Piyush Koria

Self-assembling elastin-like peptides growth factor chimeric nanoparticles for the treatment of chronic wounds

Chronic wounds are associated with poor epidermal and dermal remodeling. Previous work has shown the efficacy of keratinocyte growth factor (KGF) in reepithelialization and elastin in dermal wound healing. Here we demonstrate the fabrication of a fusion protein comprising of elastin-like peptides and KGF. This fusion protein retains the performance characteristics of KGF and elastin as evidenced by its enhancement of keratinocyte and fibroblast proliferation. It also preserved the characteristic elastin-like peptides inverse phase transitioning allowing the recombinant protein to be expressed in bacterial hosts (such as Escherichia coli) and purified rapidly and easily using inverse temperature cycling. The fusion protein self-assembled into nanoparticles at physiological temperatures. When applied to full thickness, wounds in Leprdb diabetic mice these particles enhanced reepithelialization and granulation, by 2- and 3-fold respectively, when compared to the controls. The data strongly suggests that these self-assembled nanoparticles may be beneficial in the treatment of chronic wounds resulting from diabetes or other underlying circulatory conditions.

JNK phosphorylates β-catenin and regulates adherens junctions

The c‐Jun amino‐terminal kinase (JNK) is an important player in inflammation, proliferation, and apoptosis. More recently, JNK was found to regulate cell migration by phosphorylating paxillin. Here, we report a novel role of JNK in cell adhesion. Specifically, we provide evidence that JNK binds to E‐cadherin/β‐catenin complex and phosphorylates β‐catenin at serine 37 and threonine 41, the sites also phosphorylated by GSK‐3β. Inhibition of JNK kinase activity using dominant‐negative constructs reduces phosphorylation of β‐catenin and promotes localization of E‐cadherin/β‐catenin complex to cell‐cell contact sites. Conversely, activation of JNK induces β‐catenin phosphorylation and disruption of cell contacts, which are prevented by JNK siRNA. We propose that JNK binds to β‐catenin and regulates formation of adherens junctions, ultimately controlling cell‐to‐cell adhesion.—Lee, M.‐H., Koria, P., Qu, J., Andreadis, S. T. JNK phosphorylates β‐catenin and regulates adherens junctions. FASEBJ. 23, 3874‐3883 (2009). www.fasebj.org

Delivery of Growth Factors for Tissue Regeneration and Wound Healing

Growth factors are soluble secreted proteins capable of affecting a variety of cellular processes important for tissue regeneration. Consequently, the self-healing capacity of patients can be augmented by artificially enhancing one or more processes important for healing through the application of growth factors. However, their application in clinics remains limited due to lack of robust delivery systems and biomaterial carriers. Interestingly, all clinically approved therapies involving growth factors utilize some sort of a biomaterial carrier for growth factor delivery. This suggests that biomaterial delivery systems are extremely important for successful usage of growth factors in regenerative medicine. This review outlines the role of growth factors in tissue regeneration, and their application in both pre-clinical animal models of regeneration and clinical trials is discussed. Additionally, current status of biomaterial substrates and sophisticated delivery systems such as nanoparticles for delivery of exogenous growth factors and peptides in humans are reviewed. Finally, issues and possible future research directions for growth factor therapy in regenerative medicine are discussed.

JNK regulates binding of α-catenin to adherens junctions and cell-cell adhesion

We recently reported that c‐Jun N‐termi‐nal kinase (JNK) is associated with adherens junctions and phosphorylates β‐catenin at serine 33/37 and threonine 41. Here, we report that inhibition of JNK led to formation of adherens junctions, which was accompanied by dissociation of α‐catenin from the β‐catenin/ E‐cadherin complex and increased association of α‐catenin with the cytoskeleton. Conversely, activation of JNK increased binding of α‐catenin to β‐catenin, which was blocked by the JNK inhibitor SP600125 or JNK siRNA. In addition, inhibition of JNK failed to lead to adherens junction formation in cells where α‐catenin was absent or knocked down. Conversely, introduction of α‐catenin restored the responsiveness of cells to JNK inhibition and led to cell‐cell adhesion. Experiments with domain deletion mutants showed that binding of α‐catenin to β‐catenin was required for transport of adherens junction complexes to the cell surface, while binding to actin was required for translocation to the cell‐cell contact sites. Collectively, our results suggest that JNK affects the association of α‐cate‐nin with the adherens junction complex and regulates adherens junctions.—Lee, M.‐H., Padmashali, R., Koria, P., Andreadis, S. T. JNK regulates binding of α‐catenin to adherens junctions and cell‐cell adhesion. FASEB J. 25, 613–623 (2011). www.fasebj.org

Leveraging growth factor induced macropinocytosis for targeted treatment of lung cancer.

