Optimization of electrospinning process & parameters for producing defect-free chitosan/polyethylene oxide nanofibers for bone tissue engineering.

Chitosan (CS) nanofibers were electrospun from aqueous chitosan solution using concentrated acetic acid solution as a solvent. Polyethylene oxide (PEO) with varying weight content from 10- 60 wt% was mixed with chitosan solution that acted as a plasticizer to improve spinability of the prepared chitosan solution. With the increase in PEO content from 10-50 wt% the viscosity of the resultant CS/PEO solution was decreased from 0.938 Pa-s to 0.272 Pa-s, whereas higher the concentration of acetic acid lower was the surface tension of resultant chitosan solution. It was found beadless nanofibrous chitosan mat was obtained not less than 85% acetic acid concentration, 50 wt% PEO and at 0.2 wt% NaCl and 5 wt% total polymer concentration. From field emission scanning electron microscopy (FESEM) investigation, it was observed that chitosan fibers with an average diameter of 149 nm were produced at an applied voltage of 22.5 KV, while that varied between 17.5- 25 KV. On the other hand, a minimum of 110 nm of average diameter chitosan nanofiber was obtained at a needle tip to rotor collector distance of 15 cm by the method of electrospining. In terms of solution flow rate, 0.4 mL/h was found to be optimum in obtaining defect-free electrospun fiber with lower average diameter. As a whole, smooth and uniform chitosan nanofibers were obtained from 50/50 CS/PEO solution prepared by using 90% acetic acid and electrospun at 20 kV applied voltage, 15 cm needle tip-to- rotor collector distance with 0.2 mm inner diameter needle and 0.4 mL/h feeding rate. After crosslinking with 1 wt% glutaraldehyde (GTA), the ultimate tensile strength and Young's modulus of chitosan scaffold increased upto 9.47 MPa and 147.75 MPa respectively. From MTT assay and alkaline phosphatase expression analysis upto 11 days of cell culture period it was evident that thus prepared electrospun CS scaffolds supported MG 63 cell proliferation and its differentiation into mature osteoblast.

Traversing the profile of biomimetically nanoengineered iron substituted hydroxyapatite: synthesis, characterization, property evaluation, and drug release modeling.

Even though ion substituted hydroxyapatite nanoparticles are associated with promising features for biomedical applications, green synthesis with precise control of size and shape to produce uniform nanoparticles remains elusive. To overcome this, we herein propose a room temperature, biomimetic approach to synthesize iron substituted nano-hydroxyapatite (m-HA) along with thorough physicochemical and biological evaluation. The study revealed that 10% iron could be isomorphously doped into hydroxyapatite crystal structure. Stress, strain, energy density and atomic occupancy, as a result of substitution, have been ascertained by Williamson-Hall and Rietveld analysis using X-ray diffraction data. X-Ray photoelectron spectroscopy has been employed to confirm the elemental composition, chemical state and environment of m-HA. In addition, vibrating sample magnetometer of m-HA shows a trend towards superparamagnetic behaviour. Further, fluorescence assisted cell sorting and scanning electron microscope studies confirmed increase in the cell density with increasing iron concentration. Excellent antibacterial property, enhanced biocompatibility and bioactivity have also been interestingly observed. More controlled and sustained drug release has been observed with the inclusion of iron. A mathematical model developed to elucidate drug diffusion coefficient reveals Fickian mechanism to govern the release profile up to 8 hours followed by a non-Fickian transport. With these distinct features, this versatile material holds immense potential as bone repair material for osteoporosis where targeted delivery of calcium is required, as a heating mediator in cancer treatment and as a vehicle for site specific delivery of drug.

Two-dimensional collagen-graphene as colloidal templates for biocompatible inorganic nanomaterial synthesis.

