coacervates(American Chemical Society (ACS), 2024-05) Ramírez Marrero, Isaac André; Boudreau, Luke; Hu, Weiguo; Gutzler, Rainer; Kaiser, Nadine; von Vacano, Bernhard; Konradi, Rupert; Perry, SarahHere, we explore the effect of copolymer chemistry on the phase behavior and viscoelasticity of complex coacervates. To this end, we utilized a library of methacrylate copolymers with varying charge densities and hydrophobicity. Our results show that changing the charge density and hydrophobicity drastically affects the phase behavior─with charge density dictating the salt stability and hydrophobicity controlling the polymer concentration of the coacervates. Small-amplitude oscillatory shear measurements were used to study the viscoelastic response of the coacervates, leveraging knowledge of the coacervate phase behavior in tandem with time-salt superposition to construct a series of time-salt-copolymer master curves that highlight the effects of polymer charge density and hydrophobicity. These combined data show evidence of charge-dominated and hydrophobicity-dominated regimes, allowing for an understanding of how copolymer chemistry can be used to tune the mechanical properties of complex coacervates.Publication Physical adsorption analysis of intact supported MFI zeolite membranes(2007) Hammond, KD; Tompsett, GA; Auerbach, SM; Conner, WCPublication In situ SAXS and WAXS of zeolite microwave synthesis(2007) Tompsett, GA; Panzarella, BA; Conner, WC; Bennett, S; Jones, KWPublication In situ SAXS/WAXS of zeolite microwave synthesis: NaY, NaA, and beta zeolites(2007) Panzarella, B; Tompsett, G; Conner, WC; Jones, KPublication In situ small angle x-ray scattering, wide angle x-ray scattering, and Raman spectroscopy of microwave synthesis(2006) Tompsett, GA; Panzarella, B; Conner, WC; Yngvesson, KS; Lu, F; Suib, SL; Jones, KW; Bennett, SPublication Microwave synthesis of zeolites. 2. Effect of vessel size, precursor volume, and irradiation method(2007) Panzarella, B; Tompsett, GA; Yngvesson, KS; Conner, WCPublication Apparatus for measuring physical adsorption on intact supported porous membranes(2007) Hammond, KD; Tompsett, GA; Auerbach, SM; Conner, WCPublication Encapsulation of Inorganic Nanoparticles by Anionic Emulsion Polymerization of Diethyl Methylene Malonate for Developing Hybrid Microparticles with Tailorable Composition(2024) Beltramo, Peter J.Colloidal particle self-assembly into higher-ordered structures has been of great interest due to the promise of creating metamaterials with novel macroscopic properties. The physicochemical properties of these metamaterials can be tailored to achieve composites with tunable functionalities, either by controlling the assembly morphology and/or chemistry of the colloidal building blocks. This work describes a strategy of developing microparticles with a hybrid configuration that have an inorganic and an organic part. The inorganic part comprises functional nanoparticles, which are embedded within an organic polymer particle composed of diethyl methylene malonate polymer [p(DEMM)] prepared using anionic emulsion polymerization. DEMM polymerization is initiated entirely by the presence of hydroxyl anions and the resulting particle diameter can be tuned between 300 nm and 1 micrometer by reaction pH. Inorganic nanoparticles with varying chemistry (TiO2, CdTe, ZnO) can be loaded into the p(DEMM) particle with a controlled weight fraction, as confirmed by thermogravimetric analysis. The colloidal stability of the composite microparticles is seen to be dependent on the ligand coating attached to the inorganic constituent. These results provide a synthetic groundwork for creating hybrid, stimuli-responsive microparticles.Publication Ultrasonic nozzle spray in situ mixing and microwave-assisted preparation of nanocrystalline spinel metal oxides: Nickel ferrite and zinc aluminate(2008) Nyutu, EK; Conner, WC; Auerbach, SM; Chen, CH; Suib, SLPublication Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917(2024) Lebovich, Matthew; Lora, Marcos A; Gracia-David, Jared; Andrews, LaurenEngineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.Publication Self-assembling polypeptides in complex coacervation(2024) Sathyavageeswaran, Arvind; Sabadini, Júlia Bonesso; Perry, Sarah L.Publication Design Rules for Sequestration of Viruses into Polypeptide Complex Coacervates(2023) Joshi, Pratik U.; Decker, Claire; Zeng, Xianci; Sathyavageeswaran, Arvind; Perry, Sarah L.; Heidt, Caryn L.Encapsulation is a strategy that has been used to facilitate the delivery and increase the stability of proteins and viruses. Here, we investigate the encapsulation of viruses via complex coacervation, which is a liquid–liquid phase separation resulting from the complexation of oppositely charged polymers. In particular, we utilized polypeptide-based coacervates and explored the effects of peptide chemistry, chain length, charge patterning, and hydrophobicity to better understand the effects of the coacervating polypeptides on virus incorporation. Our study utilized two nonenveloped viruses, porcine parvovirus (PPV) and human rhinovirus (HRV). PPV has a higher charge density than HRV, and they both appear to be relatively hydrophobic. These viruses were compared to characterize how the charge, hydrophobicity, and patterning of chemistry on the surface of the virus capsid affects encapsulation. Consistent with the electrostatic nature of complex coacervation, our results suggest that electrostatic effects associated with the net charge of both the virus