Human Hair Keratin-Based Hydrogels in Regenerative Medicine: Current Status and Future Directions.

Regenerative medicine (RM) is a multidisciplinary field that utilizes the inherent regenerative potential of human cells to generate functionally and physiologically acceptable human cells, tissues, and organs in vivo or ex vivo. An appropriate biomaterial scaffold with desired physicochemical properties constitutes an important component of a successful RM approach. Among various forms of biomaterials explored until the present day, hydrogels have emerged as a versatile candidate for tissue engineering and regenerative medicine (TERM) applications such as scaffolds for spatial patterning and delivering therapeutic agents, or substrates to enhance cell growth, differentiation, and migration. Although hydrogels can be prepared from a variety of synthetic polymers as well as biopolymers, the latter are preferred for their inherent biocompatibility. Specifically, keratins are fibrous proteins that have been recently explored for constructing hydrogels useful for RM purposes. The present review discusses the suitability of keratin-based biomaterials in RM, with a particular focus on human hair keratin hydrogels and their use in various RM applications.

Palladium Nanocapsules for Photothermal Therapy in the Near-Infrared II Biological Window.

Recent developments in nanomaterials with programmable optical responses and their capacity to modulate the photothermal effect induced by an extrinsic source of light have elevated plasmonic photothermal therapy (PPTT) to the status of a favored treatment for a variety of malignancies. However, the low penetration depth of near-infrared-I (NIR-I) lights and the need to expose the human body to a high laser power density in PPTT have restricted its clinical translation for cancer therapy. Most nanostructures reported to date exhibit limited performance due to (i) activity only in the NIR-I region, (ii) the use of intense laser, (iii) need of large concentration of nanomaterials, or (iv) prolonged exposure times to achieve the optimal hyperthermia state for cancer phototherapy. To overcome these shortcomings in plasmonic nanomaterials, we report a bimetallic palladium nanocapsule (Pd Ncap)─with a solid gold bead as its core and a thin, perforated palladium shell─with extinction both in the NIR-I as well as the NIR-II region for PPTT applications toward cancer therapy. The Pd Ncap demonstrated exceptional photothermal stability with a photothermal conversion efficiency of ∼49% at the NIR-II (1064 nm) wavelength region at a very low laser power density of 0.5 W/cm2. The nanocapsules were further surface-functionalized with Herceptin (Pd Ncap-Her) to target the breast cancer cell line SK-BR-3 and exploited for in vitro PPTT applications using NIR-II light. Pd Ncap-Her caused more than 98% cell death at a concentration of just 50 µg/mL and a laser power density of 0.5 W/cm2 with an output power of only 100 mW. Flow cytometric and microscopic analyses revealed that Pd Ncap-Her-induced apoptosis in the treated cancer cells during PPTT. Additionally, Pd Ncaps were found to have reactive oxygen species (ROS) scavenging ability, which can potentially reduce the damage to cells or tissues from ROS produced during PPTT. Also, Pd Ncap demonstrated excellent in vivo biocompatibility and was highly efficient in photothermally ablating tumors in mice. With a high photothermal conversion and killing efficiency at very low nanoparticle concentrations and laser power densities, the current nanostructure can operate as an effective phototherapeutic agent for the treatment of different cancers with ROS-protecting ability.

Red Blood Cell-Membrane-Coated Poly(Lactic-co-glycolic Acid) Nanoparticles for Enhanced Chemo- and Hypoxia-Activated Therapy.

