The use of the gastrointestinal tract as a site for the local delivery of DNA is an exciting prospect. In order to obtain an effective vector capable of delivering a gene of interest to target cells ...to achieve sufficient and sustained transgene expression, with minimal toxicity, we developed a new generation of filamentous bacteriophage. This particular bacteriophage was genetically engineered to display an arginine-glycine-aspartic acid (RGD) motif (an integrin-binding peptide) on the major coat protein pVIII and carry a mammalian DNA cassette. One unanticipated observation is the thermoresponsive behavior of engineered bacteriophage. This finding has led us to simplify the isolation method to purify bacteriophage particles from cell culture supernatant by low-temperature precipitation. Our results showed that, in contrast to non-surface modified, the RGD-modified bacteriophage was successfully used to deliver a transgene to mammalian cells. Our in vitro model of the human intestinal follicle-associated epithelium also demonstrated that bacteriophage particles were stable in simulated gastrointestinal fluids and able to cross the human intestinal barrier. In addition, we confirmed an adjuvant property of the engineered bacteriophage to induce nitric oxide production by macrophages. In conclusion, our study demonstrated the possibility of using bacteriophage for gene transfer in the gastrointestinal tract.
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Biocomposite cellulose-based scaffolds derived from sugarcane bagasse (SCB) were integrated with hydroxyapatite (HA) to enhance the proliferation, attachment and differentiation of preosteoblast ...cells for tissue engineering. Sugar extraction produces a lot of SCB with approximately 40% cellulose content. Cellulose fibers were successfully achieved from SCB using steam explosion and bleaching and then dissolved by 1-butyl-3-methylimidazolium chloride (BmimCl) followed by the addition of HA. Complete homogenous dissolution of cellulose in BmimCl was achieved at 7 h. Scanning electron microscopy analysis confirmed the appropriate porosities of the scaffolds for cellular activities. The prepared scaffolds exhibited a low degradation rate. The cytotoxicity of all scaffolds prepared showed no adverse toxicity to the osteoblast cells, which was higher than 70% in all scaffold types. The MTS tetrazolium assay, live/dead cell staining and alkaline phosphatase (ALP) activity results indicated that cellulose combined HA scaffolds supported bone cell proliferation and differentiation. Cell morphology revealed increasing HA concentration in the scaffolds, with enhanced growth and adhesion of osteoblast cells. Results suggested that regenerated cellulose from SCB is an ideal material in bone scaffolding applications.
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•Cellulose-based scaffolds were successfully fabricated from sugarcane bagasse.•The scaffolds promoted proliferation and differentiation of osteoblasts.•Cell adhesion and morphology displayed attachment of osteoblasts on the scaffolds.•The scaffolds showed promise as candidates for bone tissue engineering.
We investigated cellular uptake behavior and biological responses of spherical and fibrous titanate nanomaterials in human monocyte THP-1 cells. Two titanate nanofibers (TiNFs), namely TF-1 and TF-2, ...were synthesized from anatase TiO
nanoparticles (TNPs) via hydrothermal treatment. The synthesized TiNFs and TNPs were thoroughly characterized for their size, crystallinity, surface area and surface pH. TF-1 (∼2 µm in length) was amorphous with an acidic surface, while TF-2 (∼7 µm in length) was brookite with a basic surface. The results demonstrated that none of these titanate nanomaterials resulted in significant cytotoxicity, even at the highest doses tested (50 µg/ml), consistent with an absence of ROS generation and lack of change of mitochondrial membrane potential. While no cytotoxic effect was found in the titanate nanomaterials, TF-2 tended to decrease the proliferation of THP-1 cells. Furthermore, TF-2 resulted in an inflammatory cytokine response, as evidenced by dramatic induction of IL-8 and TNF-α release in TF2 but not TF-1 nor TNPs. These results suggest that shape of titanate nanomaterials plays an important role in cellular internalization, while surface pH may play a prominent role in inflammatory response in THP-1 cells.
Nanoparticles have been used as antibacterial agents in several products. To optimize their effectiveness, synthesis processes and particle modifications have been developed, creating the need for a ...rapid screening method to investigate their potencies. Owing to the opacity and insolubility of nanoparticles, a classical method to determine antibacterial activity—such as the minimum inhibitory concentration (MIC), which relies on turbidimetry—might not apply to them. In this study, we demonstrate the potential of a dye (resazurin)-based assay as an indicator of bacterial growth to rapidly screen the antibacterial activities of both organic and inorganic nanomaterials against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria. The results indicate that the resazurin-based assay successfully determine the MIC of organic lipid nanocarriers, and several inorganic nanoparticles. However, the use of resazurin require a precaution for nanoparticles with photocatalytic properties, which may cause dye degradation at higher concentrations. In this study, resazurin bleaching was observed at approximately >50 mg/ml of TiO2. In summary, the modified MIC assay with resazurin can evaluate antibacterial activity of nanomaterials, whose turbidity interferer conventional MIC assay. This modification conserves an advantage of MICs assay which are simple and reliable. This would be useful for screening of antibacterial nanomaterials.
