Nanomedicine-based strategies to improve treatment of cutaneous leishmaniasis.

Nanomedicine strategies were first adapted and successfully translated to clinical application for diseases, such as cancer and diabetes. These strategies would no doubt benefit unmet diseases needs as in the case of leishmaniasis. The latter causes skin sores in the cutaneous form and affects internal organs in the visceral form. Treatment of cutaneous leishmaniasis (CL) aims at accelerating wound healing, reducing scarring and cosmetic morbidity, preventing parasite transmission and relapse. Unfortunately, available treatments show only suboptimal effectiveness and none of them were designed specifically for this disease condition. Tissue regeneration using nano-based devices coupled with drug delivery are currently being used in clinic to address diabetic wounds. Thus, in this review, we analyse the current treatment options and attempt to critically analyse the use of nanomedicine-based strategies to address CL wounds in view of achieving scarless wound healing, targeting secondary bacterial infection and lowering drug toxicity.

Quantitative E. coli Enzyme Detection in Reporter Hydrogel-Coated Paper Using a Smartphone Camera.

There is a growing demand for rapid and sensitive detection approaches for pathogenic bacteria that can be applied by non-specialists in non-laboratory field settings. Here, the detection of the typical E. coli enzyme ß-glucuronidase using a chitosan-based sensing hydrogel-coated paper sensor and the detailed analysis of the reaction kinetics, as detected by a smartphone camera, is reported. The chromogenic reporter unit affords an intense blue color in a two-step reaction, which was analyzed using a modified Michaelis-Menten approach. This generalizable approach can be used to determine the limit of detection and comprises an invaluable tool to characterize the performance of lab-in-a-phone type approaches. For the particular system analyzed, the ratio of reaction rate and equilibrium constants of the enzyme-substrate complex are 0.3 and 0.9 pM-1h-1 for ß-glucuronidase in phosphate buffered saline and lysogeny broth, respectively. The minimal degree of substrate conversion for detection of the indigo pigment formed during the reaction is 0.15, while the minimal time required for detection in this particular system is ~2 h at an enzyme concentration of 100 nM. Therefore, this approach is applicable for quantitative lab-in-a-phone based point of care detection systems that are based on enzymatic substrate conversion via bacterial enzymes.

Proteolysis of decellularized extracellular matrices results in loss of fibronectin and cell binding activity.

Excessive inflammation in the chronic wound bed is believed to result in increased fibronectin (FN) proteolysis and poor tissue repair. However, FN fragments can prime the immune response and result in higher protease levels. The reciprocity between FN proteolysis and inflammation makes it challenging to determine the specific contribution of FN proteolysis in the extracellular matrix (ECM) on tissue responses. We studied the impact of proteolysis of decellularized extracellular matrices (dECMs) obtained from NIH 3T3 mouse fibroblasts on FN level and activity. The dECMs were treated with α chymotrypsin and proteolysis was stopped at different time points. The protease solution was obtained, the remaining dECM was scrapped and examined by immunoblotting and Bicinchoninic Acid assays. Fibronectin was 9.4 ± 1.8% of the total protein content in the dECM but was more susceptible to proteolysis. After 15 min of protease treatment there was a 67.6% and 11.1% decrease in FN and total protein, respectively, in the dECMs. Fibronectin fragments were present both in the proteolysis solution and in the dECM. Cell adhesion, spreading and actin extensions on dECMs decreased with increasing proteolysis time. Interestingly, the solutions obtained after proteolysis of the dECMs supported cell adhesion and spreading in a time dependent manner, thus demonstrating the presence of FN cell binding activity in the protease solution of dECMs. This study demonstrates the susceptibility of FN in the ECM to proteolysis and the resulting loss of cell adhesion due to the decrease of FN activity and places weight on bioengineering strategies to stabilize FN against proteolysis.

Proteolytically stabilizing fibronectin without compromising cell and gelatin binding activity.

