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In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young’s modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures. [ABSTRACT FROM AUTHOR]

The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo. [ABSTRACT FROM AUTHOR]

With greater than 500,000 orthopaedic procedures performed in the United States each year requiring a bone graft, the development of novel graft materials is necessary. We report that some porous polymer/ceramic composite scaffolds possess intrinsic osteoinductivity as shown through their capacity to induce in vivo host osteoid mineralization and in vitro stem cell osteogenesis making them attractive synthetic bone graft substitutes. It was discovered that certain low crystallinity ceramics partially dissociate into simple signaling molecules (i.e., calcium and phosphate ions) that induce stem cells to endogenously produce their own osteoinductive proteins. Review of the literature has uncovered a variety of simple signaling molecules (i.e., gases, ions, and redox reagents) capable of inducing other desirable stem cell differentiation through endogenous growth factor production. Inductive simple signaling molecules, which we have termed inducerons, represent a paradigm shift in the field of regenerative engineering where they can be utilized in place of recombinant protein growth factors. [ABSTRACT FROM AUTHOR]

Iron-oxide based contrast agents play an important role in magnetic resonance imaging (MRI) of labelled cells in vivo. Currently, a wide range of such contrast agents is available with sizes varying from several nanometers up to a few micrometers and consisting of single or multiple magnetic cores. Here, we evaluate the effectiveness of these different particles for labelling and imaging stem cells, using a mouse mesenchymal stem cell line to investigate intracellular uptake, retention and processing of nano- and microsized contrast agents. The effect of intracellular confinement on transverse relaxivity was measured by MRI at 7 T and in compliance with the principles of the ‘3Rs’, the suitability of the contrast agents for MR-based cell tracking in vivo was tested using a chick embryo model. We show that for all particles tested, relaxivity was markedly reduced following cellular internalisation, indicating that contrast agent relaxivity in colloidal suspension does not accurately predict performance in MR-based cell tracking studies. Using a bimodal imaging approach comprising fluorescence and MRI, we demonstrate that labelled MSC remain viable following in vivo transplantation and can be tracked effectively using MRI. Importantly, our data suggest that larger particles might confer advantages for longer-term imaging. [ABSTRACT FROM AUTHOR]

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in Km of enzyme in CLEAs and HM-PAL-CLEAs, Vmax of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application. [ABSTRACT FROM AUTHOR]

The primary objective of this study was to fabricate a TiO2 nanotubular surface, which could maintain hydrophilicity over time (resist aging). In order to achieve non-aging hydrophilic surfaces, anodization and annealing conditions were optimized. This is the first study to show that anodization and annealing condition affect the stability of surface hydrophilicity. Our results indicate that maintenance of hydrophilicity of the obtained TiO2 nanotubes was affected by anodization voltage and annealing temperature. Annealing sharply decreased the water contact angle (WCA) of the as-synthesized TiO2 nanotubular surface, which was correlated to improved hydrophilicity. TiO2 nanotubular surfaces are transformed to hydrophilic surfaces after annealing, regardless of annealing and anodization conditions; however, WCA measurements during aging demonstrate that surface hydrophilicity of non-anodized and 20 V anodized samples decreased after only 11 days of aging, while the 60 V anodized samples maintained their hydrophilicity over the same time period. The nanotubes obtained by 60 V anodization followed by 600 °C annealing maintained their hydrophilicity significantly longer than nanotubes which were obtained by 60 V anodization followed by 300 °C annealing. [ABSTRACT FROM AUTHOR]

Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches. [ABSTRACT FROM AUTHOR]

The chemical, mechanical, and topographical features of the extracellular matrix (ECM) have all been documented to influence cell adhesion, gene expression, migration, proliferation, and differentiation. Topography plays a key role in the architecture and functionality of various tissues in vivo, thus raising the possibility that topographic cues can be instructive when incorporated into biomaterials for regenerative applications. In the literature, there are discrepancies regarding the potential roles of nanotopography to enhance the osteogenic phenotype of mesenchymal stem cells (MSC). In this study, we used thin film substrates of poly(methyl methacrylate) (PMMA) with nanoscale gratings to investigate the influence of nanotopography on the osteogenic phenotype of MSCs, focusing in particular on their ability to produce mineral similar to native bone. Topography influenced focal adhesion size and MSC alignment, and enhanced MSC proliferation after 14 days of culture. However, the osteogenic phenotype was minimally influenced by surface topography. Specifically, alkaline phosphatase (ALP) expression was not increased on nanotopographic films, nor was calcium deposition improved after 21 days in culture. Ca: P ratios were similar to native mouse bone on films with gratings of 415 nm width and 200 nm depth (G415) and 303 nm width and 190 nm depth (G303). Notably, all surfaces had Ca∶P ratios significantly lower than G415 films. Collectively, these data suggest that, PMMA films with nanogratings are poor drivers of an osteogenic phenotype. [ABSTRACT FROM AUTHOR]

