Room-Temperature, Copper-Free, and Amine-Free Sonogashira Reaction in a Green Solvent: Synthesis of Tetraalkynylated Anthracenes and In Vitro Assessment of Their Cytotoxic Potentials.

The multifold Sonogashira coupling of a class of aryl halides with arylacetylene in the presence of an equivalent of Cs2CO3 has been accomplished using a combination of Pd(CH3CN)2Cl2 (0.5 mol %) and cataCXium A (1 mol %) under copper-free and amine-free conditions in a readily available green solvent at room temperature. The protocol was used to transform several aryl halides and alkynes to the corresponding coupled products in good to excellent yields. The rate-determining step is likely to involve the oxidative addition of Ar-X. The green protocol provides access to various valuable polycyclic aromatic hydrocarbons (PAHs) with exciting photophysical properties. Among them, six tetraalkynylated anthracenes have been tested for their anticancer properties on the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and human dermal fibroblasts (HDFs). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to find out the IC50 concentration and lethal dose. The compounds being intrinsically fluorescent, their cellular localization was checked by live cell fluorescence imaging. 4',6-Diamidino-2-phenylindole (DAPI) and propidium iodide (PI) staining was performed to check apoptosis and necrosis, respectively. All of these studies have shown that anthracene and its derivatives can induce cell death via DNA damage and apoptosis.

Silk fibroin bioscaffold from Bombyx mori and Antheraea assamensis elicits a distinct host response and macrophage activation paradigm in vivo and in vitro.

Biomaterials composed of silk fibroin from both mulberry and non-mulberry silkworm varieties have been investigated for their utility in tissue engineering and drug delivery, but these studies have largely excluded any evaluation of host immune response. The present study compares the macrophage activation response towards mulberry (Bombyx mori, BM) and non-mulberry (Antheraea assamensis, AA) silk types, individually and as a blend (BA) in a partial thickness rat abdominal wall defect model and in vitro primary murine bone marrow-derived macrophage (BMDM) assay. Biologic materials composed of liver extracellular matrix (LECM) and small intestinal submucosa (SIS) ECM that are recognized for constructive tissue remodeling, and polypropylene mesh that is associated with pro-inflammatory macrophage phenotype activation are used as controls in the animal model. The AA silk graft shows a host response similar to SIS with few foreign body multinucleate giant cells, vascularization, high CD206 expression, and high M2-like: M1-like macrophage phenotype ratio. Exposure to AA silk degradation products in vitro induces a higher arginase: iNOS ratio in both naive BMDM and pro-inflammatory activated BMDM; and higher Fizz1: iNOS ratio in pro-inflammatory activated BMDM. These data suggest that the AA silk supports a pro-remodeling macrophage response with potential therapeutic applications.

Injectable anti-cancer drug loaded silk-based hydrogel for the prevention of cancer recurrence and post-lumpectomy tissue regeneration aiding triple-negative breast cancer therapy.

A single system capable of delivering anticancer drugs and growth factors by a minimally invasive approach is in demand for effective treatment of triple-negative breast cancer (TNBC) after lumpectomy. Here, we showcase one such holistic system for TNBC therapy and its assessment via 3D in vitro lumpectomy model, a first of its kind. Firstly, Bombyx mori silk fibroin (BMSF) and Antheraea assamensis silk fibroin (AASF) blended hydrogels were prepared and biophysically characterized. Secondly, a 3D in vitro lumpectomy model was developed using MDA-MB-231 cell line to assess the efficacy of localized delivery of doxorubicin (dox) using injectable hydrogel system in terminating remaining breast cancer after lumpectomy. Additionally, we have also evaluated the adipose tissue regeneration in the lumpectomy region by delivering dexamethasone (dex) using injectable hydrogels. Rheological studies showed that the BMSF/AASF blended hydrogels exhibit viscoelasticity and injectability conducive for minimally invasive application. The developed hydrogels by virtue of its slow and sustained release of dox exerted cytotoxicity towards MDA-MB-231 cells assessed through in vitro studies. Further, dex loaded hydrogel supported adipogenic differentiation of adipose tissue derived stem cells (ADSCs), while the secreted factors were found to aid in vascularization and macrophage polarization. This was confirmed through in vitro angiogenic tube formation assay and macrophage polarization study respectively. The corroborated results vouch for potential application of this injectable hydrogels for localized anticancer drug delivery and aiding in breast reconstruction, post lumpectomy.