In this study, natural graphite was first converted to collagen-graphene composites and then used as templates for the synthesis of nanoparticles of silver, iron oxide, and hydroxyapatite. X-ray diffraction did not show any diffraction peaks of graphene in the composites after inorganic nucleation, compared to the naked composite which showed (002) and (004) peaks. Scanning electron micrographs showed lateral gluing/docking of these composites, possibly driven by an electrostatic attraction between the positive layers of one stack and negative layers of another, which became distorted after inorganic nucleation. Docking resulted in single layer-like characteristics in certain places, as seen under transmission electron microscopy, but sp2/sp3 ratios from Raman analysis inferred three-layer composite formation. Strain-induced folding of these layers into uniform clusters at the point of critical nucleation, revealed beautiful microstructures under scanning electron microscopy. Lastly, cell viability studies using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed the highest cell viability for the collagen-graphene-hydroxyapatite composites. In this manner, this study provided - to the field of nanomedicine - a new process for the synthesis of several nanoparticles (with low toxicity) of high interest for numerous medical applications.

Optimizing superparamagnetic iron oxide nanoparticles as drug carriers using an in vitro blood-brain barrier model.

In the current study, an optimized in vitro blood-brain barrier (BBB) model was established using mouse brain endothelial cells (b.End3) and astrocytes (C8-D1A). Before measuring the permeability of superparamagnetic iron oxide nanoparticle (SPION) samples, the BBB was first examined and confirmed by an immunofluorescent stain and evaluating the transendothelial electrical resistance. After such confirmation, the permeability of the following five previously synthesized SPIONs was determined using this optimized BBB model: 1) GGB (synthesized using glycine, glutamic acid, and bovine serum albumin [BSA]), 2) GGC (glycine, glutamic acid, and collagen), 3) GGP (glycine, glutamic acid, and polyvinyl alcohol), 4) BPC (BSA, polyethylene glycol, and collagen), and 5) CPB (collagen, polyvinyl alcohol, and BSA). More importantly, after the permeability test, transmission electron microscopy thin section technology was used to investigate the mechanism behind this process. Transmission electron microscopy thin section images supported the hypothesis that collagen-coated CPB SPIONs displayed better cellular uptake than glycine and glutamine acid-coated GGB SPIONs. Such experimental data demonstrated how one can modify SPIONs to better deliver drugs to the brain to treat a wide range of neurological disorders.

Anomalously augmented charge transport capabilities of biomimetically transformed collagen intercalated nanographene-based biocolloids.

Collagen microfibrils biomimetically intercalate graphitic structures in aqueous media to form graphene nanoplatelet-collagen complexes (G-Cl). Synthesized G-Cl-based stable, aqueous bionanocolloids exhibit anomalously augmented charge transportation capabilities oversimple collagen or graphene based colloids. The concentration tunable electrical transport properties of synthesized aqueous G-Cl bionanocolloids has been experimentally observed, theoretically analyzed, and mathematically modeled. A comprehensive approach to mathematically predict the electrical transport properties of simple graphene and collagen based colloids has been presented. A theoretical formulation to explain the augmented transport characteristics of the G-Cl bionanocolloids based on the physicochemical interactions among the two entities, as revealed from extensive characterizations of the G-Cl biocomplex, has also been proposed. Physical interactions between the zwitterionic amino acid molecules within the collagen triple helix with the polar water molecules and the delocalized π electrons of graphene and subsequent formation of partially charged entities has been found to be the crux mechanism behind the augmented transport phenomena. The analysis has been observed to accurately predict the degree of enhancement in transport of the concentration tunable composite colloids over the base colloids. The electrically active G-Cl bionanocolloids with concentration tunability promises find dual utility in novel gel bioelectrophoresis-based protein separation techniques and advanced surface charge modulated drug delivery using biocolloids.

In situ synthesis of hydroxyapatite nanocomposites using iron oxide nanofluids at ambient conditions.

This paper describes a simple method for the room temperature synthesis of magnetite/hydroxyapatite composite nanocomposites using ferrofluids. The in situ synthesis of magnetic-hydroxyapatite results in a homogenous distribution of the two phases as seen both in transmission electron micrographs and assembled to a micron range in the confocal micrographs. The selected area diffraction pattern analysis shows the presence of both phases of iron oxide and hydroxyapatite. To the dialyzed ferrofluid, the constituents of hydroxyapatite synthesis was added, the presence of the superparamagnetic iron oxide particles imparts directionality to the hydroxyapatite crystal growth. Electron probe microanalysis confirms the co-existence of both iron and calcium atoms. Vibrating Sample magnetometer data shows magnetization three times more than the parent ferrofluid, the local concentration of iron oxide nanoparticles affects the strength of dipolar interparticle interactions changing the energy barrier for determining the collective magnetic behavior of the sample. The limitations inherent to the use of external magnetic fields which can be circumvented by the introduction of internal magnets located in the proximity of the target by a minimal surgery or by using a superparamagnetic scaffold under the influence of externally applied magnetic field inspires us to increase the magnetization of our samples. The composite in addition shows anti-bacterial properties against the two gram (-ve) bacteria tested. This work is significant as magnetite-hydroxyapatite composites are attracting a lot of attention as adsorbents, catalysts, hyperthermia agents and even as regenerative medicine.