Herein, a membrane-coated nanocarrier for codelivery of chemotherapeutic agents, curcumin (Cur) and the hypoxia-activated molecule, tirapazamine (TPZ), has been developed. Cur and TPZ were loaded into biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) and finally coated with red blood cell (RBC) membrane by an extrusion process. Characterization of drug-loaded membrane-coated NPs (Cur+TPZ@RB) by dynamic light scattering, TEM and FESEM analyses showed that the NPs were of 105 nm size with a surface charge of -31 mV. Experimental results demonstrated long-term stability, biocompatibility and efficient cellular internalization (primarily through caveolin mediated pathway) of the Cur+TPZ@RB. Antiproliferative studies on 2D monolayer as well as hypoxic 3D multicellular spheroids (MCS) confirmed that the drug-loaded NPs were more potent than free drugs, inducing apoptosis via generation of reactive oxygen species and consequent DNA damage. Furthermore, the reduced cell migration in the scratch assay and down regulations mesenchymal markers as a result of Cur+TPZ@RB treatment suggest the potential of the present system in circumventing hypoxic solid tumors.

Plasmonic Gold Nanorattle Impregnated Chitosan Nanocarrier for Stimulus Responsive Theranostics.

Herein, a stimulus-responsive theranostic nanosystem comprising gold nanorattles (AuNRTs), having a solid octahedron core and thin porous cubic shell, encapsulated within chitosan nanocarriers (CS-AuNRT) has been reported. Due to the plasmonic AuNRTs, CS-AuNRT demonstrated unique features of near infrared (NIR) absorbance and accessible intrinsic electromagnetic "hot spots" arising due to coupling of inner solid core and outer porous shell. These properties enabled CS-AuNRTs to be used for NIR-responsive drug delivery, photothermal therapy, and surface enhanced Raman scattering (SERS) based bioimaging. Following loading of chemotherapeutic drug doxorubicin (DOX) within AuNRTs along with a phase changing material (PCM), application of NIR irradiation resulted in photothermal melting of the PCM and simultaneous payload release in the surrounding medium. Although being nontoxic themselves, CS-AuNRTs with or without loaded DOX could mount significant cell death in breast cancer cell line (MCF-7) in the presence of NIR light as external stimulus. The oxidative stress generated by DOX-loaded and empty CS-AuNRTs upon NIR irradiation were confirmed by flow-cytometric determination of intracellular reactive oxygen species (ROS). Further, the ROS-led induction of apoptosis in treated MCF-7 cells was established from characteristic nuclear fragmentation, morphological changes and membrane blebbing as observed through confocal fluorescence and scanning electron microscopy. Thus, with NIR responsive chemo-photothermal therapy and SERS based bioimaging, the present nanocarrier system holds potential for cancer theranostics.

Protein-Nanoparticle Agglomerates as a Plasmonic Magneto-Luminescent Multifunctional Nanocarrier for Imaging and Combination Therapy.

We report the fabrication of a plasmonic magneto-luminescent multifunctional nanocarrier (PML-MF nanocarrier) by lysozyme-mediated agglomeration of gold-coated iron oxide nanoparticles (IO@AuNPs) and subsequent coating of these agglomerates with BSA-stabilized gold nanoclusters (BSA-AuNCs). The agglomeration-mediated enhancement of plasmonic absorbance at the NIR biological window helped in plasmonic photothermal therapy (PPTT) by PML-MF nanocarriers. PML-MF nanocarriers demonstrated excellent in vitro bioimaging and magnetic targeting capabilities due to the strong photoluminance and superparamagnetism of the constituent AuNCs and IO@AuNPs, respectively. Moreover, these nanocarriers showed the successful loading and delivery of doxorubicin to cancer cells with a significant killing efficiency that could be synergistically improved by combining with PPTT.

Mechanistic Insight into the Antibacterial Activity of Chitosan Exfoliated MoS2 Nanosheets: Membrane Damage, Metabolic Inactivation, and Oxidative Stress.