3D Bioprinted Silk-Based In Vitro Osteochondral Model for Osteoarthritis Therapeutics.

3D bioprinting of osteochondral tissue offers unique opportunities for enabling precise pharmacological interventions in osteoarthritis (OA). The current study investigates the screening potential of anti-inflammatory drugs using bioprinted inflamed human osteochondral units. The biomimetic hierarchical geometry is bioprinted using silk-based bioinks encapsulating pre-differentiated stem cells, creating an in vitro model. Inflammation is stimulated in the model, using tumor necrosis factor-alpha and Interleukin-1 beta pro-inflammatory cytokines. The resultant degeneration, akin to OA, is flagged by key markers like sulfated glycosaminoglycan, collagen, alkaline phosphatase, and downregulation of osteochondral transcript levels. In the next step, the screening of anti-inflammatory drugs is validated using celecoxib and rhein. Consequently, in the inflamed constructs, the initial upregulation of the key inflammatory mediators (nitric oxide, Prostaglandin E2), is subsequently downregulated, post-drug treatment. In addition, catabolic markers (matrix metalloproteinases and aggrecanase-1), indicative of hypertrophic and apoptosing chondrocytes, are significantly downregulated in the treatment groups; while the transcript and protein levels required for osteochondral health are attenuated. Therefore, the in vitro model mimicks the inflammation in the early stages of OA, and corroborates a potential high-throughput platform for screening novel anti-inflammatory drugs in OA therapeutics.

Silk-Based Bioengineered Diaphyseal Cortical Bone Unit Enclosing an Implantable Bone Marrow toward Atrophic Nonunion Grafting.

Postnatal fracture healing of atrophic long bone diaphyseal nonunions remains a challenge for orthopedic surgeons. Paucity of autologous spongiosa has potentiated the use of tissue engineered bone grafts to improve success rates of bone marrow engraftment used in plate reosteosynthesis. Herein, the development and in vitro validation of a "sandwich-type" biofabricated diaphyseal cross-sectional unit, with an outer mechanically robust bioprinted cortical bone shell, encompassing an engineered bone marrow, are reported. Channelized silk fibroin blend sponges derived from Bombyx mori and Antheraea assama help in developing compartmentalized endosteum, exhibiting specialized osteoblasts (endosteal niche) and discontinuous endothelium (vascular niche). The cellular cross-talk between these two niches triggered via integrin-mediated cell adhesion, enables in preserving quiescence state of CD34+ /CD38- hematopoietic stem cells and their recycling in the engineered marrow. The outer cortical bone strut is developed through multimaterial microextrusion bioprinting strategy. Osteogenically primed mesenchymal stem cells-laden silk fibroin-nano-hydroxyapatite bioink is bioprinted alongside paramagnetic Fe-doped bioactive glass-polycaprolactone blend thermoplastic ink, reinforcing it for mechanical stability. Pulsed magnetic field actuation positively influences the osteogenic commitment and maturation of the bioprinted constructs via mechanotransductory route. Therefore, the assembled engineered marrow and bioprinted cortical shell hold promise as potential orthobiologic substitutes toward atrophic nonunion repairs.

CCL2 loaded microparticles promote acute patency in silk-based vascular grafts implanted in rat aortae.