Colloidal graphite/graphene nanostructures using collagen showing enhanced thermal conductivity.

In the present study, the exfoliation of natural graphite (GR) directly to colloidal GR/graphene (G) nanostructures using collagen (CL) was studied as a safe and scalable process, akin to numerous natural processes and hence can be termed "biomimetic". Although the exfoliation and functionalization takes place in just 1 day, it takes about 7 days for the nano GR/G flakes to stabilize. The predominantly aromatic residues of the triple helical CL forms its own special micro and nanoarchitecture in acetic acid dispersions. This, with the help of hydrophobic and electrostatic forces, interacts with GR and breaks it down to nanostructures, forming a stable colloidal dispersion. Surface enhanced Raman spectroscopy, X-ray diffraction, photoluminescence, fluorescence, and X-ray photoelectron spectroscopy of the colloid show the interaction between GR and CL on day 1 and 7. Differential interference contrast images in the liquid state clearly reveal how the GR flakes are entrapped in the CL fibrils, with a corresponding fluorescence image showing the intercalation of CL within GR. Atomic force microscopy of graphene-collagen coated on glass substrates shows an average flake size of 350 nm, and the hexagonal diffraction pattern and thickness contours of the G flakes from transmission electron microscopy confirm ≤ five layers of G. Thermal conductivity of the colloid shows an approximate 17% enhancement for a volume fraction of less than approximately 0.00005 of G. Thus, through the use of CL, this new material and process may improve the use of G in terms of biocompatibility for numerous medical applications that currently employ G, such as internally controlled drug-delivery assisted thermal ablation of carcinoma cells.

Controlling ferrofluid permeability across the blood­brain barrier model.

In the present study, an in vitro blood­brain barrier model was developed using murine brain endothelioma cells (b.End3 cells). Confirmation of the blood­brain barrier model was completed by examining the permeability of FITCDextran at increasing exposure times up to 96 h in serum-free medium and comparing such values with values from the literature. After such confirmation, the permeability of five novel ferrofluid (FF) nanoparticle samples, GGB (ferrofluids synthesized using glycine, glutamic acid and BSA), GGC (glycine, glutamic acid and collagen), GGP (glycine, glutamic acid and PVA), BPC (BSA, PEG and collagen) and CPB (collagen, PVA and BSA), was determined using this blood­brain barrier model. All of the five FF samples were characterized by zeta potential to determine their charge as well as TEM and dynamic light scattering for determining their hydrodynamic diameter. Results showed that FF coated with collagen passed more easily through the blood­brain barrier than FF coated with glycine and glutamic acid based on an increase of 4.5% in permeability. Through such experiments, diverse magnetic nanomaterials (such as FF) were identified for: (1) MRI use since they were less permeable to penetrate the blood­brain barrier to avoid neural tissue toxicity (e.g. GGB) or (2) brain drug delivery since they were more permeable to the blood­brain barrier (e.g. CPB).

Protein-Polymer Matrix Mediated Synthesis of Silver Nanoparticles.

Silver nanoparticles were synthesized in the protein-polymer matrices of two different ratios to obtain a stringent control over the morphology. UV-visible spectrophotometry showed a single plasmon resonance peak at 416nm and 418nm respectively, confirming the formation of silver nanoparticles. X-ray diffractometry confirmed that the peaks matched with that of the reference silver. Both confocal microscopy and FEG-SEM confirmed the uniform morphology of the synthesized particles dependent on the template ratio. Doubling the protein-polymer concentration results in greater stability, more nucleation sites and hence restricted growth. Photoluminescence of the sample in the doubled matrix was found to be much greater at any given wavelength, meaning the flexibility and rigidity of interacting molecules affects the luminescence signal. The interaction in turn is dependent on the proximity of the proteins and polymer in the dispersion that forms a template and dictates the synthesis.