Purpose: To provide additional functional information for tumor characterization, we investigated the use of dual-energy computed tomography for imaging murine lung tumors. Tumor blood volume and vascular permeability were quantified using gold and iodine nanoparticles. This approach was compared with a single contrast agent/single-energy CT method. Ex vivo validation studies were performed to demonstrate the accuracy of in vivo contrast agent quantification by CT. Methods: Primary lung tumors were generated in LSL-KrasG12D; p53FL/FL mice. Gold nanoparticles were injected, followed by iodine nanoparticles two days later. The gold accumulated in tumors, while the iodine provided intravascular contrast. Three dual-energy CT scans were performed–two for the single contrast agent method and one for the dual contrast agent method. Gold and iodine concentrations in each scan were calculated using a dual-energy decomposition. For each method, the tumor fractional blood volume was calculated based on iodine concentration, and tumor vascular permeability was estimated based on accumulated gold concentration. For validation, the CT-derived measurements were compared with histology and inductively-coupled plasma optical emission spectroscopy measurements of gold concentrations in tissues. Results: Dual-energy CT enabled in vivo separation of gold and iodine contrast agents and showed uptake of gold nanoparticles in the spleen, liver, and tumors. The tumor fractional blood volume measurements determined from the two imaging methods were in agreement, and a high correlation (R2 = 0.81) was found between measured fractional blood volume and histology-derived microvascular density. Vascular permeability measurements obtained from the two imaging methods agreed well with ex vivo measurements. Conclusions: Dual-energy CT using two types of nanoparticles is equivalent to the single nanoparticle method, but allows for measurement of fractional blood volume and permeability with a single scan. As confirmed by ex vivo methods, CT-derived nanoparticle concentrations are accurate. This method could play an important role in lung tumor characterization by CT. [ABSTRACT FROM AUTHOR]

Ultrasound-triggered phase transition sensitive nanodroplets with multimodal imaging functionality were prepared via premix Shirasu porous glass (SPG) membrane emulsification method. The nanodroplets with fluorescence dye DiR and SPIO nanoparticles (DiR-SPIO-NDs) had a polymer shell and a liquid perfluoropentane (PFP) core. The as-formed DiR-SPIO-NDs have a uniform size of 385±5.0 nm with PDI of 0.169±0.011. The TEM and microscopy imaging showed that the DiR-SPIO-NDs existed as core-shell spheres, and DiR and SPIO nanoparticles dispersed in the shell or core. The MTT and hemolysis studies demonstrated that the nanodroplets were biocompatible and safe. Moreover, the proposed nanodroplets exhibited significant ultrasound-triggered phase transition property under clinical diagnostic ultrasound irradiation due to the vaporization of PFP inside. Meanwhile, the high stability and R2 relaxivity of the DiR-SPIO-NDs suggested its applicability in MRI. The in vivo T2-weighted images of MRI and fluorescence images both showed that the image contrast in liver and spleen of rats and mice model were enhanced after the intravenous injection of DiR-SPIO-NDs. Furthermore, the ultrasound imaging (US) in mice tumor as well as MRI and fluorescence imaging in liver of rats and mice showed that the DiR-SPIO-NDs had long-lasting contrast ability in vivo. These in vitro and in vivo findings suggested that DiR-SPIO-NDs could potentially be a great MRI/US/fluorescence multimodal imaging contrast agent in the diagnosis of liver tissue diseases. [ABSTRACT FROM AUTHOR]