Excessive proteolytic degradation of fibronectin (FN) has been implicated in impaired tissue repair in chronic wounds. We previously reported two strategies for stabilizing FN against proteolytic degradation; the first conjugated polyethylene glycol (PEG) through cysteine residues and the second conjugated PEG chains of varying molecular weight on lysine residues. PEGylation of FN via lysine residues resulted in increased resistance to proteolysis with increasing PEG size, but an overall decrease in biological activity, as characterized by cell and gelatin binding. Our latest method to stabilize FN against proteolysis masks functional regions in the protein during lysine PEGylation. FN is PEGylated while it is bound to gelatin Sepharose beads with 2, 5, and 10 kDa PEG precursors. This results in partially PEGylated FN that is more stable than native FN and whose proteolytic stability increases with PEG molecular weight. Unlike completely PEGylated FN, partially PEGylated FN has cell adhesion, gelatin binding, and matrix assembly responses that are comparable to native FN. This is new evidence of how PEGylation variables can be used to stabilize FN while retaining its activity. The conjugates developed herein can be used to dissect molecular mechanisms mediated by FN stability and functionality, and address the problem of FN degradation in chronic wounds.

PEGylation of lysine residues improves the proteolytic stability of fibronectin while retaining biological activity.

Excessive proteolysis of fibronectin (FN) impairs tissue repair in chronic wounds. Since FN is essential in wound healing, our goal is to improve its proteolytic stability and at the same time preserve its biological activity. We have previously shown that reduced FN conjugated with polyethylene glycol (PEG) at cysteine residues is more proteolytically stable than native FN. Cysteine-PEGylated FN supported cell adhesion and migration to the same extent as native FN. However, unlike native FN, cysteine-PEGylated FN was not assembled into an extracellular matrix (ECM) when immobilized. Here, we present an alternative approach in which FN is preferentially PEGylated at lysine residues using different molecular weight PEGs. We show that lysine PEGylation does not perturb FN secondary structure. PEG molecular weight, from 2 to 10 kDa, positively correlates with FN-PEG proteolytic stability. Cell adhesion, cell spreading, and gelatin binding decrease with increasing molecular weight of PEG. The 2-kDa FN-PEG conjugate shows comparable cell adhesion to native FN and binds gelatin. Moreover, immobilized FN-PEG is assembled into ECM fibrils. In summary, lysine PEGylation of FN can be used to stabilize FN against proteolytic degradation with minimal perturbation to FN structure and retained biological activity.

A comparative study of polyethylene glycol hydrogels derivatized with the RGD peptide and the cell-binding domain of fibronectin.

The goal of our study was to compare the biological responses of cells cultured on polyethylene glycol (PEG) hydrogels functionalized with varying concentrations of the widely used adhesion peptide, RGD, and the cell-binding domain of fibronectin, III(9-10). We used Michael addition chemistry to covalently link cysteines in GRGDSPC and glutathione S-transferase (GST) tagged III(9-10) (GST-III(9-10)), to the acrylate groups in PEG diacrylate (PEGDA). Conjugation of GST-III(9-10) to PEGDA occurred through cysteine residues in GST. Ellman's reagent and immunoblotting studies demonstrated an efficiency of 90% or more for PEG conjugation of 1 µM GST-III(9-10) or GRDGSPC in 10% (wt/vol) PEGDA at 37°C for 1 h. Circular dichroism and limited proteolysis demonstrated that conjugating PEGDA to GST-III(9-10) did not significantly perturb its native secondary structure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis characterization of the wash solution of PEG hydrogels after photopolymerization demonstrated that >95% of the 1 µM GST-III(9-10) was incorporated into the PEG hydrogels after cross-linking. PEG hydrogels derivatized with 1 µM GST-III(9-10) had significantly higher cell adhesion and spreading than PEG hydrogels with 1 µM GRGDSPC. A comparable adhesion response between GRGDSPC and GST-III(9-10) was obtained when the former was at millimolar and the latter at micromolar concentration. The amount and type of conjugate in the PEG hydrogel derivative was statistically more significant than hydrogel rigidity in stimulating the biological responses observed. This report presents new evidence of the robustness of III(9-10) in mediating cell adhesion and spreading on PEG hydrogels.

Fibronectin alters the rate of formation and structure of the fibrin matrix.

