Avinash J. Patil

The fabrication of nano-hydroxyapatite on PLGA and PLGA/collagen nanofibrous composite scaffolds and their effects in osteoblastic behavior for bone tissue engineering

Bone is a nanocomposite consisting of two main components, nano-hydroxyapatite (n-HA) and Type I collagen (Col). The aim is to exploit the nano-scale functional and material characteristics of natural bone in order to modulate cellular functions for optimal bone repair in bone graft systems. Here, we present an effective and novel technique in obtaining n-HA in cognate with native apatite on electrospun nanofibers within minutes without any pre-treatment. Using an alternate calcium and phosphate (Ca-P) solution dipping method, n-HA was formed on poly(lactide-co-glycolide) acid (PLGA) and blended PLGA/Col nanofibers. The presence of the functional groups of collagen significantly hastened n-HA deposition closed to nine-fold. The quantity of n-HA impinged upon the specific surface area, whereby mineralized PLGA/Col had a greater surface area than non-mineralized PLGA/Col, whereas n-HA did not significantly improve the specific surface area of mineralized PLGA compared to pure PLGA. The novelty of the process was that n-HA on PLGA had a positive modulation on early osteoblast capture (within minutes) compared to pure PLGA. Contrary, cell capture on mineralized PLGA/Col was comparable to pure PLGA/Col. Interestingly, although n-HA impeded proliferation during the culture period (days 1, 4 and 7), the cell functionality such as alkaline phosphatase (ALP) and protein expressions were ameliorated on mineralized nanofibers. The amount of n-HA appeared to have a greater effect on the early stages of osteoblast behavior (cell attachment and proliferation) rather than the immediate/late stages (proliferation and differentiation).

Interfacial Assembly of Protein-polymer Nano-conjugates into Stimulus-Responsive Biomimetic Protocells

The mechanism of spontaneous assembly of microscale compartments is a central question for the origin of life, and has technological repercussions in diverse areas such as materials science, catalysis, biotechnology and biomedicine. Such compartments need to be semi-permeable, structurally robust and capable of housing assemblages of functional components for internalized chemical transformations. In principle, proteins should be ideal building blocks for the construction of membrane-bound compartments but protein vesicles with cell-like properties are extremely rare. Here we present an approach to the interfacial assembly of protein-based micro-compartments (proteinosomes) that are delineated by a semi-permeable, stimulus-responsive, enzymatically active, elastic membrane consisting of a closely packed monolayer of conjugated protein-polymer building blocks. The proteinosomes can be dispersed in oil or water, thermally cycled to temperatures of 70 °C, and partially dried and re-inflated without loss of structural integrity. As a consequence, they exhibit protocellular properties such as guest molecule encapsulation, selective permeability, gene-directed protein synthesis and membrane-gated internalized enzyme catalysis.

Fabrication of Mineralized Polymeric Nanofibrous Composites for Bone Graft Materials

Poly-L-lactic acid (PLLA) and PLLA/collagen (50% PLLA+50% collagen; PLLA/Col) nanofibers were fabricated using electrospinning. Mineralization of these nanofibers was processed using a modified alternating soaking method. The structural properties and morphologies of mineralized PLLA and PLLA/Col nanofibers were investigated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and contact angle measurements. Human bone-derived osteoblasts were cultured on the materials for up to 1 week to assess the biological properties of the nanofibrous composites. Cell attachment on these nanocomposites was also tested within 1 h of culture at room temperature. The mechanical properties of the cell-nanocomposite constructs were determined using tensile testing. From our results, the bone-like nano-hydroxyapatite (n-HA) was successfully deposited on the PLLA and PLLA/Col nanofibers. We observed that the formation of n-HA on PLLA/Col nanofibers was faster and significantly more uniform than on pure PLLA nanofibers. The n-HA significantly improved the hydrophilicity of PLLA/Col nanofibers. From the results of cell attachment studies, n-HA deposition enhanced the cell capture efficacy at the 20-minute time point for PLLA nanofibers. The E-modulus values for PLLA+n-HA with cells (day 1 and day 4) were significantly higher than for PLLA+n-HA without cells. Based on these observations, we have demonstrated that n-HA deposition on nanofibers is a promising strategy for early cell capture.