A novel supersaturated self-emulsifying drug delivery system (Super-SEDDS) loaded with scutellarin-phospholipid complex (SPC) was developed. The system aimed to address the limitations presented by conventional SEDDS as delivery carriers for drugs with poor water-solubility, low liposolubility and high dose. As an intermediate, SPC was first prepared based on the response surface design. The presence of amorphous scutellarin was demonstrated through differential scanning calorimetry (DSC) and X-ray diffraction (XRD), while enhanced liposolubility was confirmed through comparison with scutellarin powder via an octanol/water distribution test. On the basis of the solubility study and ternary phase diagram, Super-SEDDS containing SPC of up to 200% equilibrium solubility (Seq) was designed, which composed of ethyl oleate, Cremophor RH40 and Transcutol HP with a ratio of 60∶25∶15 (w/w%). The subsequent in vitro lipolysis study and ex vivo intestinal absorption test indicated that Super-SEDDS enhanced the cumulative dissolution from 70% to 100% and improved the intestinal absorption from 0.04 to 0.12 µg/cm2 compared with scutellarin powder. Furthermore, an in vivo study demonstrated that Super-SEDDS achieved the AUC0-t of scutellarin up to approximate 1.7-fold as scutellarin powder. It was also proved superior to SPC and the conventional SEDDS. Super-SEDDS showed great potential for expanding the usage of SEDDS and could act as an alternative to conventional SEDDS. [ABSTRACT FROM AUTHOR]

The design of optimized nanoparticles offers a promising strategy to enable DNA vaccines to cross various physiological barriers for eliciting a specific and protective mucosal immunity via intranasal administration. Here, we reported a new designed nanoparticle system through incorporating anionic liposomes (AL) into chitosan/DNA (CS/DNA) complexes. With enhanced cellular uptake, the constructed AL/CS/DNA nanoparticles can deliver the anti-caries DNA vaccine pGJA-P/VAX into nasal mucosa. TEM results showed the AL/CS/DNA had a spherical structure. High DNA loading ability and effective DNA protection against nuclease were proved by gel electrophoresis. The surface charge of the AL/CS/DNA depended strongly on pH environment, enabling the intracellular release of loaded DNA via a pH-mediated manner. In comparison to the traditional CS/DNA system, our new design rendered a higher transfection efficiency and longer residence time of the AL/CS/DNA at nasal mucosal surface. These outstanding features enable the AL/CS/DNA to induce a significantly (p<0.01) higher level of secretory IgA (SIgA) than the CS/DNA in animal study, and a longer-term mucosal immunity. On the other hand, the AL/CS/DNA exhibited minimal cytotoxicity. These results suggest that the developed nanoparticles offer a potential platform for DNA vaccine packaging and delivery for more efficient elicitation of mucosal immunity. [ABSTRACT FROM AUTHOR]

Silver and gold nanoparticles (of average size ∼20–27 nm) were incorporated in PU (Polyurethane), PCLm (Polycaprolactam), PC (polycarbonate) and PMMA (Polymethylmethaacrylate) by swelling and casting methods under ambient conditions. In the latter method the nanoparticle would be present not only on the surface, but also inside the polymer. These nanoparticles were prepared initially by using a cosolvent, THF. PU and PCLm were dissolved and swollen with THF. PC and PMMA were dissolved in CHCl3 and here the cosolvent, THF, acted as an intermediate between water and CHCl3. FTIR indicated that the interaction between the polymer and the nanoparticle was through the functional group in the polymer. The formation of E.coli biofilm on these nanocomposites under low (in a Drip flow biofilm reactor) and high shear (in a Shaker) conditions indicated that the biofilm growth was higher (twice) in the former than in the latter (ratio of shear force = 15). A positive correlation between the contact angle (of the virgin surface) and the number of colonies, carbohydrate and protein attached on it were observed. Ag nanocomposites exhibited better antibiofilm properties than Au. Bacterial attachment was highest on PC and least on PU nanocomposite. Casting method appeared to be better than swelling method in reducing the attachment (by a factor of 2). Composites reduced growth of organisms by six orders of magnitude, and protein and carbohydrate by 2–5 times. This study indicates that these nanocomposites may be suitable for implant applications. [ABSTRACT FROM AUTHOR]