and polypeptide dominated the potential for incorporating the virus into a coacervate, with clustering of charges also playing a significant role. Additionally, the hydrophobicity of a virus appears to determine the degree to which increasing the hydrophobicity of the coacervating peptides can enhance virus uptake. Nonintuitive trends in uptake were observed with regard to both charge patterning and polypeptide chain length, with these parameters having a significant effect on the range of coacervate compositions over which virus incorporation was observed. These results provide insights into biophysical mechanisms, where sequence effects can control the uptake of proteins or viruses into biological condensates and provide insights for use in formulation strategies.Publication Challenges and Opportunities Modeling the Dynamic Tumor Matrisome(2023) Peyton, Shelly R.; Platt, Manu O.; Cukierman, EdnaWe need novel strategies to target the complexity of cancer and, particularly, of metastatic disease. As an example of this complexity, certain tissues are particularly hospitable environments for metastases, whereas others do not contain fertile microenvironments to support cancer cell growth. Continuing evidence that the extracellular matrix (ECM) of tissues is one of a host of factors necessary to support cancer cell growth at both primary and secondary tissue sites is emerging. Research on cancer metastasis has largely been focused on the molecular adaptations of tumor cells in various cytokine and growth factor environments on 2-dimensional tissue culture polystyrene plates. Intravital imaging, conversely, has transformed our ability to watch, in real time, tumor cell invasion, intravasation, extravasation, and growth. Because the interstitial ECM that supports all cells in the tumor microenvironment changes over time scales outside the possible window of typical intravital imaging, bioengineers are continuously developing both simple and sophisticated in vitro controlled environments to study tumor (and other) cell interactions with this matrix. In this perspective, we focus on the cellular unit responsible for upholding the pathologic homeostasis of tumor-bearing organs, cancer-associated fibroblasts (CAFs), and their self-generated ECM. The latter, together with tumoral and other cell secreted factors, constitute the “tumor matrisome”. We share the challenges and opportunities for modeling this dynamic CAF/ECM unit, the tools and techniques available, and how the tumor matrisome is remodeled (e.g., via ECM proteases). We posit that increasing information on tumor matrisome dynamics may lead the field to alternative strategies for personalized medicine outside genomics.Publication Live Cell Lineage Tracing of Dormant Cancer Cells(2023) Kim, Hyuna; Wirasaputra, Anna; Mohammadi, Farnas; Kundu, Aritra Nath; Esteves, Jennifer; Heiser, Laura; meyer, Aaron; Peyton, ShellyBreast cancer is a leading cause of global cancer-related deaths, and metastasis is the overwhelming culprit of poor patient prognosis. The most nefarious aspect of metastasis is dormancy, a prolonged period between primary tumor resection and relapse. Current therapies are insufficient at killing dormant cells; thus, they can remain quiescent in the body for decades until eventually undergoing a phenotypic switch, resulting in metastases that are more adaptable and more drug resistant. Unfortunately, dormancy has few in vitro models, largely because lab-derived cell lines are highly proliferative. Existing models address tumor dormancy, not cellular dormancy, because tracking individual cells is technically challenging. To combat this problem, we adapted a live cell lineage approach to find and track individual dormant cells, distinguishing them from proliferative and dying cells over multiple days. We applied this approach across a range of different in vitro microenvironments. Our approach revealed that the proportion of cells that exhibited long-term quiescence was regulated by both cell intrinsic and extrinsic factors, with the most dormant cells found in 3D collagen gels. We envision that this approach will prove useful to biologists and bioengineers in the dormancy community to identify, quantify, and study dormant tumor cells.Publication Strain-stiffening Hydrogels with Dynamic, Secondary Crosslinking(2023) Sonu, K. P.; Zhou, Le; Biswas, Santidan; Klier, John; Balazs, Anna; Emrick, Todd; Peyton, ShellyHydrogels are water-swollen, typically soft networks useful as biomaterials and in other fields of biotechnology. Hydrogel networks capable of sensing and responding to external perturbations, such as light, temperature, pH, or force, are useful across a wide range of applications requiring on-demand crosslinking or dynamic changes. Thus far, although mechanophores have been widely described as strain-sensitive reactive groups, embedding this type of force-responsiveness into hydrogels is unproven. Here, we synthesized multi-functional polymers that combine a hydrophilic zwitterion with strong, permanently crosslinking alkenes and dynamically crosslinking dithiols. From these polymers, we created hydrogels that contained irreversible and strong thiol-ene crosslinks and reversible dithiol crosslinks, and they stiffened in response to strain, increasing hundreds of kPa in modulus under compression. We examined variations in polymer composition and used a constitutive model to determine how to balance the number of thiol-ene vs. dithiol crosslinks to create maximally force-responsive