Gelatin grafted poly(D,L-lactide) as an inhibitor of protein aggregation: An in vitro case study.

Amyloids are a group of proteins that are capable of forming aggregated amyloid fibrils, which is responsible for many neurodegenerative diseases including Alzheimer's disease (AD). In our previous study, synthesis and characterization of star-shaped poly(D,L-lactide)-b-gelatin (ss-pLG) have been reported. In the present work, we have extended our work to study ss-pLG against protein aggregation. To the best of our knowledge, this is the first report on the inhibition of amyloid fibrillation by protein grafted poly(D,L-lactide). Bovine serum albumin (BSA) was chosen as the model protein, which readily forms fibril under high temperature. We found that ss-pLG efficiently suppressed the fibril formation of BSA compared with gelatin (Gel), which was supported by Thioflavin T assay, circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). In addition, ss-pLG significantly curtailed amyloid-induced hemolysis. We also found that incubation of ss-pLG with neuroblastoma cells (MC65) protected the cells from fibril-induced toxicity. The rescuing efficiency of ss-pLG was better than Gel, which could be attributed to the reduced lamella thickness in branched ss-pLG. These results suggest the significance of gelatin grafting, which probably allows gelatin to interact with the key residues of the amyloidogenic core of BSA effectively.

Silica Release from Silane Cross-Linked Gelatin Based Hybrid Scaffold Affects Cell Proliferation.

Earlier, we had reported the synthesis and characterization of star-shaped poly(d,l-lactide)-b-gelatin (ss-pLG) to improve cell adhesion and proliferation, but the stability of ss-pLG scaffolds remained a persistent issue. Here we show an increase in the stability of ss-pLG using 3-glycidoxypropyl trimethoxysilane (GPTMS) as a covalent cross-linker (h-ss-pLG). The rate of cell proliferation within Hep-G2 cultured h-ss-pLG scaffolds increased until the third day, and afterward it drastically declined. Further, we identified the release of inorganic silica from GPTMS cross-linked h-ss-pLG, which may be associated with the decrease in the rate of HepG2 cell proliferation. However, the cross-linking did not affect red blood cells (RBCs) and they were completely hemocompatible. In addition, our in vivo experiments in female rats showed that the hybrid h-ss-pLG scaffolds were not degraded completely after 4 weeks, as they were covalently cross-linked with silane. These results suggest the significance of the cross-linker selection, which is one of the other key factors, and needs to be considered while designing scaffolds.

Highly selective fluorescence 'turn off' sensing of picric acid and efficient cell labelling by water-soluble luminescent anthracene-bridged poly(N-vinyl pyrrolidone).

A novel, water-soluble, luminescent anthracene-bridged AA-type bi-arm poly(N-vinylpyrrolidone) (ATC-PNVP) was synthesized using a click reaction between alkyne-terminated PNVP and 9,10-bis(azidomethyl)anthracene. The resultant anthracene-bridged PNVP (ATC-PNVP) was characterized using 1H NMR, FTIR, UV-Vis, and fluorescence spectroscopic methods and GPC analysis. ATC-PNVP showed effective fluorescence properties in an aqueous medium. It showed highly selective "turn off" sensing behaviour towards picric acid, a common nitro-aromatic explosive, with a wide linear range of detection of 0.01-0.3 mM and LOD value of 0.006 mM in water. ATC-PNVP-based paper sensors also showed very effective detection of picric acid in the concentration range 0.001-1.0 mM. Its binding with bovine serum albumin (BSA) was studied using steady-state, synchronous and 3D fluorescence spectroscopy and this study showed effective quenching of the intrinsic fluorescence of BSA and occurrence of a FRET-type interaction. Furthermore, this luminescent ATC-PNVP was efficiently used as a fluorescence microscopy labelling agent in NIH-3T3 and HeLa cells, and showed greater uptake and hence better fluorescent labelling in the cytosols of the tested cells than free 9,10-bis(azidomethyl) anthracene. The cell viability study also showed a very good biocompatible and non-toxic nature of ATC-PNVP at lower working concentrations towards each of the types of cells tested.