Plasma fibronectin is a vital component of the fibrin clot; however its role on clot structure is not clearly understood. The goal of this study was to examine the influence of fibronectin on the kinetics of formation, structural characteristics and composition of reconstituted fibrin clots or fibrin matrices. Fibrin matrices were formed by adding thrombin to 1, 2 or 4 mg/ml fibrinogen supplemented with 0-0.4 mg/ml fibronectin. The rate of fibrin matrix formation was then monitored by measuring light absorbance properties at different time points. Confocal microscopy of fluorescein conjugated fibrinogen was used to visualize the structural characteristics of fibrin matrices. The amount of fibronectin in fibrin matrices was determined through electrophoresis and immunoblotting of solubilized matrices. Fibronectin concentration positively correlated with the initial rate of fibrin matrix formation and with steady state light absorbance values of fibrin matrices. An increase in fibronectin concentration resulted in thinner and denser fibers in the fibrin matrices. Electrophoresis and immunoblotting showed that fibronectin was covalently and non-covalently bound to fibrin matrices and in the form of high molecular weight multimers. The formation of fibronectin multimers was attributed to cross-linking of fibronectin by trace amounts Factor XIIIa. These findings are novel because they link results from light absorbance studies to microcopy analyses and demonstrate an influence of fibronectin on fibrin matrix structural characteristics. This data is important in developing therapies that destabilize fibrin clots.

PEGylated human plasma fibronectin is proteolytically stable, supports cell adhesion, cell migration, focal adhesion assembly, and fibronectin fibrillogenesis.

Delayed wound healing in many chronic wounds has been linked to the degradation of fibronectin (FN) by abnormally high protease levels. We sought to develop a proteolytically stable and functionally active form of FN. For this purpose, we conjugated 3.35 kDa polyethylene glycol diacrylate (PEGDA) to human plasma fibronectin (HPFN). Conjugation of PEGDA to HPFN or HPFN PEGylation was characterized by an increase of approximately 16 kDa in the average molecular weight of PEGylated HPFN compared to native HPFN in SDS-PAGE gels. PEGylated HPFN was more resistant to α chymotrypsin or neutrophil elastase digestion than native HPFN: after 30 min incubation with α chymotrypsin, 56 and 90% of native and PEGylated HPFN respectively remained intact. PEGylated HPFN and native HPFN supported NIH 3T3 mouse fibroblast adhesion and spreading, migration and focal adhesion formation in a similar manner. Fluorescence microscopy showed that both native and PEGylated HPFN in the culture media were assembled into extracellular matrix (ECM) fibrils. Interestingly, when coated on surfaces, native but not PEGylated HPFN was assembled into the ECM of fibroblasts. The proteolytically stable PEGylated HPFN developed herein could be used to replenish FN levels in the chronic wound bed and promote tissue repair.

A combinatorial approach for directing the amount of fibronectin fibrils assembled by cells that uses surfaces derivatized with mixtures of fibronectin and cell binding domains.

Fibrillar fibronectin (FN) has the crucial role of attracting and attaching cells as well as molecules that mediate tissue repair during wound healing. A previous study demonstrated higher extracellular staining of FN fibrils in cells cultured on surfaces tethered with an equimolar mixture of a FN binding domain and FN's cell binding domain, III1-2 and III9-10 respectively, than on surfaces with III9-10 alone. The effect of varying surface amounts of III1-2 and III9-10 on the quantity of FN fibrils formed by NIH-3T3 fibroblasts was examined. GST tagged III1-2 and III9-10 were conjugated to polyurethane surfaces and ELISAs were used to identify the experimental design space or the range of concentrations of GST-III1-2 and GST-III9-10 that demarcated the limits of protein loading on the surface. When GST-III1-2 was fixed and GST-III9-10 varied within the design space, the amount of FN fibrils measured by immunoblotting detergent insoluble cell lysates was dependent on the ratio of III9-10 to III1-2. When the total protein concentration was fixed and the mixture composition of GST-III1-2 and GST-III9-10 varied such that it optimally covered the design space, a parabolic relationship between FN fibril amount and the ratio of III9-10 to III1-2 was obtained. This relationship had a maximum value when the surface was bonded to equal amounts of III1-2 and III9-10 (P<0.05). Thus the ratio of III9-10 to III1-2 can be utilized to direct the quantity of FN fibrils formed on surfaces.

Surface derivatization strategy for combinatorial analysis of cell response to mixtures of protein domains.

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

Probing the conformation of the fibronectin III1-2 domain by fluorescence resonance energy transfer.