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•This study demonstrates an efficiency of resazurin dye to modify MIC tests of Nanomaterials, of which their characteristics generally obscure turbidity of bacterial growth in MIC assay.•The resazurin based MIC assay can be successfully used to determine MICs of organic nanoparticle, and several inorganic nanoparticles.•Photocatalytic nanoparticles degrade resazurin dye with concentration dependent manner. This is accounting to limitation of resazurin application.
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•Papain was immobilized on BC by normal adsorption and cross-linked with glutaraldehyde.•The BC template provided the ability to release papain for at least 24 h.•The incorporated ...particles reduced crystallinity and mechanical property of the BC.•Stronger action of antibacterial properties was detected on BEG sample.
A bioactive wound dressing consists of a bioactive compound coated on a bio-polymer-based material. In the present study, papain was immobilized on bacterial cellulose (BC) to develop a BC film for dressing application. Pure BC and BC cross-linked with glutaraldehyde (BG) were submersed in papain solution at 25 °C and 150 rpm for 24 h. Physicochemical, morphological, loading/release properties and antibacterial activities were determined to analyze the suitability of BC loaded with papain membranes. Scanning electron microscopy/energy-dispersive X-ray spectroscopy and Fourier-transform infrared spectroscopy demonstrated that papain was immobilized successfully on the BC fibrils. The results showed that the presence of glutaraldehyde increased the amount of papain loaded onto the BC, while the releasing property of the BC membranes loaded with papain was active for at least 24 h. The incorporation with papain decreased the crystallinity of the cellulose fiber (from 64% to 55%). This also led to a reduction in the mechanical properties of the wound dressing membranes to half that of the BC. The swelling ratio of BC/papain (BE) and BC/glutaraldehyde/papain (BEG) were at 4546.7 ± 554.9% and 4296.0 ± 119.2%, respectively. The results of agar diffusion and cell growth inhibition assay indicated the antibacterial properties of the wound dressing, with tested strains of Escherichia coli (ATCC 25922), Pseudomonas aeuroginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923) being completely inhibited in the in-vitro test. These findings suggest that the BC dressing produced in this study is a promising material to be applied in the biomedical field.
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Hydroxyapatite (HA), an inorganic compound, plays an essential role in the proliferation and differentiation of bone cells. Using cellulose nanocrystals (CNCs) as green dispersants to ...improve homogenization of HA is promising in the fabrication of nanocomposite scaffolds with biocompatibility for bone tissue engineering. The HA/CNC (HC) nanoparticle suspension was incorporated in polyvinyl alcohol (PVA)-based scaffold to investigate the physical and chemical properties. The PVA/HC composites demonstrated high porous structure and swelling ability for cell attachment and a 3-fold improvement in compressive modulus compared with free HC scaffold. Moreover, the presence of HC nanoparticles has promoted the proliferation and mineralization of pre-osteoblast. Our findings could provide an effective strategy by using bio-dispersants to incorporate mineral elements into synthetic polymers for the fabrication of functional tissue engineering scaffolds.
Tilapia culture is an essential protein-rich food production sector worldwide. The emergence of pathogens is sometimes associated with fatal outbreaks in tilapia, which dramatically slow production ...and result in severe economic losses. While tilapia lake virus (TiLV) infection has been associated with fatal diseases in tilapia, the novel tilapia parvovirus (TiPV) has been recently identified and reported to be associated with mass mortality events in tilapia farming. In this study, we identified the coinfection of a novel TiPV and TiLV in multiple independent tilapia farms in Thailand, thus causing significant losses. The full-length genomes of the TiPV were characterized, and a phylogenetic analysis was performed, indicating the genetic diversity of the TiPV and multiple amino acid mutations found in the structural protein VP1. Using the in situ hybridization (ISH) technique, TiPV was localized in the gills, heart, brain, liver, pancreas, spleen, intestine, kidney, eyes, and muscles of tilapia, with evidence of cellular tropism. The TiLV localization was confirmed using a dual ISH/immunohistochemistry protocol and transmission electron microscopy, which indicated a potential association with pathological alterations. Both TiPV and TiLV were successfully propagated in tilapia brain cells and the fish cell line E-11. This study also provided an indication of the potential cellular roles of the pathological features of TiPV coinfection with TiLV in red hybrid tilapia. Since mutations in the parvovirus structural protein led to virulence and extensive infection in susceptible hosts, several point mutations in the VP1 gene with amino acid signatures of TiPV found in tilapia in Thailand warrant future observations. Although the synergism between TiPV and TiLV coinfection remains undetermined, the contributory role of these two viruses requires intensive focus. Further investigations should lead to a suitable strategy for disease control.
•Massive mortality events of TiPV coinfection with TiLV in multiple tilapia farms in Thailand were reported.•The presence of TiPV was confirmed by PCRs, virus isolation, ISH, and TEM.•Dual identification of TiPV and TiLV was confirmed with the dual ISH/IHC assay, and associated with pathological lesions.•Full-length genome and phylogenetic analysis of TiPV demonstrated genetic diversity and mutations within the VP1 gene.
