Alginate- and Chitosan-Modified Gelatin Hydrogel Microbeads for Delivery of E. coli Phages.

Bacterial infections are among the most significant health problems/concerns worldwide. A very critical concern is the rapidly increasing number of antibiotic-resistant bacteria, which requires much more effective countermeasures. As nature's antibacterial entities, bacteriophages shortly ("phages") are very important alternatives to antibiotics, having many superior features compared with antibiotics. The development of phage-carrying controlled-release formulations is still challenging due to the need to protect their activities in preparation, storage, and use, as well as the need to create more user-friendly forms by considering their application area/site/conditions. Here, we prepared gelatin hydrogel microbeads by a two-step process. Sodium alginate was included for modification within the initial recipes, and these composite microbeads were further coated with chitosan. Their swelling ratio, average diameters, and Zeta potentials were determined, and degradations in HCl were demonstrated. The target bacteria Escherichia coli (E.coli) and its specific phage (T4) were obtained from bacterial culture collections and propagated. Phages were loaded within the microbeads with a simple method. The phage release characteristics were investigated comparatively and were demonstrated here. High release rates were observed from the gelatin microbeads. It was possible to reduce the phage release rate using sodium alginate in the recipe and chitosan coating. Using these gelatin-based microbeads as phage carrier matrices-especially in lyophilized forms-significantly improved the phage stability even at room temperature. It was concluded that phage release from gelatin hydrogel microbeads could be further controlled by alginate and chitosan modifications and that user-friendly lyophilized phage formulations with a much longer shelf life could be produced.

Following hybridization on sensor/array platforms by using SPR, elipsometer and MALDI-MS.

The aim of this study is to develop a methodology in which Surface Plasmon Resonance (SPR), Ellipsometer (EM) and Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS) will be used together for detection of single-strand oligodeoxynucleotides (ssODNs) targets. A selected target-ssODNs, and its complementary, the probe-ssODNs carrying a -SH end group, a spacer arm (HS-(CH2)6-(T)15, and a non-complementary ssODNs were used. Silicone based stamps with 16 regions were prepared and used for micro-contact printing (µCP) of the probe-ssODNs on the gold coated surfaces homogeneously. A modulator-spacer molecule (6-mercapto-1-hexanol) was co-immobilized to control surface probe density, to orientate the probe-ssODNs, and to eliminate the nonspecific interactions. SPR was used successfully to follow the hybridization of the target-ssODNs with the immobilized probe-ssODNs on the platform surfaces. Complete hybridizations were achieved in 100 min. It was obtained that there was a linear relationship between relative change in delta and target concentration below 1 µm. Using imaging version of ellipsometer (IEM) allowed imaging of the surfaces and supported extra datum for the SPR results. After a very simple dehybridization protocol, MALDI-MS analysis allowed detection of the target-ssODNs hybridized on the sensor/array platforms.

The Pimpled Gold Nanosphere: A Superior Candidate for Plasmonic Photothermal Therapy.

BACKGROUND: The development of highly efficient nanoparticles to convert light to heat for anti-cancer applications is quite a challenging field of research. METHODS: In this study, we synthesized unique pimpled gold nanospheres (PGNSs) for plasmonic photothermal therapy (PPTT). The light-to-heat conversion capability of PGNSs and PPTT damage at the cellular level were investigated using a tissue phantom model. The ability of PGNSs to induce robust cellular damage was studied during cytotoxicity tests on colorectal adenocarcinoma (DLD-1) and fibroblast cell lines. Further, a numerical model of plasmonic (COMSOL Multiphysics) properties was used with the PPTT experimental assays. RESULTS: A low cytotoxic effect of thiolated polyethylene glycol (SH-PEG400-SH-) was observed which improved the biocompatibility of PGNSs to maintain 89.4% cell viability during cytometry assays (in terms of fibroblast cells for 24 hrs at a concentration of 300 µg/mL). The heat generated from the nanoparticle-mediated phantom models resulted in ΔT=30°C, ΔT=23.1°C and ΔT=21°C for the PGNSs, AuNRs, and AuNPs, respectively (at a 300 µg/mL concentration and for 325 sec). For the in vitro assays of PPTT on cancer cells, the PGNS group induced a 68.78% lethality (apoptosis) on DLD-1 cells. Fluorescence microscopy results showed the destruction of cell membranes and nuclei for the PPTT group. Experiments further revealed a penetration depth of sufficient PPTT damage in a physical tumor model after hematoxylin and eosin (H&E) staining through pathological studies (at depths of 2, 3 and 4 cm). Severe structural damages were observed in the tissue model through an 808-nm laser exposed to the PGNSs. CONCLUSION: Collectively, such results show much promise for the use of the present PGNSs and photothermal therapy for numerous anti-cancer applications.