Cell proliferation influenced by matrix compliance of gelatin grafted poly(d,l-Lactide) three dimensional scaffolds.

Surface and mechanical properties of the biomaterials are determinants of cellular responses. In our previous study, star-shaped poly(d,l-Lactide)-b-gelatin (ss-pLG) was reported for possessing improved cellular adhesion and proliferation. Here, we extended our investigation to establish the cellular compatibility of gelatin-grafted PDLLA with respect to mechanical properties of biological tissues. In this view, linear PDLLA-b-gelatin (l-pLG) was synthesized and tissue-level compatibility of 1-pLG and ss-pLG against fibroblasts (L929), myoblasts (C2C12) and preosteoblasts (MG-63) was examined. The cell proliferation of C2C12 was significantly higher within l-pLG scaffolds, whereas L929 showed intensified growth within ss-pLG scaffolds. The difference in cell proliferation may be attributed to the varying mechanical properties of scaffolds; where the stiffness of l-pLG scaffolds was notably higher than ss-pLG scaffolds, most likely due to the variable levels of gelatin grafting on the backbone of PDLLA. Therefore, gelatin grafting can be used to modulate mechanical property of the scaffolds and this study reveals the significance of the matrix stiffness to produce the successful 3D scaffolds for tissue engineering applications.

In vitro biocompatibility analysis of functionalized poly(vinyl chloride)/layered double hydroxide nanocomposites.

The aim of this study was to examine the cytotoxicity and biocompatibility of functionalized poly(vinyl chloride) (PVC)/layered double hydroxide (LDH) nanocomposites. The biocompatibility of the LDH-based nanocomposites of thiosulphate PVC (TS-PVC), thiourea PVC (TU-PVC) and sulphite PVC (S-PVC) was assessed via haemolysis and thrombogenicity tests followed by the analysis of cellular adhesion and proliferation. The MTT assay was performed on cells in direct contact with the polymeric nanocomposites to evaluate the side effects of the biomaterials. The cellular morphology of mouse mesenchymal stem cells was also analyzed after incubation with direct contact with the functionalized polymer nanocomposites for different time periods. Although the results of the haemolysis test displayed a positive influence of LDH on the functionalized PVC compared to the neat PVC, the thrombogenic property was observed to be notably decreased, which indicated improved blood compatibility. The resulting LDH samples were also studied for their performance via fluorescence imaging of cells after incubation with the materials. The LDH-based polymers exhibited an excellent level of cytocompatibility, which validates their use as biomaterials. PVC-TU/LDH-2 and PVC-S-2 were found to be notably less cytotoxic for the tested cell type. Also, the cells were found to adhere better to the entire PVC-S/LDH nanocomposite surface. The cytotoxicity test also revealed that the PVC-TU/LDH and PVC-S/LDH nanocomposites exhibited similar responses. The fluorescence-based image analysis showed that cells were spread much more on the polymer surface containing a higher LDH weight percentage. Overall, this study provides a benchmark for the biocompatibility properties of PVC/LDH nanocomposites, which may be useful for numerous applications in the biomedical and related areas.

Osteoconductive Amine-Functionalized Graphene-Poly(methyl methacrylate) Bone Cement Composite with Controlled Exothermic Polymerization.

Bone cement has found extensive usage in joint arthroplasty over the last 50 years; still, the development of bone cement with essential properties such as high fatigue resistance, lower exothermic temperature, and bioactivity has been an unsolved problem. In our present work, we have addressed all of the mentioned shortcomings of bone cement by reinforcing it with graphene (GR), graphene oxide (GO), and surface-modified amino graphene (AG) fillers. These nanocomposites have shown hypsochromic shifts, suggesting strong interactions between the filler material and the polymer matrix. AG-based nanohybrids have shown greater osteointegration and lower cytotoxicity compared to other nanohybrids as well as pristine bone cement. They have also reduced oxidative stress on cells, resulting in calcification within 20 days of the implantation of nanohybrids into the rabbits. They have significantly reduced the exothermic curing temperature to body temperature and increased the setting time to facilitate practitioners, suggesting that reaction temperature and settling time can be dynamically controlled by varying the concentration of the filler. Thermal stability and enhanced mechanical properties have been achieved in nanohybrids vis-à-vis pure bone cement. Thus, this newly developed nanocomposite can create natural bonding with bone tissues for improved bioactivity, longer sustainability, and better strength in the prosthesis.

