Kapil D. Patel

Potential of Magnetic Nanofiber Scaffolds with Mechanical and Biological Properties Applicable for Bone Regeneration

Magnetic nanofibrous scaffolds of poly(caprolactone) (PCL) incorporating magnetic nanoparticles (MNP) were produced, and their effects on physico-chemical, mechanical and biological properties were extensively addressed to find efficacy for bone regeneration purpose. MNPs 12 nm in diameter were citrated and evenly distributed in PCL solutions up to 20% and then were electrospun into nonwoven nanofibrous webs. Incorporation of MNPs greatly improved the hydrophilicity of the nanofibers. Tensile mechanical properties of the nanofibers (tensile strength, yield strength, elastic modulus and elongation) were significantly enhanced with the addition of MNPs up to 15%. In particular, the tensile strength increase was as high as ∼25 MPa at 15% MNPs vs. ∼10 MPa in pure PCL. PCL-MNP nanofibers exhibited magnetic behaviors, with a high saturation point and hysteresis loop area, which increased gradually with MNP content. The incorporation of MNPs substantially increased the degradation of the nanofibers, with a weight loss of ∼20% in pure PCL, ∼45% in 10% MNPs and ∼60% in 20% MNPs. Apatite forming ability of the nanofibers tested in vitro in simulated body fluid confirmed the substantial improvement gained by the addition of MNPs. Osteoblastic cells favored the MNPs-incorporated nanofibers with significantly improved initial cell adhesion and subsequent penetration through the nanofibers, compared to pure PCL. Alkaline phosphatase activity and expression of genes associated with bone (collagen I, osteopontin and bone sialoprotein) were significantly up-regulated in cells cultured on PCL-MNP nanofibers than those on pure PCL. PCL-MNP nanofibers subcutaneously implanted in rats exhibited minimal adverse tissue reactions, while inducing substantial neoblood vessel formation, which however, greatly limited in pure PCL. In vivo study in radial segmental defects also signified the bone regeneration ability of the PCL-MNP nanofibrous scaffolds. The magnetic, bone-bioactive, mechanical, cellular and tissue attributes of MNP-incorporated PCL nanofibers make them promising candidate scaffolds for bone regeneration.

Multifunctional Hybrid Nanocarrier: Magnetic CNTs Ensheathed with Mesoporous Silica for Drug Delivery and Imaging System

Here we communicate the development of a novel multifunctional hybrid nanomaterial, magnetic carbon nanotubes (CNTs) ensheathed with mesoporous silica, for the simultaneous applications of drug delivery and imaging. Magnetic nanoparticles (MNPs) were first decorated onto the multiwalled CNTs, which was then layered with mesoporous silica (mSiO2) to facilitate the loading of bioactive molecules to a large quantity while exerting magnetic properties. The hybrid nanomaterial showed a high mesoporosity due to the surface-layered mSiO2, and excellent magnetic properties, including magnetic resonance imaging in vitro and in vivo. The mesoporous and magnetic hybrid nanocarriers showed high loading capacity for therapeutic molecules including drug gentamicin and protein cytochrome C. In particular, genetic molecule siRNA was effectively loaded and then released over a period of days to a week. Furthermore, the hybrid nanocarriers exhibited a high cell uptake rate through magnetism, while eliciting favorable biological efficacy within the cells. This novel hybrid multifunctional nanocarrier may be potentially applicable as drug delivery and imaging systems.

Chitosan–nanobioactive glass electrophoretic coatings with bone regenerative and drug delivering potential