Cardiovascular disease is the leading cause of death worldwide, often associated with coronary artery occlusion. A common intervention for arterial blockage utilizes a vascular graft to bypass the diseased artery and restore downstream blood flow; however, current clinical options exhibit high long-term failure rates. Our goal was to develop an off-the-shelf tissue-engineered vascular graft capable of delivering a biological payload based on the monocyte recruitment factor C-C motif chemokine ligand 2 (CCL2) to induce remodeling. Bi-layered silk scaffolds consisting of an inner porous and outer electrospun layer were fabricated using a custom blend of Antherea Assama and Bombyx Mori silk (lyogel). Lyogel silk scaffolds alone (LG), and lyogel silk scaffolds containing microparticles (LGMP) were tested. The microparticles (MPs) were loaded with either CCL2 (LGMP+) or water (LGMP-). Scaffolds were implanted as abdominal aortic interposition grafts in Lewis rats for 1 and 8 weeks. 1-week implants exhibited patency rates of 50% (7/14), 100% (10/10), and 100% (5/5) in the LGMP-, LGMP+, and LG groups, respectively. The significantly higher patency rate for the LGMP+ group compared to the LGMP- group (p = 0.0188) suggests that CCL2 can prevent acute occlusion. Immunostaining of the explants revealed a significantly higher density of macrophages (CD68+ cells) within the outer vs. inner layer of LGMP- and LGMP+ constructs but not in LG constructs. After 8 weeks, there were no significant differences in patency rates between groups. All patent scaffolds at 8 weeks showed signs of remodeling; however, stenosis was observed within the majority of explants. This study demonstrated the successful fabrication of a custom blended silk scaffold functionalized with cell-mimicking microparticles to facilitate controlled delivery of a biological payload improving their in vivo performance. STATEMENT OF SIGNIFICANCE: This study outlines the development of a custom blended silk-based tissue-engineered vascular graft (TEVG) for use in arterial bypass or replacement surgery. A custom mixture of silk was formulated to improve biocompatibility and cellular binding to the tubular scaffold. Many current approaches to TEVGs include cells that encourage graft cellularization and remodeling; however, our technology incorporates a microparticle based delivery platform capable of delivering bioactive molecules that can mimic the function of seeded cells. In this study, we load the TEVGs with microparticles containing a monocyte attractant and demonstrate improved performance in terms of unobstructed blood flow versus blank microparticles. The acellular nature of this technology potentially reduces risk, increases reproducibility, and results in a more cost-effective graft when compared to cell-based options.

Functionalized Silk Vascular Grafts with Decellularized Human Wharton's Jelly Improves Remodeling via Immunomodulation in Rabbit Jugular Vein.

Cell-free polymeric tissue-engineered vascular grafts (TEVGs) have shown great promise towards clinical translation; however, their limited bioactivity and remodeling ability challenge this cause. Here, a novel cell-free bioresorbable small diameter silk TEVG system functionalized with decellularized human Wharton's jelly (dWJ) matrix is developed and successfully implanted as interposition grafts into rabbit jugular vein. Implanted TEVGs remain patent for two months and integrate with host tissue, demonstrating neo-tissue formation and constructive remodeling. Mechanistic analysis reveals that dWJ matrix is a reservoir of various immunomodulatory cytokines (Interleukin-8, 6, 10, 4 and tumor necrosis factor alpha (TNF-α)), which aids in upregulating M2 macrophage-associated genes facilitating pro-remodeling behavior. Besides, dWJ treatment to human endothelial cells upregulates the expression of functional genes (cluster of differentiation 31 (CD31), endothelial nitric oxide synthase (eNOS), and vascular endothelial (VE)-cadherin), enables faster cell migration, and elevates nitric oxide (NO) production leading to the in situ development of endothelium. The dWJ functionalized silk TEVGs support increased host cell recruitment than control, including macrophages and vascular cells. It endows superior graft remodeling in terms of a dense medial layer comprising smooth muscle cells and elevates the production of extracellular matrix proteins (collagen and elastin). Altogether, these findings suggest that dWJ functionalization imitates the usefulness of cell seeding and enables graft remodeling.

Synthesis of NNN Chiral Ruthenium Complexes and Their Cytotoxicity Studies.

The synthesis and characterization of chiral pincer-ruthenium complexes of the type (R2NNN)RuCl2 (PPh3) (R = 3-methylbutyl and 3,3-dimethylbutyl) is reported here. The cytotoxicity studies of these complexes were studied and compared with the corresponding activity of achiral complexes. The cytotoxic effect of pincer-ruthenium complexes on human dermal fibroblasts and human tongue carcinoma cells assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay displayed an inhibition of normal and cancer cell growth in a dose-dependent manner. Intracellular reactive oxygen species (ROS) level measurement, lactate dehydrogenase assay, DNA fragmentation, and necrosis studies revealed that treatment with pincer-ruthenium complexes induced a redox imbalance in SAS cells by upregulating ROS generation and caused necrotic cell death by disrupting the cellular membrane integrity.

Bio-inspired Underwater Super-Oil-Wettability for Controlling Platelet Adhesion.