Two-dimensional molybdenum disulfide (MoS2) based nanosheets functionalized or loaded with an antimicrobial agent have recently attracted attention as highly efficient antibacterial agent. MoS2 sheets act as the photothermal transducers in inducing bacterial cell death on impingement of NIR radiation or enabled cell inactivation by wrapping around the cells. However, the intrinsic ability of MoS2 to act as an effective antibacterial agent without the use of any external stimuli or antimicrobial agent is still not well explored. This study provides a detailed mechanism of antibacterial action of chitosan exfoliated MoS2 nanosheets (CS-MoS2) by deciphering the key events happening both at the membrane surface and inside the bacteria as a result of interaction of bacterial cells with the nanosheets. A simple, green, one-step process was employed for synthesizing stable and positively charged MoS2 nanosheets. The prepared nanosheets showed excellent bactericidal activity against both Gram-positive (MIC = 90 µg/mL, MBC = 120 µg/mL) and Gram-negative bacteria (MIC = 30 µg/mL, MBC = 60 µg/mL). Investigations into deciphering the mechanism of action revealed that the CS-MoS2 nanosheets interacted strongly with the bacterial cells through electrostatic interactions and caused rapid depolarization of the membranes through dent formations. On account of strong van der Waals and electrostatic forces occurring between the CS-MoS2 nanosheets and membrane phospholipid molecules, deepening of dents occurred, which resulted in complete membrane disruption and leakage of cytoplasmic contents. This led to inactivation of the bacterial respiratory pathway through inhibition of dehydrogenase enzymes and induced metabolic arrest in the cells. Simultaneously, disruption of the antioxidant defense system of the cells by increased levels of intracellular ROS subjected the cells to oxidative damage and added to the overall bactericidal action. The nanosheets also displayed antibiofilm properties and were found to be compatible with mammalian cells even at high concentrations.

Combination Therapy with MAPK-Pathway-Specific Inhibitor and Folic-Acid-Receptor-Targeted Selenium Nanoparticles Induces Synergistic Antiproliferative Response in BRAF Mutant Cancer Cells.

Combination chemotherapy, where drugs with a nonoverlapping mode of action achieve better efficacy at lower doses with reduced dose-dependent side effects, holds promise for complete remission of the tumor. Herein, we report a combination therapeutic module comprised of the folic acid receptor (FAR)-targeted SeNPs (FA-SeNPs) and MAPK-pathway inhibitor PD98059 (PD98). While aberrant signaling leading to an uncontrolled proliferation in BRAF mutation bearing cancer cells including MDAMB231 (breast cancer) and A375 (melanoma) cells was inhibited by PD98, high expression of FAR by these cells also led to selective internalization of FA-SeNPs. Consequently, the combination treatment of PD98 and FA-SeNPs demonstrated synergistic antiproliferative efficacy in MDAMB231 and A375 cells involving apoptotic modes of cell death. The selective nature of the present combination module was supported by the low off-target response in L132 noncancerous lung cells. Moreover, the size of the three-dimensional MDAMB231 spheroids was successfully reduced because of the combination therapy projecting its potential in eradicating tumors in vivo.

Synergistic Anticancer Potential of Artemisinin When Loaded with 8-Hydroxyquinoline-Surface Complexed-Zinc Ferrite Magnetofluorescent Nanoparticles and Albumin Composite.

Herein, we report the fabrication of a novel class of magnetofluorescent theranostic nanoparticles (MFTNPs) based on "surface-complexation" of zinc ferrite (ZnFe2O4) NPs with 8-hydroxyquinoline. The potential of these MFTNPs in fluorescence-based bioimaging of different cancer cells was successfully demonstrated. The superparamagnetic behavior of the MFTNPs was exploited effectively in magnetic targeting in vitro. Finally, a well-known hydrophobic antimalarial and prospective anticancer drug artemisinin was efficiently loaded into MFTNPs. Artemisinin loaded MFTNPs were observed to induce superior antiproliferative response, as compared to free drug, in cancer cells in a synergistic mechanism with combination index of 0.1 or less.

Efficient induction of apoptosis in cancer cells by paclitaxel-loaded selenium nanoparticles.