Nanocomposites with bone-bioactivity and drug eluting capacity are considered as potentially valuable coating materials for metallic bone implants. Here, we developed composite coatings of chitosan (CH)–bioactive glass nanoparticles (BGn) via cathodic electrophoretic deposition (EPD). BGn 50–100 nm in size with aminated surface were suspended with CH molecules at different ratios (5–20 wt% BGn) in aqueous medium, and EPD was performed. Uniform coatings with thicknesses of a few to tens of micrometers were produced, which was controllable by the EPD parameters (voltage, pH and time). Thermogravimetric analysis revealed the quantity of BGn within the coatings that well corresponded to that initially incorporated. Apatite forming ability of the coatings, performed in simulated body fluid, was significantly improved by the addition of BGn. Degradation of the coatings increased with increasing BGn addition. Of note, the degradation profile was almost linear with time; degradation of 5–13 wt% during 1 week became 30–40 wt% after 7 weeks at almost a constant rate. The CH–BGn coatings showed favorable cell adhesion and growth, and stimulated osteogenic differentiation. Drug loading and release capacity of the CH–BGn coatings were performed using the ampicillin antibiotic as a model drug. Ampicillin, initially incorporated within the CH–BGn suspension, was eluted from the coatings continuously over 10–11 weeks, confirming long-term drug delivering capacity. Antibacterial tests also confirmed the effects of released ampicillin using agar diffusion assay against Streptococcus mutants. The CH–BGn may be potentially useful as a coating composition for metallic implants due to the excellent bone bioactivity and cell responses, as well as the capacity for long-term drug delivery.

Mesoporous Silica-Layered Biopolymer Hybrid Nanofibrous Scaffold: A Novel Nanobiomatrix Platform for Therapeutics Delivery and Bone Regeneration

Nanoscale scaffolds that characterize high bioactivity and the ability to deliver biomolecules provide a 3D microenvironment that controls and stimulates desired cellular responses and subsequent tissue reaction. Herein novel nanofibrous hybrid scaffolds of polycaprolactone shelled with mesoporous silica (PCL@MS) were developed. In this hybrid system, the silica shell provides an active biointerface, while the 3D nanoscale fibrous structure provides cell-stimulating matrix cues suitable for bone regeneration. The electrospun PCL nanofibers were coated with MS at controlled thicknesses via a sol-gel approach. The MS shell improved surface wettability and ionic reactions, involving substantial formation of bone-like mineral apatite in body-simulated medium. The MS-layered hybrid nanofibers showed a significant improvement in mechanical properties, in terms of both tensile strength and elastic modulus, as well as in nanomechanical surface behavior, which is favorable for hard tissue repair. Attachment, growth, and proliferation of rat mesenchymal stem cells were significantly improved on the hybrid scaffolds, and their osteogenic differentiation and subsequent mineralization were highly up-regulated by the hybrid scaffolds. Furthermore, the mesoporous surface of the hybrid scaffolds enabled the loading of a series of bioactive molecules, including small drugs and proteins at high levels. The release of these molecules was sustainable over a long-term period, indicating the capability of the hybrid scaffolds to deliver therapeutic molecules. Taken together, the multifunctional hybrid nanofibrous scaffolds are considered to be promising therapeutic platforms for stimulating stem cells and for the repair and regeneration of bone.

A novel therapeutic design of microporous-structured biopolymer scaffolds for drug loading and delivery

Three-dimensional (3-D) open-channeled scaffolds of biopolymers are a promising candidate matrix for tissue engineering. When scaffolds have the capacity to deliver bioactive molecules the potential for tissue regeneration should be greatly enhanced. In order to improve drug-delivery capacity, we exploit 3-D poly(lactic acid) (PLA) scaffolds by creating microporosity within the scaffold network. Macroporous channeled PLA with a controlled pore configuration was obtained by a robotic dispensing technique. In particular, a room temperature ionic liquid (RTIL) bearing hydrophilic counter-anions, such as OTf and Cl, was introduced to the biopolymer solution at varying ratios. The RTIL-biopolymer slurry was homogenized by ultrasonication, and then solidified through the robotic dispensing process, during which the biopolymer and RTIL formed a bicontinuous interpenetrating network. After ethanol wash-out treatment the RTIL was completely removed to leave highly microporous open channels throughout the PLA network. The resultant pore size was observed to be a few micrometers (average 2.43 μm) and microporosity was determined to be ∼ 70%. The microporous surface was also shown to favor initial cell adhesion, stimulating cell anchorage on the microporous structure. Furthermore, in vivo tissue responses assessed in rat subcutaneous tissue revealed good tissue compatibility, with minimal inflammatory reactions, while gathering a larger population of fibroblastic cells than the non-microporous scaffolds, and even facilitating invasion of the cells within the microporous structure. The efficacy of the micropore networks generated within the 3-D scaffolds in loading and releasing therapeutic molecules was addressed using antibiotic sodium ampicillin and protein cytochrome C as model drugs. The microporous scaffolds exhibited significantly enhanced drug loading capacity: 4-5 times increase in ampicillin and 9-10 times increase in cytochrome C compared to the non-microporous scaffolds. The release of ampicillin loaded within the microporous scaffolds was initially fast (∼ 85% for 1 week), and was then slowed down, showing a continual release up to a month. On the other hand, cytochrome C was shown to release in a highly sustainable manner over a month, without showing an initial burst release effect. This study provides a novel insight into the generation of 3-D biopolymer scaffolds with high performance in loading and delivery of biomolecules, facilitated by the creation of microporous channels through the scaffold network. The capacity to support tissue cells while in situ delivering drug molecules makes the current scaffolds potentially useful for therapeutic tissue engineering.

