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.

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.

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.

Influence of PCL on the material properties of collagen based biocomposites and in vitro evaluation of drug release.

Formulation of biodegradable collagen-poly-ε-caprolactone (PCL) based biomaterials for the sustained release of insulin is the main objective of the present work. PCL has been employed to modulate the physico-chemical behavior of collagen to control the drug release. Designed formulations were employed to statistically optimize insulin release parameter profile at different collagen to PCL molar ratios. Circular dichroism, thermoporometry, FTIR, impedance and scanning electron microscopy techniques have been employed to investigate the effect of PCL on hydration dynamics of the collagen molecule, which in turn changes the dissolution parameters of the drug from the systems. Drug entrapment efficiency has been found to be maximum for collagen to PCL molar ratio of 1:2 (>90%). In vitro dissolution test reveals that 99% of the drug was released from composite at collagen to PCL molar ratio of 1:3 and 1:4 within 2h, which indicates that hydrophobicity of the matrix results in weak interaction between lipophilic drug and carrier materials. The least burst release was observed for collagen to PCL molar ratio at 1:2 as synergistic interactions between collagen and PCL was maximum at that particular polymer-polymer ratios. The drug release data indicates super case-II transport of drug (n>1.0).

Investigation of nuclease, proteolytic and antiproliferative effects of copper(II) complexes of thiophenylmethanamine derivatives.

Four coordinate copper(II) complexes 1, 2 and 3 of ligands based on thiophenemethylamine containing imidazole, benzimidazole and pyridine moiety have been synthesized and characterized. Complex 1 has also been crystallographically characterized. The three complexes bind to DNA non-intercalatively, though partial intercalation in the case of complex 2 cannot be ruled out. All the three complexes bring about hydroxyl radical mediated DNA cleavage in the presence of H2O2. Binding of the three copper(II) complexes to BSA lead to changes in the helicity of the protein. Among the three complexes, 2 and 3 are more effective in inhibiting the growth of cancerous MG63 cells than normal NIH3T3 cells. These two complexes promote apoptosis in MG 63 cells.

Importance of ligand structure in DNA/protein binding, mutagenicity, excision repair and nutritional aspects of chromium(III) complexes.

Chromium is extensively used in leather, chrome plating and refining industries. On one hand the occupational exposure to chromium leads to cancer, whereas on the contrary certain Cr(III) compounds have been proposed as nutritional supplements for Type II diabetes and as muscle building agents. Despite the positive outlook of chromium as a bio-essential element, there is increasing concern over the therapeutic application of Cr(III) based supplements, its bioavailability and toxicity profile. In this perspective, we discuss the role of ligand structure in mediating the interaction of chromium(III) complexes with DNA/protein, their mutagenic outcomes, adduct reparability and as nutritional supplements.

Copper(II) complexes of terpyridine derivatives: a footstep towards development of antiproliferative agent for breast cancer.

Two copper(II) complexes with terpyridyl conjugates, [Cu(meotpy)(dmp)](NO(3))(2) (1) and [Cu(bitpy)(dmp)](NO(3))(2) (2) where meotpy, bitpy and dmp stand for methoxybenzyl terpyridine, benzimidazolyl terpyridine and dimethyl phenanthroline respectively have been synthesized and characterized. Complex 1 has also been characterized crystallographically. Both the complexes have been found to bind CT-DNA intercalatively. The ability of these complexes to bring about DNA cleavage has been analyzed using gel electrophoresis. Both complexes 1 and 2 have been found to bring about hydrolytic cleavage of DNA. The cytotoxicity of both these complexes has been tested against cancerous as well as non-cancerous cell lines. Towards non-cancerous cell line complex 2 exhibited very low toxicity. On the other hand both the complexes have been found to exhibit cytotoxic effects against cancerous cell lines. Complex 2 which has lower IC(50), was found to be a potent antiproliferative agent against MCF-7 cells and was able to induce mitochondrial-mediated and caspase-dependent apoptosis with increase in G(0)/G(1) and subsequent arrest in the S phase, in cell cycle progression. Based on this study, it is hypothesized that 2 may be a suitable candidate for further evaluation as a chemopreventive and chemotherapeutic agent for human cancer.

Effect of coordinated ligands on antiproliferative activity and DNA cleavage property of three mononuclear Cu(II)-terpyridine complexes.

Three mononuclear copper(II) complexes, [Cu(tpy)Cl(2)] 1, [Cu(tpy)(NO(3))(2)(H(2)O)] 2 and [Cu(Ptpy)Cl(2)]·H(2)O·HCl 3 have been synthesised and characterized by various spectroscopic techniques and single crystal X-ray diffraction. Complexes 1 and 3 have five coordinate geometry in solid state, whereas complex 2 has six coordinate geometry. Mass spectral and EPR evidence suggest that in solution all the three complexes exist predominantly as a four coordinate species. Molecular modelling and DNA cleavage studies indicate that complexes 1 and 2 are DNA minor groove binders, whereas 3 is an intercalator. All the three complexes show nuclease activity in the presence of hydrogen peroxide. The three complexes have been found to be cytotoxic towards A549 lung adenocarcinoma cells.

Enhancing collagen stability through nanostructures containing chromium(III) oxide.

Stabilization of collagen for various applications employs chemicals such as aldehydes, metal ions, polyphenols, etc. Stability against enzymatic, thermal and mechanical degradation is required for a range of biomedical applications. The premise of this research is to explore the use of nanoparticles with suitable functionalization/encapsulation to crosslink with collagen, such that the three dimensional architecture had the desired stability. Collagen solution prepared as per standard protocols is treated with chromium(III) oxide nanoparticules encapsulated within a polymeric matrix (polystyrene-block-polyacrylic acid copolymer). Selectivity towards encapsulation was ensured by the reaction in dimethyl sulfoxide, where the PS groups popped out and encapsulated the Cr(2)O(3). Subsequently when immersed in aqueous solution, PAA units popped up to react with functional groups of collagen. The interaction with collagen was monitored through techniques such as CD, FTIR, viscosity measurements, stress analysis. CD studies and FTIR showed no degradation of collagen. Thermal stability was enhanced upon interaction of nanostructures with collagen. Self-assembly of collagen was delayed but not inhibited, indicating a compete binding of the metal oxide encapsulated polymer to collagen. Metal oxide nanoparticles encapsulated within a polymeric matrix could provide thermal and mechanical stability to collagen. The formed fibrils of collagen could serve as ideal material for various smart applications such as slow/sustained drug release. The study is also relevant to the leather industry in that the nanostructures can diffuse through the highly networked collagen fibre bundles in skin matrix easily, thus overcoming the rate limiting step of diffusion.