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.

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.

In vitro and in vivo evaluation of pirfenidone loaded acrylamide grafted pullulan-poly(vinyl alcohol) interpenetrating polymer networks.

The aim of present study was to develop controlled release formulation of pirfenidone using acrylamide grafted pullulan. Interpenetrating polymer network (IPN) microspheres were prepared using acrylamide grafted pullulan and PVA utilizing glutaraldehyde assisted water-in-oil emulsion crosslinking method. IPN microspheres were characterized by FTIR, solid state 13C NMR and XRD spectroscopy. In vitro enzymatic degradation study showed 34.30% degradation after 24 h with degradation rate constant of 0.0088 min-1. In vitro biocompatibility test showed no changes in cellular morphology and cell adherence to microspheres, indicating its biocompatible nature. The release exponent value of all formulations was less than 0.45, indicating the release mechanism to be Fickian diffusion. Finally, in vivo pharmacokinetic study showed longer Tmax (1.16 h) and greater AUC value (10037.76 ng h/mL,) as compared to Pirfenex® (Tmax = 0.5 h; AUC = 4310.45 ng h/mL,). The results indicated that the prepared formulation could successfully control the drug release for prolonged time period.

Therapeutically Effective Controlled Release Formulation of Pirfenidone from Nontoxic Biocompatible Carboxymethyl Pullulan-Poly(vinyl alcohol) Interpenetrating Polymer Networks.

The present study was conducted to develop therapeutically effective controlled release formulation of pirfenidone (PFD) and explore the possibility to reduce the total administered dose and dosing regimen. For this purpose, pH-sensitive biomaterial was prepared by inducing carboxymethyl group on pullulan by Williamson ether synthesis reaction, and further, interpenetrating polymeric network microspheres were prepared by glutaraldehyde-assisted water-in-oil (w/o) emulsion cross-linking method, which showed higher swelling ratio in acidic and basic pH. The formation of microspheres was confirmed by different spectral characterization techniques, and thermal kinetic study indicated the formation of thermally stable microspheres. Cell viability and biocompatibility studies on hepatocellular carcinoma (HepG2) cell showed the polymeric matrix to be biocompatible. In vitro dissolution of optimized formulation (F5) showed releases of 54.09 and 76.37% in 0.1 N HCl after 2 h and phosphate buffer (pH 6.8) up to 8 h, respectively. In vivo performances of prepared microsphere and marketed product of PFD were compared in rabbit. T max (time taken to reach peak plasma concentration) was found to be achieved at 0.83 h, compared to 0.5 h for Pirfenex with no significant difference complementing the immediate action, while area under curve was significantly greater for optimized formulation (9768 ± 1300 ng h/mL) compared to Pirfenex (4311 ± 110 ng h/mL), complementing the sustained action. In vivo pharmacokinetic study suggested that the prepared microsphere could be a potential candidate for therapeutically effective controlled delivery of PFD used in dyspnea and cough management due to idiopathic pulmonary fibrosis.

Synthesis and characterization of a non-cytotoxic and biocompatible acrylamide grafted pullulan - Application in pH responsive controlled drug delivery.

The aim of present study was to develop a pH responsive rate controlling polymer by acrylamide grafting onto pullulan. Grafting was performed using free radical induced microwave assisted irradiation technique using ceric ammonium nitrate as free radical inducer. Acrylamide grafted pullulan (Aam-g-pull) was characterized by Fourier transform infrared spectroscopy, solid state 13C nuclear magnetic resonance and field emission scanning electron microscopy. In vitro enzymatic degradation of Aam-g-pull showed degradation of 22.45% after 8 h with degradation rate constant (k) of 0.019 min-1. In vitro cytotoxicity test did not show cell viability below 80% on HepG2 cell line. Pirfenidone tablets were prepared by utilizing wet granulation method using Aam-g-pull as the only rate controlling polymer. The tablets were characterized in terms of in-process quality control parameters like weight variation, hardness, assay, and in vitro dissolution study. The dissolution study showed that the cumulative drug release in phosphate buffer pH 6.8 (rel3 h = 44.12 ±â€¯0.56%) got a significant jump as compared to the release in 0.1 N hydrochloric acid (rel2 h = 26.78 ±â€¯0.23%), confirming the material to be pH responsive. Aam-g-pull can be used as pH responsive rate controlling polymer.

Simultaneous and controlled release of two different bioactive small molecules from nature inspired single material.

Extended and controlled release of more than a single bioactive molecule, simultaneously, from the same biocompatible matrix is challenging to achieve. However, this is important for combating various severe challenges (drug resistance, improved efficacy, etc.) related to drug delivery. In the recent past, the meta-stable trapped air (in the lotus leaf inspired artificial interfaces), which attributed to the extreme water repellency in biomimicked heirarchical (consisted of micro/nano features) interfaces, was unprecedentedly exploited for addressing multiple relevant aspects related to drug delivery (e.g., multiple drug release, tunable drug release, dose control through post-loading of drug molecules, etc.). A biocompatible polymeric material that is (a) synthesized using a one-step covalent and featured gelation of a single polymer and (b) capable of tailoring with a wide range of water wettabilities, was exploited for post loading both hydrophilic and hydrophobic small molecules from a wide variety (less polar, more polar, nonpolar) of organic solvents. Such small molecules loaded polymeric materials continued to display durable nature-inspired bulk wettability and provided simultaneous co-release of two different bioactive drug molecules (i.e., doxorubicin (DOX, anticancer drug) and tetracycline (TC, antibacterial drug)), over 6 months. Moreover, the release extent (from hours to months) of these small molecules was successfully tuned by controlling the water wettability of the single porous polymeric material. The released drug molecules remained bioactive and capable of inhibiting the proliferation of cancer cells (MG-63 (human osteosarcoma) and MDA-MB-231 (human breast adenocarcinoma)) and microorganisms (S. aureus and E. coli). These results provide a facile basis for developing a more potent and multifunctional drug release system for prospective biomedical applications.

Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc.

Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col Iα 1, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.