Synthesis, Characterization, and In V ivo Toxicological Evaluation of Copper (II) Oxide Containing Herbometallic Siddha Nanocomplex "Thamira Parpam".

"Thamira parpam" (TP), a copper-based herbometallic oxide (copper (II) oxide) nanodrug has been used in Siddha medicine for centuries because of its anti-ulcerogenic property. However, the physicochemical properties and in vivo toxicity of TP still remain elusive. Rigorous clinical translation requires deciphering these vital properties. We have synthesized TP following a gold standard protocol in the traditional Siddha methodology. We assessed the size, phase, elemental constituents, and thermal stability of TP by SEM and TEM, XRD, EPR, and EDAX analyses, respectively. The results depicted the conversion of metallic copper into copper (II) oxide in the final stages of TP preparation and exhibited nanodimensions ranging between 10 and 50 nm. The XPS spectra revealed the presence of oxygen-deficient state and a carbonaceous coating was found on the surface of TP using TEM analysis. In vivo safety was studied in rat toxicity models by adopting OECD guidelines. Body weight changes, feed, and water intake were unaltered upon TP administration. Hematological, biochemical profiling, and histopathological findings also suggested its nontoxic nature with no abnormalities in major organs and its functions. Interestingly, we found that the metal toxicity could have been subdued because of the carbonaceous coating around the nanoparticle copper (II) oxide, confirming that the drug is safe at a low dose. Overall, our study has enlightened the safety of TP supporting the use of Siddha formulations.

Dendrimer-Functionalized Metal Oxide Nanoparticle-Mediated Self-Assembled Collagen Scaffold for Skin Regenerative Application: Function of Metal in Metal Oxides.

Functionalized metal oxide nanoparticles cross-linked collagen scaffolds are widely used in skin regenerative applications because of their enhanced physicochemical and biocompatibility properties. From the safety clinical trials point of view, there are no reports that have compared the effects of functionalized metal oxide nanoparticles mediated collagen scaffolds for in vivo skin regenerative applications. In this work, triethoxysilane-poly (amido amine) dendrimer generation 3 (TES-PAMAM-G3 or G3)-functionalized spherical shape metal oxide nanoparticles (MO NPs: ZnO, TiO2, Fe3O4, CeO2, and SiO2, size: 12-25 nm) cross-linked collagen scaffolds were prepared by using a self-assembly method. Triple helical conformation, pore size, mechanical strength, and in vitro cell viability of MO-TES-PAMAM-G3-collagen scaffolds were studied through different methods. The in vivo skin regenerative proficiency of MO-TES-PAMAM-G3-collagen scaffolds was analyzed by implanting the scaffold on wounds in Wistar albino rats. The results demonstrated that MO-TES-PAMAM-G3-collagen scaffold showed superior skin regeneration properties than other scaffolds. The skin regenerative efficiency of MO NPs followed the order ZnO > TiO2 > CeO2 > SiO2 > Fe3O4 NPs. This result can be attributed to higher mechanical strength, cell viability, and better antibacterial activity of ZnO-TES-PAMAM-G3-collagen scaffold that leads to accelerate the skin regenerative properties in comparison to other metal oxide based collagen scaffolds.

Chromium-catechin complex, synthesis and toxicity check using bacterial models.

Chromium-catechin complex was synthesized by reacting [Cr(H2O)6]2+ (hexa-aqua) with catechin as a ligand. Toxicity studies were carried out for the complex using bacterial models for safer application of this complex in the future as a drug. Chromium-catechin complex was characterized using ESI Mass spectrometry, electronic spectroscopy, FT-IR spectroscopy and cyclic voltammetry. The complex was found mildly inhibitory towards B. subtilis with the mode of action being oxidative damage, targeting cell membrane. The complex was supportive towards E. coli, which was evident from the growth profile and inhibition studies. SEM analysis supported the results of membrane integrity studies, where the bacterial liposomes upon treatment with the complex revealed slight morphological changes in the case of B. subtilis, without any change in the case of E. coli. The toxicity studies on chromium-catechin complex using bacterial model saves time, as well as resources by providing quick and reliable results, which could ease up the work to be done in future with higher group of organisms like animal model. Therefore, in the future, this complex can be used as an antidiabetic drug after performing toxicity studies with animal model.

