Direct monitoring of protease activity using an integrated microchip coated with multilayered fluorogenic nanofilms.

Proteases play an essential role in the four sequential but overlapping phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. In chronic wounds, excessive protease secretion damages the newly formed extracellular matrix, thereby delaying or preventing the normal healing process. Peptide-based fluorogenic sensors provide a visual platform to sense and analyze protease activity through changes in the fluorescence intensity. Here, we have developed an integrated microfluidic chip coated with multilayered fluorogenic nanofilms that can directly monitor protease activity. Fluorogenic protease sensors were chemically conjugated to polymer films coated on the surface of parallel microfluidic channels. Capillary flow layer-by-layer (CF-LbL) was used for film assembly and combined with subsequent sensor modification to establish a novel platform sensing technology. The benefits of our platform include facile fabrication and processing, controllable film nanostructure, small sample volume, and high sensitivity. We observed increased fluorescence of the LbL nanofilms when they were exposed to model recombinant proteases, confirming their responsiveness to protease activity. Increases in the nanofilms' fluorescence intensity were also observed during incubation with liquid extracted from murine infected wounds, demonstrating the potential of these films to provide real-time, in situ information about protease activity levels.

Engineering Helical Modular Polypeptide-Based Hydrogels as Synthetic Extracellular Matrices for Cell Culture.

Expanding the toolkit of modular and functional synthetic material systems for biomimetic extracellular matrices (ECMs) is needed for achieving more predictable and characterizable cell culture. In the present study, we engineered a synthetic hydrogel system incorporating poly(γ-propargyl-l-glutamate) (PPLG), an N-carboxy anhydride polypeptide with a unique α-helical secondary structure. PPLG macromers were cross-linked into poly(ethylene glycol) (PEG) networks to form hybrid polypeptide-PEG hydrogels. We compared the properties of PPLG-PEG to systems where the PPLG macromers were replaced with 8-arm PEG or poly(γ-propargyl-d,l-glutamate) (PPDLG), which has a flexible random-coil conformation. We evaluated each hydrogel system as synthetic ECMs for two-dimensional (2D) endothelial cell culture. Cells on PPLG-PEG displayed superior attachment and spreading at comparable adhesion ligand incorporation concentrations, demonstrating the unique benefit of combining the more rigid and hydrophobic α-helical PPLG within the more flexible and hydrophilic PEG matrix. The modular PPLG macromer is a promising building block for developing other types of PPLG-based hydrogels with favorable and tunable properties.

Development and Application of the Metalloprotease Activity Multiplexed Bead-Based Immunoassay (MAMBI).

Metalloproteinases (MMPs) are zinc-dependent endopeptidases that cleave various proteins to regulate normal and diseased cellular functions, and as such, they play significant roles in human tissue development, homeostasis, and the pathogenesis of many diseases, including cancers, endometriosis, arthritis, etc. Most MMPs are produced as zymogenic latent enzymes that must be cleaved to activate their catalytic regions, and localized endogenous protein inhibitors further regulate activity. Accordingly, they operate within recursive networks to degrade extracellular matrix proteins and regulate cell signaling by cleaving growth factors and receptors at the cell surface and in the local pericellular environment. Thus, high-resolution information about the concentrations of specific active MMPs, revealing their intricate regulatory networks, may improve disease diagnosis and treatment. Here, we introduce a new and readily mastered method for measuring MMP activities in a multiplex fashion. We integrate aspects of activity-based enzyme labeling with commercial high-throughput, multiplexed protein quantification to yield the metalloproteinase activity multiplexed bead-based immunoassay (MAMBI). Assays of recombinant active MMP-1, -2, -3, -7, -8, -9, -12, and -13 establish the sensitivity and selectivity of MAMBI detection. Levels of active native MMPs are similarly characterized in conditioned cell culture medium, menstrual effluent, and uterine tissue. In a single MAMBI (5 µL), we achieve sensitivities equal to those from leading single-plex MMP activity detection strategies (e.g., 10-15 M for MMP-1). We also demonstrate high-throughput inhibitor screening via the MAMBI approach in complex, patient-derived samples.

Chemoproteomics of matrix metalloproteases in a model of cartilage degeneration suggests functional biomarkers associated with posttraumatic osteoarthritis.

Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of many diseases including osteoarthritis (OA), which involves progressive proteolytic degradation of cartilage. Clinical success of OA interventions that target MMPs has been limited by a lack of information about the presence and activity of specific disease-related proteases. We therefore developed a chemoproteomics approach based on MS to characterize the release and activity of MMPs in an in vitro model of the early inflammatory phase of posttraumatic OA (PTOA). We designed and synthesized chemical activity-based probes (ABPs) to identify active MMPs in bovine cartilage explants cultured for 30 days with the proinflammatory cytokine, interleukin-1α. Using these probes in an activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT) approach, we identified active MMP-1, -2, -3, -7, -9, -12, and -13 in the medium after 10 days of culture, the time at which irreversible proteolysis of the collagen network in the explant was detected using proteolytic activation of FRET-quenched MMP substrates. Total MMP levels were quantified by shotgun proteomics, which, taken with ABPP-MudPIT data, indicated the presence of predominantly inactive MMPs in the culture medium. The selectivity of the ABPP-MudPIT approach was further validated by detection of specific endogenous MMPs activated de novo with 4-aminophenylmurcuric acetate. The utility of the new ABPP-MudPIT approach for detecting molecular biomarkers of PTOA disease initiation and potential targets for therapeutics motivates possible application in other diseases involving MMP activity.

Engineering cell aggregates through incorporated polymeric microparticles.

Ex vivo cell aggregates must overcome significant limitations in the transport of nutrients, drugs, and signaling proteins compared to vascularized native tissue. Further, engineered extracellular environments often fail to sufficiently replicate tethered signaling cues and the complex architecture of native tissue. Co-cultures of cells with microparticles (MPs) is a growing field directed towards overcoming many of these challenges by providing local and controlled presentation of both soluble and tethered proteins and small molecules. Further, co-cultured MPs offer a mechanism to better control aggregate architecture and even to report key characteristics of the local microenvironment such as pH or oxygen levels. Herein, we provide a brief introduction to established and developing strategies for MP production including the choice of MP materials, fabrication techniques, and techniques for incorporating additional functionality. In all cases, we emphasize the specific utility of each approach to form MPs useful for applications in cell aggregate co-culture. We review established techniques to integrate cells and MPs. We highlight those strategies that promote targeted heterogeneity or homogeneity, and we describe approaches to engineer cell-particle and particle-particle interactions that enhance aggregate stability and biological response. Finally, we review advances in key application areas of MP aggregates and future areas of development. STATEMENT OF SIGNIFICANT: Cell-scaled polymer microparticles (MPs) integrated into cellular aggregates have been shown to be a powerful tool to direct cell response. MPs have supported the development of healthy cartilage, islets, nerves, and vasculature by the maintenance of soluble gradients as well as by the local presentation of tethered cues and diffusing proteins and small molecules. MPs integrated with pluripotent stem cells have directed in vivo expansion and differentiation. Looking forward, MPs are expected to support both the characterization and development of in vitro tissue systems for applications such as drug testing platforms. However, useful co-cultures must be designed keeping in mind the limitations and attributes of each material strategy within the context of the overall tissue biology. The present review integrates prospectives from materials development, drug delivery, and tissue engineering to provide a toolbox for the development and application of MPs useful for long-term co-culture within cell aggregates.

Label-free, high-throughput holographic screening and enumeration of tumor cells in blood.

We introduce inline digital holographic microscopy (in-line DHM) as a label-free technique for detecting tumor cells in blood. The optimized DHM platform fingerprints every cell flowing through a microchannel at 10 000 cells per second, based on three features - size, maximum intensity and mean intensity. To identify tumor cells in a background of blood cells, we developed robust gating criteria using machine-learning approaches. We established classifiers from the features extracted from 100 000-cell training sets consisting of red blood cells, peripheral blood mononuclear cells and tumor cell lines. The optimized classifier was then applied to targeted features of a single cell in a mixed cell population to make quantitative cell-type predictions. We tested our classification system with tumor cells spiked at different levels into a background of lysed blood that contained predominantly peripheral blood mononuclear cells. Results show that our holographic screening method can readily detect as few as 10 tumor cells per mL, and can identify tumor cells at a false positive rate of at most 0.001%. This purely optical approach obviates the need for antibody labeling and allows large volumes of sample to be quickly processed. Moreover, our in-line DHM approach can be combined with existing circulation tumor cell enrichment strategies, making it a promising tool for label-free analysis of liquid-biopsy samples.