Luminescent mesoporous nanoreservoirs for the effective loading and intracellular delivery of therapeutic drugs

Development of biocompatible and multifunctional nanocarriers is important for the therapeutic efficacy of drug molecules in the treatment of disease and tissue repair. A novel nanocarrier of luminescent hollowed mesoporous silica (L-hMS) was explored for the loading and controlled delivery of drugs. For the synthesis of L-hMS, self-activated luminescence hydroxyapatite (LHA) was used as a template. Different thicknesses (∼ 7-62 nm) of mesoporous silica shell were obtained by varying the volume of silica precursor and the subsequent removal of the LHA core, which resulted in hollow-cored (size of ∼ 40 nm × 10 nm) mesoporous silica nanoreservoirs, L-hMS. While the silica shell provided a highly mesoporous structure, enabling an effective loading of drug molecules, the luminescent property of LHA was also well preserved in both the silica-shelled and the hollow-cored nanocarriers. Doxorubicin (DOX), used as a model drug, was shown to be effectively loaded onto the mesopore structure and within the hollow space of the nanoreservoir. The DOX release was fairly pH-dependent, occurring more rapidly at pH 5.3 than at pH 7.4, and a long-term sustainable delivery over the test period of 2weeks was observed. The nanoreservoir exhibited favorable cell compatibility with low cytotoxicity and excellent cell uptake efficiency (over 90%). Treatment of HeLa cells with DOX-loaded L-hMS elicited a sufficient degree of biological efficacy of DOX, as confirmed in the DOX-induced apoptotic behaviors, including stimulation in caspase-3 expression, and was even more effective than the direct DOX treatment. Overall, the newly developed L-hMS nanoreservoirs may be potentially useful as a multifunctional (luminescent, mesoporous and biocompatible) carrier system to effectively load and sustainably deliver small molecules, including anticancer drugs.

Nanostructured Biointerfacing of Metals with Carbon Nanotube/Chitosan Hybrids by Electrodeposition for Cell Stimulation and Therapeutics Delivery

Exploring the biological interfaces of metallic implants has been an important issue in achieving biofunctional success. Here we develop a biointerface with nanotopological features and bioactive composition, comprising a carbon nanotube (CNT) and chitosan (Chi) hybrid, via an electrophoretic deposition (EPD). The physicochemical properties, in vitro biocompatibility, and protein delivering capacity of the decorated nanohybrid layer were investigated, to address its potential usefulness as bone regenerating implants. Over a wide compositional range, the nanostructured hybrid interfaces were successfully formed with varying thicknesses, depending on the electrodeposition parameters. CNT-Chi hybrid interfaces showed a time-sequenced degradation in saline water, and a rapid induction of hydroxyapatite mineral in a simulated body fluid. The nanostructured hybrid substrates stimulated the initial adhesion events of the osteoblastic cells, including cell adhesion rate, spreading behaviors, and expression of adhesive proteins. The nanostructured hybrid interfaces significantly improved the adsorption of protein molecules, which was enabled by the surface charge interaction, and increased surface area of the nanotopology. Furthermore, the incorporated protein was released at a highly sustained rate, profiling a diffusion-controlled pattern over a couple of weeks, suggesting the possible usefulness as a protein delivery device. Collectively, the nanostructured hybrid CNT-Chi layer, implemented by an electrodeposition, is considered a biocompatible, cell-stimulating, and protein-delivering biointerface of metallic implants.