Polymethyl methacrylate (PMMA) grafted collagen scaffold reinforced by PdO-TiO2 nanocomposites.

In the past few decades, the design and fabrication of bio-scaffolds exhibiting structural stability in long-term and biocompatibility has received much attention in the field of tissue engineering application. In this direction, we have synthesized different mole ratio of PdO-TiO2 nanocomposites (1:1, 2:1 and 3:1 of Pd:Ti, size 5-11nm, 7-16 nm and 9-22 nm) through a simple single step sol-gel method. The obtained nanocomposites of different sizes were assimilated into poly (methyl methacrylate) grafted collagen biopolymer (g-PMMA-Collagen), resulting in a PdO-TiO2-g-PMMA-Collagen based scaffold. Physico-chemical properties and biocompatibility of g-PMMA-Collagen/PdO-TiO2-g-PMMA-Collagen scaffolds were analysed by using various techniques such as XRD, FT-IR, TGA, DSC, Universal Testing Machine, MTT, Alkaline phosphatase, Alizarin Red S staining assay and the obtained results were compared against pure collagen scaffold. Our results suggest that the incorporation of 1:1 mol ratio PdO-TiO2 nanocomposite (Size, 5-11 nm) offers a higher thermal stability (83.45 °C) and mechanical strength (Young's modulus 105.57 MPa) than the pure collagen scaffold (71.64 °C, 11.67 MPa). The PdO-TiO2 endowed scaffolds were not toxic to MG 63 cells (human osteosarcoma) and enhanced the ALP activity on the scaffolds during in vitro osteogenic differentiation. This work provides a new approach for mechanical reinforcing and enhanced osteogenic activity of collagen scaffolds without affecting its conformation or biocompatibility, an aspect that possibly makes them ideal for bone tissue engineering applications.

Photocrosslinking of collagen using Ru(II)-polypyridyl complex functionalized gold nanoparticles.

Collagen, an extracellular matrix protein, has been used for diverse biological applications due to its clinically safe in nature and for the development of various biomedical devices. As the ECM protein is prone to degradation process, it is necessary to stabilize the collagen. In the present study, we have carried out the stabilization of collagen using newly synthesized gold nanoparticles conjugated with Ru(II) complexes (NCs) possessing different ligand environment. From the DLS measurements, the size of the nanoparticles varies from 20 ±â€¯6 nm. Fibrillation assay studies show that the NCs in the presence of photo-irradiation delays the fibrillation process significantly, while in the presence of persulfate, the acceleration in fibrillation process occurs. Circular dichroic and infra-red spectroscopic studies reveal that no alteration in triple helical structure observed for the photo-irradiated samples. SDS-PAGE analysis data reveal that the NCs facilitate the collagen crosslinks and hinders the enzymatic digestion, while neither Au-NPs nor Ru(II) complexes alone did not impart any stability to the collagen. The results from this study help us to understand the photochemical reaction of nanoparticle conjugate on collagen crosslinking and might be helpful in developing new photocatalyst for corneal application.

Effect of functionalized gold nanoparticle on collagen stabilization for tissue engineering application.