AIM: To develop selenium nanoparticles (SeNPs)-based delivery systems for paclitaxel (PTX) and assess their antiproliferative efficacy against cancer cells in vitro with potential mechanistic insight. METHODS: Pluronic F-127 stabilized SeNPs were prepared and characterized. Effects of PTX-loaded SeNPs on lung (A549), breast (MCF7), cervical (HeLa) and colon (HT29) cancer cells were studied by viability assay complemented with flow-cytometric analyses of cell cycle, apoptosis, mitochondrial membrane potential, intracellular reactive oxygen species and caspase activity. RESULTS: PTX-loaded SeNPs demonstrated significant antiproliferative activity against cancer cells. Cell cycle analyses of PTX-SeNPs treated cells established G2/M phase arrest in a dose-dependent manner leading to apoptosis. Further investigation revealed disruption of mitochondrial membrane potential orchestrated with induction of reactive oxygen species leading to the activation of caspases, key players of apoptotic cell death. CONCLUSION: Efficient induction of apoptosis in various cancer cells by PTX-loaded SeNPs, with appropriate future studies, might lead to potential anticancer strategies.

Studying in vitro phagocytosis of apoptotic cancer cells by recombinant GMCSF-treated RAW 264.7 macrophages.

Granulocyte macrophage colony stimulating factor (GMCSF), a therapeutically important cytokine that helps in the proliferation of macrophages, was recombinantly expressed in E. coli BL21 and purified as a GST-tagged protein. Cell viability assay demonstrated significant enhancement in proliferation of RAW 264.7 (murine macrophage) in presence of GMCSF. In vitro activation of macrophages was carried out by lipopolysaccharide (LPS) or pyrogallol and probed by the generation of reactive oxygen species (ROS). Following the induction of apoptosis in A549 lung cancer cells with anticancer drug cisplatin (at 25µM), apoptotic cancer cells were effectively phagocytosed by the recombinant GMCSF-treated and exogenously activated RAW 264.7 cells as observed in fluorescence microscopic images. The current findings attribute possible role of GMCSF as adjuvant in scavenging treated cancer cells.

Tobacco phytaspase: Successful expression in a heterologous system.

Phytaspase, a plant serine protease, has been demonstrated to play an important role in the programmed cell death of various plants. Phytaspase is synthesized as an inactive proenzyme containing an N-terminal signal peptide followed by a pro-domain and a mature protease catalytic domain. Pre-prophytaspase autocatalytically processes itself into a pro-domain and an active mature phytaspase enzyme. We have recently demonstrated the successful expression of mature phytaspase from tobacco in a bacterial system. Herein, we focus on the expression of pre-prophytaspase as a GST-tag fusion and on its purification by affinity chromatography.

Biomimetically crystallized protease resistant zinc phosphate decorated with gold atomic clusters for bioimaging.

A luminescent probe synthesised via biomimetic crystallization of zinc phosphate in the presence of protein fragment stabilised gold (Au) nanoclusters is reported. The engineered probe - with Au nanoclusters assembled on the crystal - was protease resistant and offered efficient bioimaging and uptake studies.

Keratin mediated attachment of stem cells to augment cardiomyogenic lineage commitment.

The objective of this work was to develop a simple surface modification technique using keratin derived from human hair for efficient cardiomyogenic lineage commitment of human mesenchymal stem cells (hMSCs). Keratin was extracted from discarded human hair containing both the acidic and basic components along with the heterodimers. The extracted keratin was adsorbed to conventional tissue culture polystyrene surfaces at different concentration. Keratin solution of 500µg/ml yielded a well coated layer of 12±1nm thickness with minimal agglomeration. The keratin coated surfaces promoted cell attachment and proliferation. Large increases in the mRNA expression of known cardiomyocyte genes such as cardiac actinin, cardiac troponin and ß-myosin heavy chain were observed. Immunostaining revealed increased expression of sarcomeric α-actinin and tropomyosin whereas Western blots confirmed higher expression of tropomyosin and myocyte enhancer factor 2C in cells on the keratin coated surface than on the non-coated surface. Keratin promoted DNA demethylation of the Atp2a2 and Nkx2.5 genes thereby elucidating the importance of epigenetic changes as a possible molecular mechanism underlying the increased differentiation. A global gene expression analysis revealed a significant alteration in the expression of genes involved in pathways associated in cardiomyogenic commitment including cytokine and chemokine signaling, cell-cell and cell-matrix interactions, Wnt signaling, MAPK signaling, TGF-ß signaling and FGF signaling pathways among others. Thus, adsorption of keratin offers a facile and affordable yet potent route for inducing cardiomyogenic lineage commitment of stem cells with important implications in developing xeno-free strategies in cardiovascular regenerative medicine.

Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease.

Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587µM and 4.67×103M-1min-1, respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value of doxorubicin. This is the first report of functional expression of mature phytaspase in bacterial system as well as its transfection to sensitize A549 cells at lower doxorubicin concentration.

Protein-Based Multifunctional Nanocarriers for Imaging, Photothermal Therapy, and Anticancer Drug Delivery.

We report a simple approach for fabricating plasmonic and magneto-luminescent multifunctional nanocarriers (MFNCs) by assembling gold nanorods, iron oxide nanoparticles, and gold nanoclusters within BSA nanoparticles. The MFNCs showed self-tracking capability through single- and two-photon imaging, and the potential for magnetic targeting in vitro. Appreciable T2-relaxivity exhibited by the MFNCs indicated favorable conditions for magnetic resonance imaging. In addition to successful plasmonic-photothermal therapy of cancer cells (HeLa) in vitro, the MFNCs demonstrated efficient loading and delivery of doxorubicin to HeLa cells leading to significant cell death. The present MFNCs with their multimodal imaging and therapeutic capabilities could be eminent candidates for cancer theranostics.

Recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF): Possibility of nanoparticle-mediated delivery in cancer immunotherapy.

Most of the cancer treatment strategies from chemotherapy to radiotherapy render cancer cells apoptotic and these apoptotic cancer cells accumulate at the tumor sites. The accumulation of apoptotic cancer cells often result in inflammation and autoimmune responses causing serious health implications. Macrophages, which are effective immune combatants, can help in the clearance of these deleterious occupants. Granulocyte macrophage colony stimulating factor (GM-CSF) is a key cytokine, modulator of immune system and responsible for growth and differentiation of granulocytes and macrophages. In this regard, supply of recombinant GM-CSF can enhance the capability of macrophages for clearance of apoptotic cancer cells. However, delivery of the cytokine in vivo can suffer from certain disadvantages like faster depletion, less stability and low targeting efficiency. We believe that the stability and sustained release of GM-CSF can be improved through its encapsulation inside appropriately designed nanoparticles.

Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively.

Functional characterization of recombinant human granulocyte colony stimulating factor (hGMCSF) immobilized onto silica nanoparticles.

OBJECTIVES: Granulocyte macrophage colony stimulating factor (GMCSF), an important therapeutic cytokine, was immobilized onto silica nanoparticles. Maintenance of structural integrity and biological performance in immobilized cytokine was assessed to augment its applicability in possible biomedical implications. RESULTS: Following its cloning and expression in E. coli, the recombinant human GMCSF (hGMCSF) was purified as a GST-tagged protein corresponding to a 42 kDa band on SDS-PAGE. The purified cytokine was immobilized onto biocompatible silica nanoparticles (~129.4 nm) by adsorption and the binding was confirmed by dynamic light scattering and infrared spectroscopy. Maximum binding of hGMCSF was at 6.4 µg mg(-1) silica nanoparticles. Efficient release of the cytokine from the nanoparticles with its structural integrity intact was deduced from circular dichroism spectroscopy. hGMCSF-immobilized silica nanoparticles efficiently increased the proliferation of RAW 264.7 macrophage cells with 50 % increase in proliferation at 600 ng hGMCSF µg(-1) silica nanoparticles. CONCLUSIONS: Silica nanoparticles successfully immobilized hGMCSF maintaining its structural integrity. The release of the immobilized cytokine from silica nanoparticles resulted in the increased proliferation of macrophages indicating the potential of the system in future applications.