Tailored Chemical Properties of 4-Arm Star Shaped Poly(d,l-lactide) as Cell Adhesive Three-Dimensional Scaffolds.

Biodegradable poly(lactic acid) (PLA) is widely used to fabricate 3D scaffolds for tissue regeneration. However, PLA lacks cell adhering functional moieties, which limit its successful application in tissue engineering. Herein, we have tailored the cell adhesive properties of star shaped poly(d,l-lactide) (ss-PDLLA) by grafting gelatin to their 4 arms. Grafting of gelatin on PDLLA backbone was confirmed by 1H NMR and FTIR. The synthesized star shaped poly(d,l-lactide)-b-gelatin (ss-pLG) exhibited enhanced wettability and protein adsorption. The modification also facilitated better cell adhesion and proliferation on their respective polymer coated 2D substrates, compared to their respective unmodified ss-PDLLA. Further, 3D scaffolds were fabricated from gelatin grafted and unmodified polymers. The fabricated scaffolds were shown to be cytocompatible to 3T3-L1 cells and hemocompatible to red blood cells (RBCs). Cell proliferation was increased up to 2.5-fold in ss-pLG scaffolds compared to ss-PDLLA scaffolds. Furthermore, a significant increase in cell number reveals a high degree of infiltration of cells into the scaffolds, forming a viable and healthy 3D interconnected cell community. In addition to that, burst release of docetaxal (DTX) was observed from ss-pLG scaffolds. Hence, this new system of grafting polymers followed by fabricating 3D scaffolds could be utilized as a successful approach in a variety of applications where cell-containing depots are used.

Brominated Graphene as Mimetic Peroxidase for Sulfide Ion Recognition.

Brominated graphene (GBR) with ∼3% bromine content has shown novel peroxidase mimetic activity toward 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2. Optimum activity has been observed at pH 4.48 and after a minimum ∼30 min of equilibration time. Among the different analytes studied using the sensor combining TMB, H2O2, and GBR in phosphate buffer of pH 4.48, the S2- ion has effectively shown a short duration of sensing (∼2 min) within the detection range of 0.04-1 mM. A calibration curve for S2- ion estimation has been constructed with the experimental linearity in 0.04-0.4 mM range and having the limit of detection (LOD) value of 25.3 µM. A standard addition experiment has validated the method. A paper strip sensor has been fabricated for successful detection of S2- ion.

Methotrexate-Loaded Four-Arm Star Amphiphilic Block Copolymer Elicits CD8+ T Cell Response against a Highly Aggressive and Metastatic Experimental Lymphoma.

We have synthesized a well-defined four-arm star amphiphilic block copolymer [poly(DLLA)-b-poly(NVP)]4 [star-(PDLLA-b-PNVP)4] that consists of D,L-lactide (DLLA) and N-vinylpyrrolidone (NVP) via the combination of ring-opening polymerization (ROP) and xanthate-mediated reversible addition-fragmentation chain transfer (RAFT) polymerization. Synthesis of the polymer was verified by 1H NMR spectroscopy and gel permeation chromatography (GPC). The amphiphilic four-arm star block copolymer forms spherical micelles in water as demonstrated by transmission electron microscopy (TEM) and 1H NMR spectroscopy. Pyrene acts as a probe to ascertain the critical micellar concentration (cmc) by using fluorescence spectroscopy. Methotrexate (MTX)-loaded polymeric micelles of star-(PDLLA15-b-PNVP10)4 amphiphilic block copolymer were prepared and characterized by fluorescence and TEM studies. Star-(PDLLA15-b-PNVP10)4 copolymer was found to be significantly effective with respect to inhibition of proliferation and lysis of human and murine lymphoma cells. The amphiphilic block copolymer causes cell death in parental and MTX-resistant Dalton lymphoma (DL) and Raji cells. The formulation does not cause hemolysis in red blood cells and is tolerant to lymphocytes compared to free MTX. Therapy with MTX-loaded star-(PDLLA15-b-PNVP10)4 amphiphilic block copolymer micelles prolongs the life span of animals with neoplasia by reducing the tumor load, preventing metastasis and augmenting CD8+ T cell-mediated adaptive immune responses.