Functionalization of nanoparticle with specific groups is one of the most straightforward strategies to induce structural stability and specific cell responses from collagen based biomaterials. The effect of functionalised nanoparticles on triple helical conformational changes in collagen has not been understood well. For understanding the role of functionalization on collagen conformation, gold nanoparticles (Au NPs) prepared through wet chemical methods and functionalized with organic molecules (F-AuNPs) such as self-assembled monolayer (SAM), (3-aminopropyl) triethoxysilane (APTES), Polysaccharides (pectin and chitosan) and Poly(amido amine) PAMAM dendrimer (G0), were characterised and their interaction with collagen was studied. Protein conformational changes assessed by circular dichroism spectroscopy (CD) reveals that triple helical conformation of collagen was retained in presence of functionalized gold nanoparticle. The biocompatibility of functionalized gold nanoparticle was analysed against keratinocytes (HaCaT) cell by using (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) MTT assay. The result suggested that dendrimer functionalized gold nanoparticle exhibited higher cell viability when compared to other molecules functionalized gold nanoparticles studied. Based on the outcome of this study it can be envisioned that dendrimer functionalized gold nanoparticle mediated collagen materials are highly suitable for tissue engineering and cosmetic application.

Collagen-ZnO Scaffolds for Wound Healing Applications: Role of Dendrimer Functionalization and Nanoparticle Morphology.

Functionalized nanoparticle cross-linked collagen scaffolds offer improved properties to biomaterials and regenerated tissues, as influence of nanoparticle shape on collagen scaffold has received little attention. The present study evaluates the role of ZnO nanoparticle shape (sphere, rod, hexagonal, needle, flower, star, circular disk, doughnut, and cube) on collagen self-assembly. The nanoparticle was prepared by using coprecipitation method and subsequently functionalized with triethoxysilane poly(amidoamine) dendrimer generation 1 (TES-PAMAM-G1 or G1) on the nanoparticle surface. The self-assembly process of collagen, facilitated by EDC-NHS cross-linking, led to stable ZnO-TES-PAMAM-G1-collagen scaffolds. Physicochemical properties and biocompatibility of scaffolds were analyzed to determine the thermal, mechanical and pore size transformation and cell viability, etc. and obtained results compared against collagen scaffolds with/without EDC-NHS cross-linking. In vivo wound healing activity of ZnO-TES-PAMAM-G1-collagen scaffolds was tested on Albino rats that were subjected to excisional wounds and results were compared with control and collagen scaffold. Our findings suggested that the functionalized nanostructure mediated collagen scaffolds exhibited higher thermal (91.2 ± 0.3 °C) and mechanical stability (130.23-305.45 ± 0.1-2.0 MPa) than collagen scaffold (77.36 ± 0.5 °C and 7.96 ± 0.8 MPa). The result of in vivo wound healing study indicated that spherical shape of ZnO-TES-PAMAM -G1 NPs cross-linked collagen scaffold showed enhanced re-epithelization and faster collagen deposition than other scaffolds probably owing to their higher surface area, which led to higher grafting density on the surface. This work provides a new approach for designing nanoparticle mediated collagen scaffold for wound healing application.

Novel mononuclear Cu (II) terpyridine complexes: Impact of fused ring thiophene and thiazole head groups towards DNA/BSA interaction, cleavage and antiproliferative activity on HepG2 and triple negative CAL-51 cell line.

Two mononuclear copper (II) terpyridine complexes namely, [Cu(Btptpy) (ClO4)](ClO4) 1, and [Cu(Bttpy) (ClO4)](ClO4) 2, (Btptpy (L1) = 4'-(Benzothiophene)-2,2':6',2″-terpyridine, Bttpy (L2) = 4'-(Benzylthiazolyl)-2,2':6',2″-terpyridine) have been synthesized and characterized. Single crystal X-ray diffraction shows that, both ligands belong to monoclinic crystal system with space group P21/c (L1) and P21/n (L2). Absorption spectral titration, DNA melting study, circular dichroism and viscosity measurement reveal that, complex 1 and 2 bind with DNA through intercalation. In addition, interaction between the two copper (II) complexes and bovine serum albumin (BSA) has been studied by fluorescence titration, circular dichroism and their protease activity has been investigated using SDS-PAGE gel electrophoresis. Agarose (AGE) and SDS-PAGE gel electrophoresis reveals both complexes have good nucleolytic and proteolytic property in the presence of additive hydrogen peroxide. Both complexes shows remarkable cytotoxic property against triple negative CAL-51 human breast cancer cell line and hepatocellular carcinoma (HepG2) cancer cell lines and bears very less cytotoxicity towards liver normal cell line (Changs). DCF-DA and TBRAS assay also supported that complex 1 and 2 induces elevated level of reactive oxygen species (ROS) and oxidative stress in cancer cells than normal cell line. Furthermore, FACS analysis confirms complex 1 and 2 brings apoptosis by growth phase cell cycle arrest.