Comparison study of ferrofluid and powder iron oxide nanoparticle permeability across the blood-brain barrier.

In the present study, the permeability of 11 different iron oxide nanoparticle (IONP) samples (eight fluids and three powders) was determined using an in vitro blood-brain barrier model. Importantly, the results showed that the ferrofluid formulations were statistically more permeable than the IONP powder formulations at the blood-brain barrier, suggesting a role for the presently studied in situ synthesized ferrofluid formulations using poly(vinyl) alcohol, bovine serum albumin, collagen, glutamic acid, graphene, and their combinations as materials which can cross the blood-brain barrier to deliver drugs or have other neurological therapeutic efficacy. Conversely, the results showed the least permeability across the blood-brain barrier for the IONP with collagen formulation, suggesting a role as a magnetic resonance imaging contrast agent but limiting IONP passage across the blood-brain barrier. Further analysis of the data yielded several trends of note, with little correlation between permeability and fluid zeta potential, but a larger correlation between permeability and fluid particle size (with the smaller particle sizes having larger permeability). Such results lay the foundation for simple modification of iron oxide nanoparticle formulations to either promote or inhibit passage across the blood-brain barrier, and deserve further investigation for a wide range of applications.

A novel biomimetic material--glycine-PVA ferrofluid that crosses the blood-brain barrier.

The present study describes the efficacy of the glycine-poly(vinyl)alcohol ferrofluid as a contrast enhancer for Magnetic resonance imaging. Interaction between inorganic minerals and biomolecules are extremely unique and interesting. The results demonstrate that ferro fluids can be a good contrast enhancer for Magnetic Resonance Imaging as seen in the very first results after MRI studies.

Fluorimetric assay of interaction of protein with ferrofluids.

Magnetic iron oxide nanoparticles are inherently biocompatible and are amenable to post synthesis surface modification, making them excellent candidates for many important applications. If the above can be achieved in a single-step i.e., in situ synthesis and functionalization, the results are expected to be more dramatic for sensitive detection of biomolecules. For any application, it is necessary to confer a high level of binding specificity through surface chemistry, which can be introduced by using biological moieties that possess lock-and-key interactions, like those observed in antibody-antigen and enzyme-substrate recognition. In this paper, we have synthesized water based ferrofluids with serum albumin, the major protein component of blood. A series of other ferrofluids using different biocompatible polymers have also been studied with respect to their size determined by transmission electron microscopy, magnetic behavior with the aid of vibrating sample magnetometry and binding capability to bovine serum albumin by quenching of its native fluorescence. From our results, it can be inferred that binding has taken place between magnetic particles and biomolecules, the binding constants of which indirectly reveal the efficiency of the interaction.

Aqueous ferrofluids as templates for magnetic hydroxyapatite nanocomposites.

Poly (vinyl) alcohol stabilized aqueous ferrofluids (PVA-ff) were used as nanotemplates for the crystallization of calcium hydroxyapatite (HAp). Four sets of PVA-ff-HAp nanocomposites were synthesized using 20, 40, 60 and 80 ml of PVA-ff for the same initial constituents of HAp. Various physico-chemical analyses suggest that the HAp lattice structure accommodates PVA-ff to a certain extent, beyond which the magnetic intra-molecular interactions predominate and PVA-ff starts to be pushed out of the HAp matrix. The in situ incorporation of PVA-ff during HAp synthesis results in a novel magnetic biomaterial with potential applications as targeted delivery vehicles.

Microwave irradiation enhances kinetics of the biomimetic process of hydroxyapatite nanocomposites.

In situ synthesized hydroxyapatite-poly(vinyl) alcohol nanocomposite was subjected to microwave irradiation, post synthesis. Interestingly, the aging time of 1 week required for the normal biomimetic process was reduced to 1 h post microwave irradiation, as characterized by x-ray powder diffraction and transmission electron microscopy. The surface topography shows the tendency of tubules to cross-link with the help of microwave energy. The microwave energy seems to provide a directional pull to the polymer chains which could have led to an enhancement of the kinetics of phase formation.