Targeted therapy focused on highly expressed growth factor receptors is increasingly becoming popular for the treatment of lung cancer. Cancer cells exhibit higher levels of macropinocytosis than the normally quiescent non-cancerous cells, which can further be enhanced by growth factors. Here, we show the targeted enhancement of macropinocytosis in lung cancer cells for the delivery of the mitochondriotoxic peptide (KLAKLAK)2 using keratinocyte growth factor (KGF). We report the formation of a nanoparticle (NP) comprising of two chimeric fusion proteins, both fused to elastin-like polypeptide (ELP), (KLAKLAK)2-ELP and KGF-ELP. We show that (KLAKLAK)2-ELP nanoparticles are internalized via macropinocytosis and its internalization is facilitated by the interaction of the ELP domain with cell surface heparin sulfate proteoglycans. This internalization leads to mitochondrial depolarization and subsequent cell death. Also, we demonstrate that KGF-ELP selectively enhances macropinocytosis in cancer cells expressing high levels of the keratinocyte growth factor receptor (KGFR). Finally, the heterogeneous NPs consisting of (KLAKLAK)2-ELP and KGF-ELP selectively kill KGFR-expressing lung cancer cells. Hence, this multipronged approach of targeting highly active processes and receptors in cancer cells will be tremendously selective in the treatment of both early-stage and advanced-stage lung cancers, thereby improving patient’s prognosis and survival rate.

Microfluidics for T‐ Lymphocyte Cell Separation and Inflammation Monitoring in Burn Patients

Severe burns result in T lymphocyte specific immunologic changes. In addition to decreased levels of circulating lymphocytes, changes in cytokine secretion and receptor expression also take place. Our finer understanding of the inflammatory response has led to the development of immune‐targeted therapeutics, requiring specialized gene‐expression monitoring. The emerging field of bio‐micro‐electromechanical systems can be used to isolate highly pure T lymphocytes in a clinically relevant and timely manner for downstream genomic analysis. Blood samples from healthy volunteers and burn‐injured patients were introduced into microfluidic devices developed in our laboratory. Utilizing cell‐affinity chromatography for positive selection of T lymphocytes, the devices served as a platform for RNA extraction and downstream cytokine analysis via quantitative real‐time polymerase chain reaction (PCR). From a 0.5‐mL whole blood sample, the microfluidic devices captured highly pure T lymphocytes from healthy volunteers and burn‐injured patients. Cell capture was of sufficient quantity, and extracted RNA was of sufficient quality, for evaluating the gene expression of cytokines: interferon‐gamma, interleukin‐2, interleukin‐4, and interleukin‐10. Microfluidics is a useful tool in processing blood from burn‐injured patients. Though in its very early stages of development, cell‐specific information obtained by this platform/technology will likely be an important component of near‐patient molecular diagnostics and personalized medicine. Clin Trans Sci 2011; Volume 4: 63–68

Dynamic visualization of photothermal heating by gold nanocages using thermoresponsive elastin like polypeptides.

Understanding how plasmonic nanoparticles collectively generate heat upon exposure to light and thus increase the local temperature of the surrounding medium is critical for many applications such as plasmon-assisted microfluidics, plasmonic tweezers, and photothermal cancer therapy. Reliable temperature manipulation requires the capability to spatially and dynamically analyze local temperature profiles as a function of nanoparticle concentration and laser intensity. In this work, we present a novel method for visualization of local temperature increase using elastin-like polypeptides (ELP). We also propose a robust algorithm that allows the construction of reliable calibration curves using known boundary conditions and Boltzmann sigmoid fit applied to the ELP solutions temperature-absorption transfer function. Using this technique, for the first time, we successfully demonstrated how surface and volume distribution of the nano-heaters affect collective heat generation. This approach allows the visualization of dynamic 2D-temperature profiles and simultaneously enables the measurement of specific temperature at any point in a 2D-map. The experimental setup is compatible with conventional optical microscopy and requires no specialized hardware or complex sample preparation. Finally, the real time visualization of plasmonic heating offers an opportunity to control outcomes of thermo-plasmonics which enables a myriad of practical applications.

Proliferative activity of Elastin-like-Peptides Depends on Charge and Phase transition.

Elastin-like-peptides (ELPs) are stimulus-responsive protein-based polymers and are attractive biomaterials due to their biocompatibility and unique properties. This study shows that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications.

Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein‐2 (BMP‐2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP‐2 is necessary, here we demonstrate the viability of an elastin‐like peptide (ELP) fusion protein containing BMP‐2 for delivery of the BMP‐2. This fusion protein retains the performance characteristics of both the BMP‐2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self‐aggregating nanoparticles at human‐body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage‐efficient and location‐precise noncytotoxic delivery vehicle for BMP‐2 in bone treatment.

Growth factor functionalized biomaterial for drug delivery and tissue regeneration

Elastin-like polypeptides are a class of naturally derived and non-immunogenic biomaterials that are widely used in drug delivery and tissue engineering. Elastin-like polypeptides undergo temperature-mediated inverse phase transitioning, which allows them to be purified in a relatively simple manner from bacterial expression hosts. Being able to genetically encode elastin-like polypeptides allows for the incorporation of bioactive peptides, thereby functionalizing them. Here, we report the synthesis of a biologically active epidermal growth factor–elastin-like polypeptide fusion protein that could aid in wound healing. Epidermal growth factor plays a crucial role in wound healing by inducing cell proliferation and migration. The use of exogenous epidermal growth factor has seen success in the treatment of acute wounds, but has seen relatively minimal success in chronic wounds because the method of delivery does not prevent it from diffusing away from the application site. Our data show that epidermal growth factor–elastin-like polypeptide retained the biological activity of epidermal growth factor and the phase transitioning property of elastin-like polypeptide. Furthermore, the ability of the epidermal growth factor–elastin-like polypeptide to self-assemble near physiological temperatures could allow for the formation of drug depots at the wound site and minimize diffusion, increasing the bioavailability of epidermal growth factor and enhancing tissue regeneration.