Role of nanoparticle size in self-assemble processes of collagen for tissue engineering application.

Nanoparticle mediated extracellular matrix may offer new and improved biomaterial to wound healing and tissue engineering applications. However, influence of nanoparticle size in extracellular matrix is still unclear. In this work, we synthesized different size of silver nanoparticles (AgNPs) comprising of 10nm, 35nm and 55nm using nutraceuticals (pectin) as reducing as well as stabilization agents through microwave irradiation method. Synthesized Ag-pectin nanoparticles were assimilated in the self-assemble process of collagen leading to fabricated collagen-Ag-pectin nanoparticle based scaffolds. Physico-chemical properties and biocompatibility of scaffolds were analyzed through FT-IR, SEM, DSC, mechanical strength analyzer, antibacterial activity and MTT assay. Our results suggested that 10nm sized Ag-pectin nanoparticles significantly increased the denaturation temperature (57.83°C) and mechanical strength (0.045MPa) in comparison with native collagen (50.29°C and 0.011MPa). The in vitro biocompatibility assay reveals that, collagen-Ag-pectin nanoparticle based scaffold provided higher antibacterial activity against to Gram positive and Gram negative as well as enhanced cell viability toward keratinocytes. This work opens up a possibility of employing the pectin caged silver nanoparticles to develop collagen-based nanoconstructs for biomedical applications.

Nanorod mediated collagen scaffolds as extra cellular matrix mimics.

Creating collagen scaffolds that mimic extracellular matrices without using toxic exogenous materials remains a big challenge. A new strategy to create scaffolds through end-to-end crosslinking through functionalized nanorods leading to well-designed architecture is presented here. Self-assembled scaffolds with a denaturation temperature of 110 °C, porosity of 70%, pore size of 0.32 µm and Young's modulus of 231 MPa were developed largely driven by imine bonding between 3-mercapto-1-propanal (MPA) functionalized ZnO nanorods and collagen. The mechanical properties obtained were much higher than that of native collagen, collagen-MPA, collagen-3-mercapto-1-propanol (3MPOH) or collagen- 3-MPOH-ZnO, clearly bringing out the relevance of nanorod mediated assembly of fibrous networks. This new strategy has led to scaffolds with mechanical properties much higher than earlier reports and can provide support for cell growth and facilitation of cell attachment.

Dual utility of a novel, copper enhanced laccase from Trichoderma aureoviridae.

Ever since the ability of laccase to oxidize non-phenolic lignin models was described, the oxidative degradation reactions catalyzed by laccase have been widely studied for paper pulp production or detoxification of aromatic pollutants. The viability of developing eco-friendly, laccase aided industrial processes has been explored. Here, we report the isolation and screening of fungi to explore their lignolytic ability on solid media using various substrates as indicators. The promising fungus was cultivated in submerged and solid state conditions. The crude enzyme obtained yielded elevated activity at 75°C and pH 9.0. Addition of CuSO4 increased the activity by almost 25% proving that Cu(2+) catalytically enhances the action of laccases. Decolorization studies were carried out using industrial dye, Remazol Brilliant Blue R (CI 61200) on solid and liquid medium. Visual decolorization was observed within 2 days of inoculation on solid media whereas, liquid medium incorporated with varying concentrations of dye solution showed a final level of decolorization of up to 76%. Bamboo degradation studies revealed a decrease in lignin content by 51 and 43% within a month. To the best of our knowledge, this study for the first time reports that Trichoderma aureoviridae can produce lignolytic enzyme and degrade lignin.

Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen) on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

Go natural and smarter: fenugreek as a hydration designer of collagen based biomaterials.

Collagen-based biomaterials have received considerable attention for smarter biomedical applications due to their inherent superior mechano-biological properties. However, accumulating evidence suggests that water, as a probe liquid bound in collagen, might be investigated to explore the influence of additives on the static and dynamic solvation behavior of collagen. The structure and dynamics of water near the surface/interface of collagen-fenugreek composites were demonstrated via circular dichroic spectroscopy, thermoporometry and impedimetric measurements to enlighten about the configuration-function relationship of collagen. Thermodynamic parameters of the composites signify the fenugreek concentration dependent structural robustness of collagen. Thermodynamic parameters such as free energies for unfolding, enthalpies, entropies and activation energies indicate that the residual structure modulates the stability of the denatured state up to 22 kcal mol(-1) and the parameters correlate with structural data for collagen complexed with fenugreek. The association constant of fenugreek is found to be 0.5807 M(-1). The binding of fenugreek influences rearrangement of the collagen-water network, resulting in the transition from a disordered (high entropy) unbound state to a structured (lower entropy) bound state. Fenugreek concentration plays a crucial role in shaping up the free energy that governs the folding, structure and stability of collagen. Dielectric data emphasize the effect of hydrophobic and hydrophilic clusters on the side chain motion constraints. The thermoporometry technique probes the pore size distributions of the composites. These methods provide insights into the role of excluded volume, chain stiffness and stability of a new collagen-galactomannan based composite, expanding its utility in "smart biomaterial applications".

Fluorescent nanonetworks: a novel bioalley for collagen scaffolds and tissue engineering.

Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis. For this, a designed synthesis of nanoparticles - using starch as a template and a reflux process, which would provide a highly anisotropic (star shaped) nanoparticle, with large surface area was adopted. Anisotropy associated decrease in Morin temperature and superparamagnetic behavior was observed. Polysaccharide on the nanoparticle surface provided aqueous stability and low cytotoxicity. Starch coated nanoparticles was utilized to build polysaccharide - collagen crosslinks, which supplemented natural crosslinks in collagen, without disturbing the conformation of collagen. The resulting fibrillar lamellae showed a striking resemblance to native lamellae, but had a melting and denaturation temperature higher than native collagen. The biocompatibility and superparamagnetism of the nanoparticles also come handy in the development of stable collagen constructs for various biomedical applications, including that of MRI contrast agents.

DNA/protein interaction and cytotoxic activity of imidazole terpyridine derived Cu(II)/Zn(II) metal complexes.

Two imidazole terpyridine (itpy) based complexes, [Cu(itpy)(OAc)(H2O)]NO3·H2O (1) and [Zn(itpy)(OAc)]OAc (2) have been synthesised and characterized. The crystal structure of complex 1 shows distorted octahedral geometry with an anti-parallel stacking arrangement. The interactions of the two complexes with Calf thymus DNA (ctDNA) have been studied using absorption titration and circular dichroism. Complex 1 shows coordinate binding to DNA bases, and complex 2 shows an intercalative mode of binding with DNA. Complex 1 cleaves the DNA via an oxidative pathway in the presence of additives, because of the presence of a redox active copper(II) centre. However, complex 2 cleaves DNA hydrolytically. Interactions of the two complexes with bovine serum albumin have been studied using fluorescence quenching and circular dichroism experiments. Circular dichroic analysis reveals that both the complexes strongly influence the secondary structure of the protein. Fluorescence quenching experiments indicate that there are different binding sites for complexes 1 and 2 on the protein. Furthermore, the complexes show potential cytotoxicity towards the A549 lung cancer cell line. Both the complexes have been found to induce apoptosis.