Bactericidal effect of iron oxide nanoparticles on Staphylococcus aureus.

In order to study the effects of iron oxide (IO) nanoparticles on Staphylococcus aureus, IO nanoparticles were synthesized via a novel matrix-mediated method using polyvinyl alcohol (PVA). The IO nanoparticles were characterized by transmission electron microscopy and dynamic light scattering. Further, S. aureus were grown in the presence of three different IO nanoparticle concentrations for four, 12, and 24 hours. Live/dead assays were performed and the results provide evidence that IO/PVA nanoparticles inhibited S. aureus growth at the highest concentration (3 mg/mL) at all time points.

Non-destructive evaluation of mechanical properties of poly (vinyl) alcohol-hydroxyapatite nanocomposites.

Hydroxyapatite-poly (vinyl) alcohol nanocomposite powder was synthesized using varying poly (vinyl) alcohol concentrations. The dried powder was compacted into micro-porous disks at a load of 4 tons. The disks were sintered at 1200 degrees C to evolve porous nanocomposites. Size and shape of the pores observed in the scanning electron micrographs were quantified by using image processing software. Ultrasound velocity measurements were done to evaluate mechanical properties non-destructively.

Composition dependent structural modulations in transparent poly(vinyl alcohol) hydrogels.

Transparent and stable Poly(vinyl alcohol) hydrogels were synthesized from polymer aqueous solution without resorting to a mixed solvent such as dimethyl sulfoxide and water. Contrary to the reported methods involving hydrogen bond induced physical crosslinking by repeated freeze-thawing at -20 degrees C, the present process demonstrates the gelation taking place at relatively higher temperature, i.e. 0 degrees C. While maintaining transparency in all the synthesized hydrogels, the present paper reports systematic structural and morphological variations in the hydrogels as a function of polymer concentration.

Mesenchymal cell response to nanosized biphasic calcium phosphate composites.

Biphasic calcium phosphate nanoparticles comprising both hydroxyapatite (HA) and beta polymorph of tricalcium phosphate (beta-TCP) have been synthesized together by a polymer matrix mediated process. The process, based on in situ mineralization of poly (vinyl alcohol), exerts a good control over the morphological features of biphasic nanoparticles. By controlling the reaction chemistry (Ca:P ratios), nanobioceramic particles having three different HA/beta-TCP ratios of 50:50, 55:45 and 60:40 respectively. As the two constituents of biphasic system (HA and beta-TCP) facilitate series of signaling cascades in osteoblast division and differentiation, the adhesion and differentiation properties of mesenchymal cells (MSCs) derived from bone marrow has been studied.

Biomimetically synthesized polymer-hydroxyapatite sheet like nano-composite.

Biomimetic internal architecture is proving valuable for multi-tissue and structural tissue interface engineering. A biomimetic method developed for the in situ synthesis of hydroxyapatite in poly (vinyl) alcohol, produces nanocomposites in the form of fabric-like sheets, when subjected to lyophilisation. The surface reactive hydroxyl groups of poly (vinyl) alcohol encourage site-specific coupling of hydroxyapatite. This results in improved mechanical properties, which may be better for biomedical applications when compared to a mere blend of the above two.

Synthesis and sintering of biomimetic hydroxyapatite nanoparticles for biomedical applications.

Synthesis of monodisperse nanoparticles with uniform morphology and narrow size distribution as achieved by nature is a challenge to materials scientists. Mimicking the process of biomineralization has led to the development of biomolecules mediated synthesis of nanoparticles that overcomes many of the problems associated with nanoparticle synthesis. Termed as biomimetics this paradigm shift in the philosophy of synthesis of materials is very advantageous for the design-based synthesis of nanoparticles. The effect of concentration of a protein named bovine serum albumin on particle size, morphology and degree of crystallinity of biomimetically synthesized hydroxyapatite particles, has been studied. Results establish 0.5% protein as the required concentration to produce 30-40 nm sized hydroxyapatite particles with an optimum degree of crystallinity as required for biomedical applications. These particles synthesized under certain stringent conditions are found to have stoichiometric calcium:phosphorus ratio of 1.67 and exhibit restricted grain growth during sintering.