Cell Isolation and Recovery Using Hollow Glass Microspheres Coated with Nanolayered Films for Applications in Resource-Limited Settings.

Established cell isolation and purification techniques such as fluorescence-activated cell sorting (FACS), isolation through magnetic micro/nanoparticles, and recovery via microfluidic devices have limited application as disposable technologies appropriate for point-of-care use in remote areas where lab equipment as well as electrical, magnetic, and optical sources are restricted. We report a simple yet effective method for cell isolation and recovery that requires neither specialized lab equipment nor any form of power source. Specifically, self-floating hollow glass microspheres were coated with an enzymatically degradable nanolayered film and conjugated with antibodies to allow both fast capture and release of subpopulations of cells from a cell mixture. Targeted cells were captured by the microspheres and allowed to float to the top of the hosting liquid, thereby isolating targeted cells. To minimize nonspecific adhesion of untargeted cells and to enhance the purity of the isolated cell population, an antifouling polymer brush layer was grafted onto the nanolayered film. Using the EpCAM-expressing cancer cell line PC-3 in blood as a model system, we have demonstrated the isolation and recovery of cancer cells without compromising cell viability or proliferative potential. The whole process takes less than 1 h. To support the rational extension of this platform technology, we introduce extensive characterization of the critical design parameters: film formation and degradation, grafting with a poly(ethylene glycol) (PEG) sheath, and introducing functional antibodies. Our approach is expected to overcome practical hurdles and provide viable targeted cells for downstream analyses in resource-limited settings.

Label-free fingerprinting of tumor cells in bulk flow using inline digital holographic microscopy.

Large-scale and label-free phenotyping of cells holds great promise in medicine, especially in cancer diagnostics and prognosis. Here, we introduce inline digital holography microscopy for volumetric imaging of cells in bulk flow and fingerprinting of flowing tumor cells based on two metrics, in-focus scattered intensity and cell diameter. Using planar distribution of immobilized particles, we identify the optimal recording distance and microscope objective magnification that minimizes the error in measurement of particle position, size and scattered intensity. Using the optimized conditions and the two metrics, we demonstrate the capacity to enumerate and fingerprint more than 100,000 cells. Finally, we highlight the power of our label-free and high throughput technology by characterizing breast tumor cell lines with different metastatic potentials and distinguishing drug resistant ovarian cancer cells from their parental cell line.

A benchtop capillary flow layer-by-layer (CF-LbL) platform for rapid assembly and screening of biodegradable nanolayered films.

Capillary flow layer-by-layer (CF-LbL) is a microfluidic platform for high throughput preparation and screening of nanolayered polymer films. Using a simple benchtop version of CF-LbL, we systematically studied the effects of various flow conditions and channel geometries on the thickness and surface roughness of the resulting films. We also investigated the biocompatibility and degradation behaviors of a series of enzymatically-degradable films made from naturally derived polymers, i.e. either alginate or hyaluronic acid as the anionic species and poly-l-arginine as the positive species. Furthermore, using one optimized film formulation for coating on the inside walls of a microfluidic chip, we successfully demonstrated the ability of this film to capture and rapidly release cancer cells from whole blood. This simple platform is expected to be a powerful tool to increase the accessibility of the LbL film assembly to a broader scientific community.

Uncharged Helical Modular Polypeptide Hydrogels for Cellular Scaffolds.

Grafted synthetic polypeptides hold appeal for extending the range of biophysical properties achievable in synthetic extracellular matrix (ECM) hydrogels. Here, N-carboxyanhydride polypeptide, poly(γ-propargyl-l-glutamate) (PPLG) macromers were generated by fully grafting the "clickable" side chains with mixtures of short polyethylene glycol (PEG) chains terminated with inert (-OH) or reactive (maleimide and/or norbornene) groups, then reacting a fraction of these groups with an RGD cell attachment motif. A panel of synthetic hydrogels was then created by cross-linking the PPLG macromers with a 4-arm PEG star molecule. Compared to well-established PEG-only hydrogels, gels containing PPLG exhibited dramatically less dependence on swelling as a function of cross-link density. Further, PPLG-containing gels, which retain an α-helical chain conformation, were more effective than standard PEG gels in fostering attachment of a human mesenchymal stem cell (hMSC) line for a given concentration of RGD in the gel. These favorable properties of PPLG-containing PEG hydrogels suggest they may find broad use in synthetic ECM.

Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.