Bone cement based nanohybrid as a super biomaterial for bone healing.

A novel nanohybrid based on bone cement has been developed which is capable of healing fractured bone in 30 days, one-third of the time required for the natural healing process. Nanohybrids of bone cement based on poly(methyl methacrylate) (PMMA), currently used as a grouting material in joint replacement surgery, were prepared by simple mixing with organically modified layered silicates of varying chemical compositions. The temperature arising from exothermic polymerization in one of the nanohybrids is 12 °C lower than that in pure bone cement, thus circumventing the reported cell necrosis that occurs during implantation with pure bone cement. The thermal stability and mechanical superiority of this nanohybrid were verified in terms of its higher degradation temperature, better stiffness, superior toughness, and significantly higher fatigue resistance compared with pure bone cement; these properties make it appropriate for use as an implant material. The biocompatibility and bioactivity of the nanohybrid were confirmed using cell adhesion, cell viability, and fluorescence imaging studies. Osteoconductivity and bone bonding properties were monitored in vivo in rabbits through radiographic imaging and histopathological studies of growing bone and muscle near the surgery site. The observed dissimilarity of the properties of two different nanoclays used as fillers were visualized through interactions measured using spectroscopic techniques. Studies of the influence of different elements on bioactivity showed a higher efficiency for the nanoclay containing greater amounts of iron.

Layered double hydroxide induced advancement in joint prosthesis using bone cement: the effect of metal substitution.

Poly(methyl methacrylate) based bone cement and its nanocomposites with layered double hydroxide (LDH) have been developed with greater mechanical strength and biocompatibility as a grouting material for total joint arthroplasty. Bivalent magnesium has been replaced with trivalent aluminium with various mole ratios, keeping the layered pattern of the LDH intact, to cater for the effect of varying substitution on the property enhancement of the nanocomposites. The intercalation of polymer inside the LDH layers makes them disordered and mechanically stiffer and tougher by more than 100%. The thermal stability of bone cement has increased by more than 30 °C in the presence of 1 wt% of nanoLDH, homogenously distributed in the bone cement matrix by creating an inorganic thermal barrier out of the LDH dispersion. The improvement in the properties of the nanocomposites has been explained in terms of the strong interaction between nanoLDH and polymer. The superior bioactivity and biocompatibility of the nanocomposites, as compared to pure bone cement, has been established through hemolysis assay, cell adhesion, MTT assay and cell proliferation using fluorescence imaging. The developed nanocomposites have been used as a grouting material and significant improvements have been achieved in fatigue behaviour with gradual increment of Al substitution in the Mg : Al mole ratio in nanoLDH, demonstrating the real use of the material in the biomedical area. In vivo experiments on rabbits clearly revealed the superior efficacy of bone cement nanocomposites, over pure bone cement and a blank.

Bone cement/layered double hydroxide nanocomposites as potential biomaterials for joint implant.

Poly(methyl methacrylate)-based bone cement and layered double hydroxide (LDH) nanocomposites have been used as a grouting material for total joint arthroplasty. Few weight percentage of nanoLDH was uniformly dispersed in the bone cement matrix to have adequate interaction with matrix polymer. Mechanical strength, stiffness, toughness, and fatigue resistance of the nanocomposites are found to be higher than that of pure bone cement. Nanocomposites are thermally stable as compared to pristine bone cement. Direct mixing of the nanoLDH without any organic solvent makes these nanocomposites biocompatible. Biocompatibility was evaluated and compared with that of commercial bone cement by measuring hydrophilic nature, hemolysis assay, thrombosis assay, and deposition of apatite in simulated body fluid immersion. Finally, the viability of human osteoblast cells on the above developed nanocomposites was testified for actual biocompatibility. The experiment showed better cell growth in nanocomposites as compared to pure bone cement. Thus, these nanocomposites are found to be better grouting material than bone cement.