Control promotion and prevention of platelet adhesion are important for various biomedical applications. In the past, surface topography and chemical modifications have been commonly utilized for tailoring the promotion and prevention of platelet adhesion. Recently, lotus-leaf-inspired superhydrophobicity has appeared as an efficient avenue to prevent platelet adhesion. However, such extreme water repellent interfaces fail to perform upon prolonged and continuous exposure to aqueous phase. In this communication, the strategic use of a catalyst-free 1,4-conjugate addition reaction between amine and acrylate allowed us to investigate the impact of two distinct underwater oil-wettability on platelet adhesion activity. While underwater superoleophobicity inhibited platelet-adhesion, a highly aggregated fibrous network of adhered platelets was observed on underwater superoleophilic coating. Further, this biocompatible and haemocompatible underwater superoleophobic multilayer coating was deposited on a commercially available catheter tube to examine its potential towards the prevention of platelet attachment.

Bioactive three-dimensional silk composite in vitro tumoroid model for high throughput screening of anticancer drugs.

HYPOTHESIS: Modeling three-dimensional (3D) in vitro culture systems recapitulating spatiotemporal characteristics of native tumor-mass has shown tremendous potential as a pre-clinical tool for drug screening. However, their applications in clinical settings are still limited due to inappropriate recapitulation of tumor topography, culture instability, and poor durability of niche support. EXPERIMENTS: Here, we have fabricated a bio-active silk composite scaffold assimilating tunable silk from Bombyx mori and - arginine-glycine-aspartate (RGD) rich silk from Antheraea assama to provide a better 3D-matrix for breast (MCF 7) and liver (HepG2) tumoroids. Cellular mechanisms underlying physiological adaptations in 3D constructs and subsequent drug responses were compared with conventional monolayer and multicellular spheroid culture. FINDINGS: Silk composite matrix assists prolonged growth and high metabolic activity (Cytochrome P450 reductase) in breast and liver 3D-tumoroids. Enhanced stemness expression (Cell surface adhesion receptor; CD44, Aldehyde dehydrogenase 1) and epithelial-mesenchymal-transition markers (E-cadherin, Vimentin) at transcript and protein levels demonstrate that bio-active matrix-assisted 3D environment augmenting metastatic potential in tumoroids. Together, enhanced secretion of Transforming growth factor ß (TGFß), anchorage-independency, and colony-forming potential of cells in the 3D-tumoroids further corroborates the aggressive behavior of cells. Moreover, the multilayered 3D-tumoroids exhibit decreased sensitivity to some known anticancer drugs (Doxorubicin and Paclitaxel). In conclusion, the bio-active silk composite matrix offers an advantage in developing robust and sustainable 3D tumoroids for a high-throughput drug screening platform.

A Heart-Breast Cancer-on-a-Chip Platform for Disease Modeling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-ß 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

Extracellular Vesicles Enhance the Remodeling of Cell-Free Silk Vascular Scaffolds in Rat Aortae.

Vascular tissue engineering is aimed at developing regenerative vascular grafts to restore tissue function by bypassing or replacing defective arterial segments with tubular biodegradable scaffolds. Scaffolds are often combined with stem or progenitor cells to prevent acute thrombosis and initiate scaffold remodeling. However, there are limitations to cell-based technologies regarding safety and clinical translation. Extracellular vesicles (EVs) are nanosized particles released by most cell types, including stem and progenitor cells, that serve to transmit protein and RNA cargo to target cells throughout the body. EVs have been shown to replicate the therapeutic effect of their parent cells; therefore, EVs derived from stem or progenitor cells may serve as a more translatable, cell-free, therapeutic base for vascular scaffolds. Our study aims to determine if EV incorporation provides a positive effect on graft patency and remodeling in vivo. We first assessed the effect of human adipose-derived mesenchymal stem cell (hADMSC) EVs on vascular cells using in vitro bioassays. We then developed an EV-functionalized vascular graft by vacuum-seeding EVs into porous silk-based tubular scaffolds. These constructs were implanted as aortic interposition grafts in Lewis rats, and their remodeling capacity was compared to that observed for hADMSC-seeded and blank (non-seeded) controls. The EV group demonstrated improved patency (100%) compared to the hADMSC (56%) and blank controls (82%) following eight weeks in vivo. The EV group also produced significantly more elastin (126.46%) and collagen (44.59%) compared to the blank group, while the hADMSC group failed to produce significantly more elastin (57.64%) or collagen (11.21%) compared to the blank group. Qualitative staining of the explanted neo-tissue revealed improved endothelium formation, increased smooth muscle cell infiltration, and reduced macrophage numbers in the EV group compared to the controls, which aids in explaining this group's favorable pre-clinical outcomes.