Self-assembly of bio–inorganic nanohybrids using organoclay building blocks

Routine and reliable syntheses of self-assembled hybrid materials with tuneable functionalities are urgently required if novel functional nanostructures are to be developed for real-life applications and economic commercialization. This Highlight presents an overview of recent progress in the synthesis and use of a new class of mesolamellar magnesium organophyllosilicate clays containing covalently linked organic functionalities. We focus in particular on strategies of template-directed synthesis, exfoliation, fractionation and re-assembly involving aminopropyl-functionalized organoclays, and illustrate how combinations of these approaches can be used for the fabrication of novel functional hybrid materials with complex morphological form and structure. Examples of mesolamellar intercalation of functional biomolecules such as enzymes, DNA and drugs, nanoscale wrapping of isolated proteins, enzymes and polynucleotides, and engineering of photoactive cell membrane/organoclay superstructures are presented.

Preparation of high quality nanowires by tobacco mosaic virus templating of gold nanoparticles

A protocol for the preparation of stable suspensions of well-defined metallized tobacco mosaic virus (TMV) nanorods in high yields and with uniform coatings is reported. Inorganic growth on the virion surface was controlled by adding aliquots of gold precursor (HAuCl4) and reducing agent (NaBH4) incrementally over five addition–reduction cycles to produce hybrid anisotropic nanostructures consisting of densely packed gold nanoparticles. Several novel steps, including addition of ethanol after the first addition–reduction cycle and wrapping of the TMV/Au nanohybrids with poly-L-lysine, were introduced into the protocol to ensure high homogeneity and stability in the nanowire suspension. Control of the interparticle spacing in the metallized nanostructures was achieved by restricting the number of reaction cycles used to less than four iterations. The importance of bio-mediated reduction was probed using laser light scattering microscopy. The results indicated that short incubation times were consistent with increased nanoparticle monodispersity and high fidelity replication.

Fabrication of functional protein–organoclay lamellar nanocomposites by biomolecule-induced assembly of exfoliated aminopropyl-functionalized magnesium phyllosilicates

An aminopropyl-functionalized magnesium phyllosilicate clay was synthesized and used as an exfoliated precursor for the intercalation of myoglobin (Mb), haemoglobin (Hb) or glucose oxidase (GOx). Intercalation was achieved by exfoliation of the as-synthesized organoclay in water followed by room temperature re-assembly of the lamellar phase in the presence of negatively charged protein molecules. X-Ray diffraction data of the resulting nanocomposites showed an expansion of the interlayer d001 spacing from 1.6 nm in the parent organoclay to 4.3 and 6.4 nm for samples prepared with Mb and GOx, respectively, indicating that these biomolecules were successfully incorporated into the gallery regions. In both cases, FTIR and circular dichroism spectroscopies showed that secondary structures of the intercalated biomolecules were preserved. In addition, UV-vis spectroscopy revealed that the redox and CO/O2 binding properties of the intercalated Mb, as well as the enzymatic activity of GOx, were retained in the protein–organoclay nanocomposites. Significantly, the relative catalytic activities of GOx between pH values of 3 to 10, and up to temperatures of 65 °C, were higher in the hybrid nanocomposites compared with the native enzyme in solution. Similar experiments with Hb produced composites consisting of structurally and functionally intact protein molecules immobilized within a disordered matrix of organoclay lamellae.

Design and Construction of Higher-Order Structure and Function in Proteinosome-Based Protocells

The design and construction of higher-order structure and function in proteinosome microcompartments enclosed by a cross-linked membrane of amphiphilic bovine serum albumin/poly(N-isopropylacrylamide) (BSA-NH2/PNIPAAm) nanoconjugates is described. Three structure/function relationships are investigated: (i) differential chemical cross-linking for the control of membrane disassembly and regulated release of encapsulated genetic polymers; (ii) enzyme-mediated hydrogel structuring of the internal microenvironment to increase mechanical robustness and generate a molecularly crowded reaction environment; and (iii) self-production of a membrane-enclosing outer hydrogel wall for generating protease-resistant forms of the protein-polymer protocells. Our results highlight the potential of integrating aspects of supramolecular and polymer chemistry into the design and construction of novel bioinspired microcompartments as a step toward small-scale materials systems based on synthetic cellularity.