Theranostic potential of gold nanoparticle-protein agglomerates.

Owing to the ever-increasing applications, glittered with astonishing success of gold nanoparticles (Au NPs) in biomedical research as diagnostic and therapeutic agents, the study of Au NP-protein interaction seems critical for maximizing their theranostic efficiency, and thus demands comprehensive understanding. The mutual interaction of Au NPs and proteins at physiological conditions may result in the aggregation of protein, which can ultimately lead to the formation of Au NP-protein agglomerates. In the present article, we try to appreciate the plausible steps involved in the Au NP-induced aggregation of proteins and also the importance of the proteins' three-dimensional structures in the process. The Au NP-protein agglomerates can potentially be exploited for efficient loading and subsequent release of various therapeutically important molecules, including anticancer drugs, with the unique opportunity of incorporating hydrophilic as well as hydrophobic drugs in the same nanocarrier system. Moreover, the Au NP-protein agglomerates can act as 'self-diagnostic' systems, allowing investigation of the conformational state of the associated protein(s) as well as the protein-protein or protein-Au NP interaction within the agglomerates. Furthermore, the potential of these Au NP-protein agglomerates as a novel platform for multifunctional theranostic application along with exciting future-possibilities is highlighted here.

Single-walled carbon nanotubes increase pandemic influenza A H1N1 virus infectivity of lung epithelial cells.

BACKGROUND: Airborne exposure to nanomaterials from unintended occupational or environmental exposures or as a consequence of product use may lead to adverse health effects. Numerous studies have focused on single-walled carbon nanotubes (SWCNTs) and their ability to cause pulmonary injury related to fibrosis, and cancer; however few studies have addressed their impact on infectious agents, particularly viruses that are known for causing severe disease. Here we have demonstrated the ability of pristine SWCNTs of diverse electronic structure to increase the susceptibility of small airway epithelial cells (SAEC) to pandemic influenza A H1N1 infection and discerned potential mechanisms of action driving this response. METHODS: Small airway epithelial cells (SAEC) were exposed to three types of SWCNTs with varying electronic structure (SG65, SG76, CG200) followed by infection with A/Mexico/4108/2009 (pH1N1). Cells were then assayed for viral infectivity by immunofluorescence and viral titers. We quantified mRNA and protein levels of targets involved in inflammation and anti-viral activity (INFß1, IL-8, RANTES/CCL5, IFIT2, IFIT3, ST3GAL4, ST6GAL1, IL-10), localized sialic acid receptors, and assessed mitochondrial function. Hyperspectral imaging analysis was performed to map the SWCNTs and virus particles in fixed SAEC preparations. We additionally performed characterization analysis to monitor SWCNT aggregate size and structure under biological conditions using dynamic light scattering (DLS), static light scattering (SLS). RESULTS: Based on data from viral titer and immunofluorescence assays, we report that pre-treatment of SAEC with SWCNTs significantly enhances viral infectivity that is not dependent on SWCNT electronic structure and aggregate size within the range of 106 nm - 243 nm. We further provide evidence to support that this noted effect on infectivity is not likely due to direct interaction of the virus and nanoparticles, but rather a combination of suppression of pro-inflammatory (RANTES) and anti-viral (IFIT2, IFIT3) gene/protein expression, impaired mitochondrial function and modulation of viral receptors by SWCNTs. CONCLUSIONS: Results of this work reveal the potential for SWCNTs to increase susceptibility to viral infections as a mechanism of adverse effect. These data highlight the importance of investigating the ability of carbon-nanomaterials to modulate the immune system, including impacts on anti-viral mechanisms in lung cells, thereby increasing susceptibility to infectious agents.