Photo-Electro Active Nanocomposite Silk Hydrogel for Spatiotemporal Controlled Release of Chemotherapeutics: An In Vivo Approach toward Suppressing Solid Tumor Growth.

Conventional systemic chemotherapeutic regimens suffer from challenges such as nonspecificity, shorter half-life, clearance of drugs, and dose-limiting toxicity. Localized delivery of chemotherapeutic drugs through noninvasive spatiotemporally controllable stimuli-responsive drug delivery systems could overcome these drawbacks while utilizing drugs approved for cancer treatment. In this regard, we developed photoelectro active nanocomposite silk-based drug delivery systems (DDS) exhibiting on-demand drug release in vivo. A functionally modified single-walled carbon nanotube loaded with doxorubicin (DOX) was embedded within a cross-linker free silk hydrogel. The resultant nanocomposite silk hydrogel showed electrical field responsiveness and near-infrared (NIR) laser-induced hyperthermal effect. The remote application of these stimuli in tandem or independent manner led to the increased thermal and electrical conductivity of nanocomposite hydrogel, which effectively triggered the intermittent on-demand drug release. In a proof-of-concept in vivo tumor regression study, the nanocomposite hydrogel was administered in a minimally invasive way at the periphery of the tumor by covering most of it. During the 21-day study, drastic tumor regression was recorded upon regular stimulation of nanocomposite hydrogel with simultaneous or individual external application of an electric field and NIR laser. Tumor cell death marker expression analysis uncovered the induction of apoptosis in tumor cells leading to its shrinkage. Heart ultrasound and histology revealed no cardiotoxicity associated with localized DOX treatment. To our knowledge, this is also the first report to show the simultaneous application of electric field and NIR laser in vivo for localized tumor therapy, and our results suggested that such strategy might have high clinical translational potential.

Glucose-methanol-based fed-batch fermentation for the production of recombinant human interferon gamma (rhIFN-γ) and evaluation of its antitumor potential.

Squamous cell carcinoma (SCC) is nonmelanoma skin cancer, which is very common in patients having T-cell immunosuppressant drugs. Anticancerous agents such as cytokines showed effective response on SCC. Human interferon-gamma (hIFN-γ), a type II cytokines, are having potent antiproliferative and immunomodulatory effects. In the current study, the fed-batch cultivation of recombinant Pichia pastoris was carried out, and its effect on cell biomass production, recombinant human interferon-gamma (rhIFN-γ) production, and the overflow metabolites was estimated. P. pastoris GS115 strain coexpressed with 6-phosphogluconolactonase (SOL3) and ribulose-phosphate 3-epimerase (RPE1) gene (GS115/rhIFN-γ/SR) resulted in 60 mg L-1 of rhIFN-γ production, which was twofold higher as compared with the production from GS115/rhIFN-γ strain. The antiproliferative potential of rhIFN-γ was examined on the human squamous carcinoma (A431) cell lines. Cells treated with 80 ng mL-1 of rhIFN-γ exhibited 50% growth inhibition by enhancing the production of intracellular reactive oxygen species levels and disrupting membrane integrity. Our findings highlight a state of art process development strategy for the high-level production of rhIFN-γ and its potential application as a therapeutic drug in SCC therapy.

Drug Delivery of Anticancer Drugs from Injectable 3D Porous Silk Scaffold for Prevention of Gastric Cancer Growth and Recurrence.

Localized cancer chemotherapy through injectable hydrogels is a next-generation advanced substitute for the currently operational systemic route of drug administration. Recently, several hydrogels have been developed for prospective drug delivery applications; however, no in vitro disease model is available to evaluate its long-term bioactivity in real time. In this regard, we have designed a porous silk scaffold that provides a single platform to accommodate both the soft hydrogel and cancer cells together. The stomach cancer (AGS) cells were seeded in the periphery of the silk scaffold, where they sit in the pores and form three-dimensional (3D) spheroids. Furthermore, the anticancer drug cisplatin-loaded nanocomposite injectable silk hydrogel was filled in the central cavity of the scaffold to evaluate its 11 day extended bioactivity. Such an arrangement keeps the released cisplatin in close contact with the spheroids for its sustained therapeutic effects. In an attempt to model cancer recurrence, the AGS cells were reseeded on the second day of treatment. Our data revealed that the shelf life and cytotoxic effects of cisplatin, which was explicitly releasing out from the nanocomposite silk hydrogel, were considerably enhanced. Hence, the reseeded AGS cells did not survive further on the scaffold, which also indicates its ability to inhibit cancer relapse. Conclusively, the current work showed a possible way to evaluate the long-term efficacy and bioactivity of the injectable hydrogel system in vitro for sustained drug delivery application.