Red tilapia (Oreochromis sp.), one of the important freshwater fish species in fish farming in Thailand, has for long been suffering from a serious bacterial disease named epizootic ulcerative ...syndrome and hemorrhagic septicemia. The disease is mainly caused by Aeromonas veronii. Vaccine is proposed to be a major impact tool for sustainable control and prevention strategies. Vaccination by immersion has many benefits over injection. However, the conventional immersion method suffers from a low potency due to the inefficient uptake of antigens across mucosal tissue. Here, we developed a chitosan-polymer based nanovaccine together with an efficient delivery vehicle to enhance the immunogenicity of immersion vaccination, increasing bioavailability and inducing local immune responses during transit to mucosal inductive immune sites. The physiochemical properties of nanovaccine, which was modified on surface particle by using a mucoadhesive polymer, were assessed for size, zeta potential, and particle distribution. Our study demonstrated by SEM image and microscopic fluorescence image that nanovaccine greatly increased the binding and penetrating ability into gills when compared with formalin killed vaccine. The nano-sized particles were well dispersed in water and trapped in core nanoparticle as confirmed by TEM image. The efficacy of vaccine was performed by immersion challenge with virulent A.veronii after 30 days post vaccination in tilapia. The result revealed a high level of mortality in the control, empty-polymeric nanovaccine and formalin killed bacterin vaccine groups. A high relative percentage survival (RPS) of vaccinated fish was noted with chitosan-polymer based nanovaccine. Our studies indicated that this chitosan-polymer based nanovaccine derived from cell fragments and supernatant was the improved version of the conventional formalin killed vaccine. The chitosan polymer based particle could increase the efficacy of nanovaccine toward the target mucosal membrane and enhance protection against A. veronii infection in red tilapia.
•As immersion vaccination, chitosan-polymer based nanovaccine have been successfully developed.•Chitosan polymer-mediated cationic bacterin nanovaccine demonstrated effective mucoadhesive properties.•Chitosan-polymer based nanovaccine is proven to be able to immunize red tilapia against Aeromonas veronii.
Limitations of achieving highly sensitive and stable surface-enhanced Raman scattering (SERS) substrate greatly concern the suitable method for fabrication of large-area plasmonic nanostructures. ...Herein we report a simple approach using template-based synthesis to create a highly ordered two-dimensional array of gold-silver alloy nanowires, followed by the controlled dealloying process. This particular step of mild acid etching (15%v/v nitric acid for 5 min) allowed the formation of Raman hot spots on the nanowire tips while maintaining the integrity of highly active alloy composition and rigid nanowire array structure. Full consideration of SERS substrate performance was accomplished using 4-mercaptobenzoic acid (4-MBA) as a probe molecule. Exceedingly higher SERS signal (150-fold) can be achieved with respect to typical gold film substrate. Moreover, an excellent stability of SERS substrate was also determined for over 3 months storage time. In contrast to the previous studies which stability improvement was accomplished at a cost of sensitivity reduction, the simultaneous improvement of sensitivity and stability makes the controlled dealloying process an excellent choice of SERS substrate fabrication. In addition, uniformity and reproducibility studies indicated satisfactory results with the acceptable values of relative standard deviation.
Self-calcifying lipid nanocarrier for bone tissue engineering Chaiin, Poowadon; Yostaworakul, Jakarwan; Rungnim, Chompoonut ...
Biochimica et biophysica acta. General subjects,
February 2022, 2022-02-00, 20220201, Letnik:
1866, Številka:
2
Journal Article
Recenzirano
A nanoemulsion with specific surface properties (such as charge and functional groups) can initiate the deposition of calcium phosphate (CaP) on its surface, leading to formation of CaP nanoparticles ...with a lipid core. The lipid core can carry lipophilic compounds based on the function of the nanoemulsion. Therefore, a dual purpose nanoemulsion of lipid nanoparticles (LNPs) exhibiting self-calcifying and carrier abilities can be developed.
We employed an emulsification process to formulate LNPs with a specific charged surface. The LNPs were tested for their ability to calcify in simulated body fluid and encapsulate cholecalciferol (a model of active compound). The self-calcifying LNP was successfully fabricated using the emulsification process and stabilized using a mixture of polysorbate 80 and polysorbate 20.
The LNPs incubated in simulated body fluid bound to calcium and phosphate, subsequently forming CaP on the particle surface and resulting in approximately 180-nm CaP spheres with a lipid core. The LNPs facilitated calcium phosphate deposition in the collagen scaffolds. In addition, LNPs can be used as carriers of lipophilic compounds without impeding the self-calcifying ability.
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•A dual purpose lipid nanoparticles was successfully fabricated with self-calcifying ability and function as a carrier of lipophilic compounds.•Nano emulsion with a specific surface charge can attach Ca2+ and PO43−.•Lipid NPs show self-calcifying ability in simulated body fluid.•Lipid NPs encapsulate vitamin D3 with high encapsulation efficiency (90.70 ± 0.82%) and a slow release profile.•Lipid NPs adhere to and facilitate deposition of calcium phosphate on collagen scaffolds.
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