networks. These strain-stiffening hydrogels represent potential biomaterials that benefit from the mechano-responsive behavior needed for emerging applications in areas such as tissue engineering.Publication Tenascin-C activation of lung fibroblasts in a 3D synthetic lung extracellular matrix mimic(2023) Kundu, Aritra; Dougan, Carey; Mahmoud, Samar; Panagiotou, Alexi; Richbourg, Nathan; Irakoze, Ninette; Peyton, ShellyThe lung extracellular matrix (ECM) maintains the structural integrity of the tissue and regulates the phenotype and functions of resident fibroblasts. Lung-metastatic breast cancer alters these cell-ECM interactions, promoting fibroblast activation. There is a need for bio-instructive ECM models that contain the ECM composition and biomechanics of the lung to study these cell-matrix interactions in vitro. Here, we developed a synthetic, bioactive hydrogel that mimics the native lung modulus and includes a representative distribution of the most abundant ECM peptide motifs responsible for integrin binding and matrix metalloproteinase (MMP)-mediated degradation in the lung, which enables quiescent culture of human lung fibroblasts (HLFs). Stimulation with transforming growth factor β1 (TGF-β1), metastatic breast cancer conditioned media (CM), or tenascin-C-derived integrin-binding peptide activated hydrogel-encapsulated HLFs, demonstrating multiple environmental methods to activate HLFs in a lung ECM mimicking hydrogel. We propose this lung hydrogel platform as a tunable, synthetic approach to studying the independent and combinatorial effects of ECM in regulating fibroblast quiescence and activation.Publication Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity(2023) Raman, Vishnu; Howell, Lars M.; Bloom, Shoshana M. K.; Hall, Christopher L.; Wetherby, Victoria E.; Minter, Lisa M.; Kulkarni, Ashish A.; Forbes, Neil S.Introduction: Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. Methods: To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. Results: We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. Discussion: This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.Publication Synthetic, Living Materials to Model the Tumor Microenvironment(2023) Peyton, Shelly; Chow, Lesley; Finley, Stacey; Ford Versypt, Ashlee; hill, Reginald; Kemp, Melissa; Langer, Ellen; McGuigan, Alison; Meyer, Aaron; Seidlits, Stephanie; Roy, Krishnendu; Mumenthaler, ShannonLiving materials, which are either made of and by living cells or synthetic with programmable elements catered to cells within, are environmentally responsive and can self-repair, allowing for controlled and predictable interactions with biological systems. Such features can also be achieved in purely synthetic materials by using chemical approaches to create dynamic and responsive materials that can undergo programmed changes, that can be remodelled by cells in a predictive way, sense their microenvironment and report back, or respond to remote triggers to rearrange in physical or chemical ways. In this Review, we discuss synthetic approaches to design such cell- and environment-responsive living materials, with a particular focus on their application in cancer. We highlight how synthetic and systems biology approaches can be implemented in the design of synthetic living materials and outline key cancer-related applications, including modelling of tumour heterogeneity, the tumour microenvironment and tumour evolution in response to therapy. Finally, we emphasize the importance of inclusive designs that should be based on an understanding of how health and disease manifest and impact humans from all racial and ethnic backgrounds, skin colors, sex, and genders.Publication How could and do microwaves influence chemistry at interfaces?(2008) Conner, WC; Tompsett, GAPublication Polymer-based Microfluidic Device for On-chip Counter-diffusive Crystallization and In Situ X-ray Crystallography at Room Temperature(2023) Saha, Sarthak; Özden, Can; Samkutty, Alfred; Russi, Silvia; Cohen, Aina; Stratton, Margaret M; Perry, Sarah L.Proteins are long chains of amino acid residues that perform a myriad of functions in living organisms, including enzymatic reactions, signalling, and maintaining structural integrity. Protein function is determined directly by the protein structure. X-ray crystallography is the primary technique for determining the 3D structure of proteins, and facilitates understanding the effects of protein structure on function. The first step towards structure determination is crystallizing the protein of interest. We have developed a centrifugally-actuated microfluidic device that incorporates the fluid handling and metering necessary for protein crystallization. Liquid handling takes advantage of surface forces to control fluid flow and enable metering, without the need of any fluidic or pump connections. Our approach requires only the simple steps of pipetting the crystallization reagents into the device followed by either spinning or shaking to set up counter diffusive protein crystallization trials. The use of thin, UV-curable polymers with a high level of X-ray transparency allows for in situ X-ray crystallography, eliminating the manual handling of fragile protein crystals and streamlining the process of protein structure analysis. We demonstrate the utility of our device using hen egg white lysozyme as a model system, followed by the crystallization and in situ, room temperature structural analysis of the hub domain of calcium-calmodulin dependent kinase II (CaMKIIβ).