The inhibitory effect of silk sericin against ultraviolet-induced melanogenesis and its potential use in cosmeceutics as an anti-hyperpigmentation compound.

Ultraviolet radiation (UVR)-induced redox imbalance in melanocytes triggers the activation of tyrosinase that results in melanogenesis and its related skin disorders. Supplementation of biological reductants or anti-tyrosinase compounds inhibits such melanogenesis. Silk sericin (SS), a globular protein, is known to possess antioxidant and anti-tyrosinase activities along with other biological attributes. However, its inhibitory activity against UVR-induced melanogenesis has yet to be explored. In the current study, we have scientifically explored the inhibitory activity of SS against UVR-induced melanogenesis. Anti-tyrosinase activity of SS was assessed using mushroom tyrosinase, showing that Antheraea assamensis sericin (AAS) and Philosamia ricini sericin (PRS) inhibited 50% of its activity. Inhibitory activity of SS against UVR-induced melanogenesis was assessed by measuring the cellular melanin content, intracellular tyrosinase activity, and reactive oxygen species (ROS) levels in mouse melanoma. SS pretreatment significantly reduced cellular melanin and ROS production in UV irradiated melanocytes compared with SS untreated cells. AAS treatment before UVA or UVB irradiation significantly inhibited tyrosinase activity. Rheological studies showed that the skin care formulation prepared by the addition of AAS to the basic formulation minimally affected its flow properties. Altogether, our results validate that AAS efficiently inhibited UVR-induced melanogenesis and it could be used as a potential antioxidant molecule in skin care cosmeceutics.

Inhibitory role of silk cocoon extract against elastase, hyaluronidase and UV radiation-induced matrix metalloproteinase expression in human dermal fibroblasts and keratinocytes.

Topical delivery of potent antioxidants maintain the redox balance of the skin, which leads to the downregulation of matrix metalloproteinase (MMP) expression and prevents UV radiation-induced photoaging. In this study, we aimed at investigating the inhibitory role of silk cocoon extract (SCE) isolated from the Antheraea assamensis (AA), Bombyx mori (BM), and Philosamia ricini (PR) silk varieties against UV radiation-induced MMP expression. Incubation of elastase and hyaluronidase with Antheraea assamensis silk cocoon extract (AASCE) caused 50% inhibition of activity. The assessment of total collagen content using the Sirius red assay showed that AASCE (10 µg mL-1) and Philosamia ricini silk cocoon extract (PRSCE at 100 µg mL-1 concentration) post-treatment significantly enhanced the total collagen content in UVA1 and UVB irradiated HDF cells, whereas BM silk cocoon extract (BMSCE at 100 µg mL-1 concentration) post-treatment significantly enhanced the total collagen content in UVA1-irradiated HDF cells. Gene expression studies revealed AASCE and PRSCE post-treatment downregulated the expression of interleukin (IL)-6, MMP-1 and upregulated procollagen genes in UV irradiated HDF cells. Gelatin zymography studies with AASCE post-treatment downregulated the release of MMP-2 and MMP-9 by HaCaT cells. The overall results validate AASCE efficiently shielding UV radiation-induced collagen and elastin degradation by downregulation of MMP expression, substantiating its further use as a potent antioxidant complement in skin care formulations.

Fiber-Reinforced Silk Composite for Enhanced Urokinase Production Using High-Density Perfusion Culture and Bioactive Molecule Supplementation.