The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs), enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissue autofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellular and deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNP luminescence on the excitation intensity results in dramatic signal reduction at (∼1 cm) depth in biological tissue. Here, we report on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal application niches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on single UCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified the optical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scattering and absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable at skin depths up to 400 µm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imaging in the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement. [ABSTRACT FROM AUTHOR]

While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates. [ABSTRACT FROM AUTHOR]

Objectives: Gold nanoparticles (AuNPs) of 21 nm have been previously well characterized in vitro for their capacity to target macrophages via active uptake. However, the short-term impact of such AuNPs on physiological systems, in particular resident macrophages located in fat tissue in vivo, is largely unknown. This project investigated the distribution, organ toxicity and changes in inflammatory cytokines within the adipose tissue after mice were exposed to AuNPs. Methods: Male C57BL/6 mice were injected intraperitoneally (IP) with a single dose of AuNPs (7.85 μg AuNPs/g). Body weight and energy intake were recorded daily. Tissues were collected at 1 h, 24 h and 72 h post-injection to test for organ toxicity. AuNP distribution was examined using electron microscopy. Proinflammatory cytokine expression and macrophage number within the abdominal fat pad were determined using real-time PCR. Results: At 72 hours post AuNP injection, daily energy intake and body weight were found to be similar between Control and AuNP treated mice. However, fat mass was significantly smaller in AuNP-treated mice. Following IP injection, AuNPs rapidly accumulated within the abdominal fat tissue and some were seen in the liver. A reduction in TNFα and IL-6 mRNA levels in the fat were observed from 1 h to 72 h post AuNP injection, with no observable changes in macrophage number. There was no detectable toxicity to vital organs (liver and kidney). Conclusion: Our 21 nm spherical AuNPs caused no measurable organ or cell toxicity in mice, but were correlated with significant fat loss and inhibition of inflammatory effects. With the growing incidence of obesity and obesity-related diseases, our findings offer a new avenue for the potential development of gold nanoparticles as a therapeutic agent in the treatment of such disorders. [ABSTRACT FROM AUTHOR]

Vascular targeting of malignant tissues with systemically injected nanoparticles (NPs) holds promise in molecular imaging and anti-angiogenic therapies. Here, a computational model is presented to predict the development of tumor neovasculature over time and the specific, vascular accumulation of blood-borne NPs. A multidimensional tumor-growth model is integrated with a mesoscale formulation for the NP adhesion to blood vessel walls. The fraction of injected NPs depositing within the diseased vasculature and their spatial distribution is computed as a function of tumor stage, from 0 to day 24 post-tumor inception. As the malignant mass grows in size, average blood flow and shear rates increase within the tumor neovasculature, reaching values comparable with those measured in healthy, pre-existing vessels already at 10 days. The NP vascular affinity, interpreted as the likelihood for a blood-borne NP to firmly adhere to the vessel walls, is a fundamental parameter in this analysis and depends on NP size and ligand density, and vascular receptor expression. For high vascular affinities, NPs tend to accumulate mostly at the inlet tumor vessels leaving the inner and outer vasculature depleted of NPs. For low vascular affinities, NPs distribute quite uniformly intra-tumorally but exhibit low accumulation doses. It is shown that an optimal vascular affinity can be identified providing the proper balance between accumulation dose and uniform spatial distribution of the NPs. This balance depends on the stage of tumor development (vascularity and endothelial receptor expression) and the NP properties (size, ligand density and ligand-receptor molecular affinity). Also, it is demonstrated that for insufficiently developed vascular networks, NPs are transported preferentially through the healthy, pre-existing vessels, thus bypassing the tumor mass. The computational tool described here can effectively select an optimal NP formulation presenting high accumulation doses and uniform spatial intra-tumor distributions as a function of the development stage of the malignancy. [ABSTRACT FROM AUTHOR]

We present an electrode, based on structurally controlled nanowires, as a first step towards developing a useful nanostructured device for neurophysiological measurements in vivo. The sensing part of the electrode is made of a metal film deposited on top of an array of epitaxially grown gallium phosphide nanowires. We achieved the first functional testing of the nanowire-based electrode by performing acute in vivo recordings in the rat cerebral cortex and withstanding multiple brain implantations. Due to the controllable geometry of the nanowires, this type of electrode can be used as a model system for further analysis of the functional properties of nanostructured neuronal interfaces in vivo. [ABSTRACT FROM AUTHOR]