Magnesium and calcium organophyllosilicates: synthesis and in vitro cytotoxicity study.

Synthesis of multifunctional hybrid nanomaterials for biomedical applications has received great attention. Herein, we examine the potential toxicity of organophyllosilicates on cells from different organs such as A549 (lung epithelial cancer), HT-29 (colon epithelial cancer), MRC-5 (lung fibroblast) and CCD-986sk (skin fibroblast) cells. For this, aminopropyl functionalized magnesium phyllosilicate (AMP clay) and aminopropyl functionalized calcium phyllosilicate (ACP clay) were prepared using one-pot direct sol-gel method. Toxic effects of these organoclays on normal fibroblast and tumor cells were examined under varying concentrations and exposure times. MTT and LDH assays indicated that both organoclays had little cytotoxicity in all of the cells tested at concentrations as high as 500 μg/mL. Even at high concentration (1000 μg/mL), the toxicity of both organoclays on cell viability and membrane damage was not severe and appeared to be cell type specific. In addition, organoclays did not induce apoptosis at concentrations as high as 1000 μg/mL.

Polymer/nucleotide droplets as bio-inspired functional micro-compartments

Using a range of physical methods, we describe the formation, structure, stability, physical properties and uptake behavior of condensed liquid micro-droplets prepared by electrostatically induced complexation of poly(diallyldimethylammonium) chloride (PDDA) and adenosine triphosphate (ATP) in water. Depending on the PDDA monomer: ATP molar ratio, positively charged or charge-neutral droplets are produced spontaneously by simple mixing. The former are typically 60–600 nm in mean size and stable with respect to sedimentation up to temperatures of 85 °C, whilst the latter grow into droplets several tens of micrometres in diameter that coalesce into a macroscopic coacervate phase. Coacervation is inhibited at pH values less than 3 and at high ionic strength, confirming the importance of charge interactions in droplet formation and stability. The droplet interior is structurally homogeneous with no surrounding membrane, comprises dynamically fluctuating domains of partially desolvated polymer/nucleotide complexes, and has a dielectric constant considerably lower than water. As a consequence, dye molecules, porphyrin macrocycles, inorganic nanoparticles or globular proteins can be sequestered from the external water phase into the droplets to produce PDDA/ATP droplets comprising supramolecular J-aggregate nanostructures, magnetically responsive deformable fluids, or soft compartments with potential storage and release properties.

Influence of polymer co-intercalation on guest release from aminopropyl-functionalized magnesium phyllosilicate mesolamellar nanocomposites

Protonation of the interlayer aminopropyl groups of a synthesized organo-functionalized 2 : 1 trioctahedral magnesium phyllosilicate was used to prepare exfoliated cationic organoclay dispersions that were subsequently re-assembled in the presence of anionic guest molecules, such as ibuprofen, epigallocatechin gallate (EGCG), poly(styrene sulfonate) (PSS), poly(acrylic acid) (PAA), or polymethyl-acrylamidopropanesulfonic acid (PMAPSA), to produce a range of novel intercalated layered nanocomposites. Re-assembled organoclays containing ibuprofen or epigallocatechin gallate co-intercalated with various types of polymer molecules were also prepared. In each case, X-ray diffraction studies confirmed the formation of intercalated or co-intercalated lamellar nanocomposites with expanded interlayer (d001) spacings. We demonstrate that pH dependent oxidation of EGCG is curtailed when intercalated within the organoclay interlayers. Significantly, the ibuprofen release profiles in water or simulated gastric fluid (pH = 2) indicated that extraction of the biomolecule into solution could be delayed or accelerated depending on the type of polymer co-intercalated. Whereas co-intercalation of PSS increased the rate of ibuprofen release, extraction of the drug from nanocomposites containing PMAPSA was significantly reduced. In contrast, PAA-containing ibuprofen/organoclay nanocomposites showed release profiles that were only marginally reduced compared with those for ibuprofen alone. Steric and electrostatic interactions between the entrapped polymer and drug molecules are discussed to account for these observations.