Urokinase plasminogen activator (uPA) has been extensively used as a thrombolytic drug in cases of myocardial infarction, thromboembolism, and ischemic brain stroke. Media optimization and high-density perfusion culture are the decisive factors that facilitate enhanced urokinase production in a conditioned medium. In this study, we have aimed for a high-density perfusion culture of HT1080, a human fibrosarcoma cell line, by formulating optimal media for enhanced urokinase productivity. Four scaffold variants were fabricated from silk fibroin and microfibers of Bombyx mori (BM) and Antheraea assamensis (AA) and physico-chemically characterized. Field emission scanning electron microscopy studies revealed a heterogeneous distribution of pores with interconnected networks supporting cell infiltration, attachment, and long-term viability. AA-based fiber-reinforced scaffolds (ASAF) demonstrated superior mechanical strength, integral stability, and increased cell proliferation as compared to pure silk scaffolds. Media formulation was accomplished by limiting serum concentration (2% FBS) and supplementing with 20 µg/mL arginine and 20 ng/mL TGF-ß1 to retain the stationary phase of cells and augment the urokinase production. A perfusion bioreactor culture of HT1080-laden scaffolds in the presence of formulated media was performed for improving the production of urokinase, with a maximum activity of 432 U/L. Also, gene expression analysis revealed that the individual silk scaffolds have different effects on regulating the expression of plasminogen activator urokinase and plasminogen activator urokinase receptor. In brief, our results suggest that a perfusion bioreactor culture of HT1080-laden ASAF scaffolds in formulated media promotes an increased urokinase production, such that it can be further used as a novel 3D matrix platform for industrial production of the lifesaving uPA drug.

Injectable Carbon Nanotube Impregnated Silk Based Multifunctional Hydrogel for Localized Targeted and On-Demand Anticancer Drug Delivery.

The major limitations of traditional methods of anticancer drug delivery include systemic distribution and frequent administration intravenously. To address these issues, in our present approach, we have fabricated a nano hybrid silk hydrogel system for localized, targeted, and on-demand delivery of anticancer drugs. The hybrid system contains a blend of two varieties of silk protein and doxorubicin (DOX)-loaded folic acid functionalized single-walled carbon nanotubes (SWCNT-FA/DOX). Owing to the single-walled carbon nanotube (SWCNT) incorporation, the mechanical strength of the hybrid silk hydrogel composite enhanced significantly. A slow and sustained DOX release was recorded over a 14 day study. The amount of DOX released was determined by concentration of the SWCNT-FA/DOX payload, rate of silk degradation, pH of the released medium, and incubation temperature. The intermittent exposure of near-infrared light to the hybrid gel system stimulated on-demand DOX release. The in vitro studies demonstrated the active targeting of SWCNT-FA/DOX to folic acid receptor-positive (FR+ve) cancer cells. The silk hydrogel, being viscoelastic in nature, is easily injectable to the targeted site. Hence, the developed silk hybrid gel system may allow its near or intratumoral implantation, where it may act as a depot for anticancer drug-loaded nanoparticles. The sustained, targeted, and external-stimuli-dependent DOX released at the localized tumor site is expected to reduce its systemic side effects and show an efficient way to treat the cancer.

Decellularized Caprine Conchal Cartilage toward Repair and Regeneration of Damaged Cartilage.

Repair and regeneration of nasal and auricular cartilage thrust significant challenges in reconstructive surgery. The burgeoning clinical requirement is yet to endorse a satisfactory cartilage replacement matrix. In this regard, we have bioengineered cross-linked decellularized caprine conchal cartilage (DC) as biocompatible, durable, and nontoxic matrices. The DC matrices exhibited reduced DNA and sulfated glycosaminoglycan (sGAG) with a minimal effect on the collagen content. Further, histology and scanning electron micrographs revealed a significant loss of cellular bodies and the presence of a compact matrix consisting of intricate collagen fibers, when compared to unprocessed matrices. An in vitro biological assessment of the matrices exhibited an increased chondrocyte proliferation and viability with a significantly higher DNA, sGAG, and total collagen content. The matrices showed a 3-fold increase in the expression of cartilage-specific genes, namely, aggrecan, collagen II, and sox-9, and exhibited a minimal in vitro immunogenicity. Further, an in vivo assessment was performed by xenografting these caprine matrices in a rabbit model. The retrieved matrices showed a well-organized structural and cellular orientation with extracellular matrix formation after 3 months of implantation. No significant infiltration of plasma cells, macrophages, lymphocytes, and immature fibroblasts was recorded. Therefore, these affordable, resourceful, xenocompatible matrices offer a potential alternate in the repair and regeneration of nasal and auricular cartilages.