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III(1) and III(2), which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III(1-2) has a compact conformation, we constructed CIIIY, a conformational sensor of III(1-2) based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III(1-2).

Structural organization of the cytoskeleton in SV40 human corneal epithelial cells cultured on nano- and microscale grooves.

The basement membrane of human corneal epithelial cells (HCECs) has a three-dimensional nanoscale architecture, which includes pores, bumps and fibers that may influence cell-substrate adhesion and spreading in the overlying cells. We previously demonstrated that nano- and microscale groove and ridge patterns influence the morphological response and the adhesive response of HCECs to a nominal wall shear stress. Cell-substrate adhesion is mediated by adhesion receptors that bind to extracellular matrix components and anchor the cytoskeleton (CSK) of cells to extracellular elements. Here we investigate the CSK organization in SV40-transformed HCECs grown on nano- and microscale groove and ridge patterns. X-ray lithography was used to fabricate uniform groove and ridge patterns with features ranging in size from 200 nm to 2 microm grooves. Scanning electron microscopy and transmission electron microscopy were used to investigate CSK structure and the distribution of -beta1 integrin adhesion receptors. CSK elements aligned with the patterns; however, the spatial organization of these elements was influenced by feature size. Larger CSK bundles lay on top of the ridges and ran parallel to the patterns, whereas smaller CSK bundles, whose width was proportional to the groove size, spanned the grooves. -Beta1 integrins co-localized with the CSK and had a higher density at the poles of aligned spindle-shaped cells. Differences in organization seen on the different topographical feature sizes may be indicative of differences in extracellular matrix organization. This may explain, in part, previous observations regarding the dependence of cell adhesive responses on the size of topographic features in the substrate.

Nano- and microscale holes modulate cell-substrate adhesion, cytoskeletal organization, and -beta1 integrin localization in SV40 human corneal epithelial cells.

Human corneal epithelial cells (HCECs) interface with a basement membrane in vivo that possesses complex nanoscale topographic features. We report that synthetic substrates patterned with nano- and microscale holes differentially modulate the proliferation, shape and adhesion of SV40 human corneal epithelial cells (SV40-HCECs) as a function of feature size: 1) Cell proliferation was inhibited on nanoscale features (features size less than 800 nm in pitch) compared to microscale features or planar substrates in identical culture conditions. 2) Cells on nanoscale holes had a stellate morphology compared to those on microscale features that were more evenly spread. 3) Cells adhered more to nanoscale features than to microscale features when exposed to shear stress in a laminar flow chamber. Transmission electron microscopy showed that cells cultured on the 400 nm pitch patterns had longer and more numerous filopodia and retraction fibers than cells cultured on the 1600 nm pitch patterns. Immunogold labeling of -beta1 integrins revealed that these receptors were localized at the cell periphery and in the aforementioned cytoskeletal elements. Our findings indicate that surface discontinuities and the activation of mechanochemical cell signaling mechanisms may contribute to the observed responses exhibited by SV40-HCECs cultured on nano- and microscale topography.

Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells.

The basement membrane possesses a rich 3-dimensional nanoscale topography that provides a physical stimulus, which may modulate cell-substratum adhesion. We have investigated the strength of cell-substratum adhesion on nanoscale topographic features of a similar scale to that of the native basement membrane. SV40 human corneal epithelial cells were challenged by well-defined fluid shear, and cell detachment was monitored. We created silicon substrata with uniform grooves and ridges having pitch dimensions of 400-4000 nm using X-ray lithography. F-actin labeling of cells that had been incubated for 24 hours revealed that the percentage of aligned and elongated cells on the patterned surfaces was the same regardless of pitch dimension. In contrast, at the highest fluid shear, a biphasic trend in cell adhesion was observed with cells being most adherent to the smaller features. The 400 nm pitch had the highest percentage of adherent cells at the end of the adhesion assay. The effect of substratum topography was lost for the largest features evaluated, the 4000 nm pitch. Qualitative and quantitative analyses of the cells during and after flow indicated that the aligned and elongated cells on the 400 nm pitch were more tightly adhered compared to aligned cells on the larger patterns. Selected experiments with primary cultured human corneal epithelial cells produced similar results to the SV40 human corneal epithelial cells. These findings have relevance to interpretation of cell-biomaterial interactions in tissue engineering and prosthetic design.