Charge instability of symmetry broken dipolar states in quadrupolar and octupolar triphenylamine derivatives.

The quadrupolar and octupolar cyano triphenylamines shows symmetry broken dipolar charge transfer state, however, its stability can be controlled by the rotation of N-C bond of amino and phenylene moiety.

DNA condensation by copper(II) complexes and their anti-proliferative effect on cancerous and normal fibroblast cells.

In our search towards copper(II) based anticancer compounds, copper(II) complexes [Cu(bitpy)2](ClO4)21, [Cu(bitpy)(phen)](NO3)22 and [Cu(bitpy)(NO3)](NO3) 3 were synthesized and characterized. All the three complexes contain the tridentate ligand bitpy, which bears biologically relevant benzimidazolyl head group, as one of the ligands. Because of the presence of the planar benzimidazolyl group in the bitpy ligand, the complexes exhibited intercalative mode of binding with DNA. The DNA binding constant, K(b), for complexes 1, 2 and 3 were determined to be (1.84 ± 0.32) × 10(4), (1.83 ± 0.57) × 10(4) and (1.87 ± 0.21) × 10(4) M(-1) respectively. All the three complexes possessed DNA condensing ability. The DNA condensing ability of the complexes was in the order 2 > 1 > 3. The DNA condensation induced by these three complexes was found to be reversed in the presence of 1 M NaCl. In vitro cytotoxicity of three complexes was tested against osteosarcoma MG63 cell line as well as normal fibroblast NIH3T3 cell line by MTT reduction assay. Complexes 1 and 2 were found to be highly toxic towards MG63 than NIH3T3 cell line and both these complexes brought about cell death in the MG-63 cell line due to apoptosis. Whereas, complex 3 exhibited almost equal toxic effect towards both MG63 and NIH3T3 cell lines. Based on the fact that both complexes 1 and 2 brought about reversible condensation of DNA and induced apoptosis in osteosarcoma MG-63 cell line, it is hypothesized that they might possess potential pharmaceutical applications.

Fabrication of solid collagen nanoparticles using electrospray deposition.

Collagen is a promising biomaterial for drug delivery due to advantages including high biocompatibility and biodegradable property. However, transforming collagen into solid nanoparticles is difficult, although the solid dosage form is advantageous for some administration routes including pulmonary and oral drug delivery. In this study, collagen solid nanoparticles are prepared in one-step using electrospray deposition under ambient temperature and pressure conditions. Although collagen molecules formed micron-sized aggregates in acetic acid solutions spontaneously, electrospraying the collagen solutions resulted in formation of nanofibers. Solid nanoparticles were obtained by increasing conductivity of the solution and/or inducing structural perturbation of the collagen molecules using salts. The ability of solid collagen particles as a drug carrier was demonstrated by incorporating theophylline as a model drug using a coaxial spray technique. Release of theophylline was controlled by cross-linking collagen molecules. Electrospray deposition was proved to be a powerful method for producing solid collagen nanoparticles for drug delivery.

Elucidation of hydration dynamics of locust bean gum-collagen composites by impedance and thermoporometry.

The intricacy of the different parameters involved in the hydration dynamics of collagen influences its performance as biomaterials. This work presents the molecular motions of collagen originating from the solvents and locust bean gum (LBG), which reveal the changes in solvation dynamics of the biopolymers affecting the surface as well as interfacial properties. Water, as a probe liquid bound in collagen has been investigated using a combination of thermoporometry, ATR-FTIR, circular dichroic spectroscopy, dielectric spectroscopy and SEM to explore the influence of LBG on collagen with respect to static and dynamic behaviour. The relaxation process of collagen in the frequency range of 0.01 Hz to 10(5)Hz and thermoporometry results indicate that the interfacial hydration dynamics are dependent on the applied concentration of LBG. This investigation explicitly reflects the rearrangements of the structural water clusters around the charged amino acids of collagen. These results can be employed to redesign the approach towards the development of collagen based biomaterials.