Novel magnetic nanocomposite injectables: calcium phosphate cements impregnated with ultrafine magnetic nanoparticles for bone regeneration

Self-setting calcium phosphate cements (CPCs) are some of the few injectables of bioceramic materials applicable to bone repair. Incorporation of nanoparticulates to CPCs is considered a promising process that can improve the mechanical and biological properties of CPCs. Here, we added magnetic nanoparticles (MNPs) at small contents, and examined their effects on the physico-chemical, mechanical, and in vitro biological properties of the CPC composition. The MNPs were coated with a silica layer and had an average size of ∼80 nm. The addition of MNPs did not alter the setting reaction, but dramatically changed the crystal shape and size of the transformed hydroxyapatite, from large plate-like crystals to small needle-like crystals. Mechanical strength was significantly improved from 22 MPa to 33 MPa with the addition of only 0.1% MNPs. A striking difference in cell adhesion of rat bone marrow stromal cells was noticed with the MNPs-incorporation. The cells adhered and spread more actively to the MNP-added CPCs than to the pure CPCs with an approximately 2.5-fold and 4.5-fold increase in cell adhesion and in cell spreading area, respectively. The cell proliferative potential lasted for a longer period of up to 14 days where osteogenic differentiation, determined by alkaline phosphatase activity, was also substantially stimulated by the MNP-incorporation. The results indicate that only a small incorporation of ultrafine MNPs changed the properties of CPCs dramatically, making them more suitable for use in cell culture and repair of bone tissues.

Progress in Nanotheranostics Based on Mesoporous Silica Nanomaterial Platforms

Theranostics based on nanoparticles (NPs) is a promising paradigm in nanomedicine. Mesoporous silica nanoparticle (MSN)-based systems offer unique characteristics to enable multimodal imaging or simultaneous diagnosis and therapy. They include large surface area and volume, tunable pore size, functionalizable surface, and acceptable biological safety. Hybridization with other NPs and chemical modification can further potentiate the multifunctionality of MSN-based systems toward translation. Here, we update the recent progress on MSN-based systems for theranostic purposes. We discuss various synthetic approaches used to construct the theranostic platforms either via intrinsic chemistry or extrinsic combination. These include defect generation in the silica structure, encapsulation of diagnostic NPs within silica, their assembly on the silica surface, and direct conjugation of dye chemicals. Collectively, in vitro and in vivo results demonstrate that multimodal imaging capacities can be integrated with the therapeutic functions of these MSN systems for therapy. With further improvement in bioimaging sensitivity and targeting specificity, the multifunctional MSN-based theranostic systems will find many clinical applications in the near future.

Development of biocompatible apatite nanorod-based drug-delivery system with in situ fluorescence imaging capacity

Biocompatible and multifunctional nanocarriers significantly improve the therapeutic and diagnostic efficacy of the cargo molecules. Here we report a novel biocompatible apatite nanocrystal-based delivery system with in situ imaging capacity. A nanorod (40 nm × 10 nm) produced by a citrate-involved sol-gel process expressed a strong blue emission at 427 nm under fluorescence microscopy. The CO2˙- radical impurities present in the apatite crystal lattice account for the fluorescent behavior. The fluorescent nanorods exhibited excellent cell viability (over 90% up to ∼100 μg ml-1 concentration for both osteoblasts and osteoclasts). The nanorods loaded alendronate drug at a high efficiency (10%) and released over 3 days, while enabling in situ fluorescence imaging within the cells. The fluorescent apatite was further hybridized onto the surface of mesoporous nanospheres, aimed at improving the drug-delivery capacity. Small interfering RNA (siRNA) gene, encoding Plekho-1, was effectively loaded to the hybrid nanocarrier, and was sustainably released over 5 days. The siRNA-loaded nanocarrier exhibited excellent osteoblastic uptake (96% efficiency) and gene-silencing effect, suppressing Plekho-1 down to ∼18%, while preserving intracellular fluorescence signals. Taken as a whole, the self-fluorescent nature of apatite nanorods is believed to find potential and versatile applications as biocompatible drug-delivery and in situ imaging systems.