In vivo imaging/detection of MRSA bacterial infections in mice using fluorescence labelled polymeric nanoparticles carrying vancomycin as the targeting agent.

This study aims to develop fluorescence labelled polymeric nanoparticle (NP) carrying vancomycin as the targeting agent for in vivo imaging of Methicillin-resistant Staphylococcus aureus bacterial infections in animal models. Maleimide functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] as the main was carrier matrix to prepare the NPs. A fluorescence probe, namely, poly[9,9'-bis (6″-N,N,N-trimethylammonium) hexyl) fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide] was encapsulated within these NPs by ultrasonication successfully. UV-Vis spectro- photometry of the NPs showed the characteristic shifting on the peak of conjugated polymers indicating successful packaging of this compound with lipid bilayers in nanoscales. Zeta-sizer and TEM analysis showed that the prepared NPs have a diameter of 80-100 nm in a narrow size distribution. Thiolated vancomycin was synthesized and attached to the NPs as the targeting agent. FTIR and MALDI-TOF spectroscopy analysis confirmed the immobilization. The specific targeting properties of the vancomycin conjugated NPs to the target bacteria were first confirmed in in vitro bacterial cultures in which Escherichia coli was the non-target bacteria - using confocal microscopy and TEM. Imaging of bacterial infections in vivo was investigated in mice model using a non-invasive live animal fluorescence imaging technique. The results confirmed that bacterial infections can be detected using these novel polymeric NPs carrying fluorescence probes for imaging and vancomycin as the targeting agent - in vivo successfully.

Stem cells combined 3D electrospun nanofibrous and macrochannelled matrices: a preliminary approach in repair of rat cranial bones.

Repair of cranial bone defects is an important problem in the clinical area. The use of scaffolds combined with stem cells has become a focus in the reconstruction of critical-sized bone defects. Electrospinning became a very attracting method in the preparation of tissue engineering scaffolds in the last decade, due to the unique nanofibrous structure of the electrospun matrices. However, they have a limitation for three dimensional (3D) applications, due to their two-dimensional structure and pore size which is smaller than a cellular diameter which cannot allow cell migration within the structure. In this study, electrospun poly(ε-caprolactone) (PCL) membranes were spirally wounded to prepare 3D matrices composed of nanofibers and macrochannels. Mesenchymal stromal/stem cells were injected inside the scaffolds after the constructs were implanted in the cranial bone defects in rats. New bone formation, vascularisation and intramembranous ossification of the critical size calvarial defect were accelerated by using mesenchymal stem cells combined 3D spiral-wounded electrospun matrices.

Critical-size alveolar defect treatment via TGF-ß3 and BMP-2 releasing hybrid constructs.

In the present study a combination of Transforming Growth Factor Beta 3 (TGF-ß3) and Bone Morphogenetic Protein-2 (BMP-2) loaded gelatin films sandwiched between poly (L-lactide) (PLLA)/poly (ε-caprolactone) (PCL) matrices were produced to enhance bone formation in alveolar bone defects. Osteogenic properties of tissue constructs were tested in alveolar bone defect model in rats. Bone healing was assessed by osteogenic gene expression levels of bone sialoprotein (BSP), alkaline phosphatase (ALP), osteonectin (ON, SPARC), osteocalcin (OC), runt-related transcription factor 2 (RUNX2), bone specific alkaline phosphatase (BALP) activity, histomorphometry and microtomography. Increase in osteogenic gene expression levels and BALP activity results showed that new bone formation was significantly accelerated in TGF-ß3 + BMP-2 loaded scaffold group compared to growth factor free and only BMP-2 loaded groups. The micro-computed tomography (µ-CT) data from the 4th months revealed that (TGF-ß3+ BMP-2) loaded scaffolds displayed increased bone formation and was able to fulfill 84% of the defect area (p < 0.05). Accelerated bone formation in the S-GF-B-T group compared to that of the S-GF group at the end of the 4th month was further verified via histomorphometric analysis (p = 0.008). Gene expression, BALP activity, microtomography and histomorphometry analysis indicated that (TGF-ß3 + BMP-2) loaded PLLA/PCL scaffolds increased the new bone formation. BMP-2 loaded scaffolds were less effective than combination of TGF-ß3 and BMP-2 loaded scaffolds. These findings demonstrated that focusing on the PLLA/PCL hybrid scaffolds combined with (TGF-ß3 + BMP-2) may lay the groundwork for future therapy-oriented efforts to enhance bone formation in alveolar defects.

Bacterial detection using bacteriophages and gold nanorods by following time-dependent changes in Raman spectral signals.

This study attemps to develop bacterial detection strategies using bacteriophages and gold nanorods (GNRs) by Raman spectral analysis. Escherichia coli was selected as the target and its specific phage was used as the bioprobe. Target bacteria and phages were propagated/purified by traditional techniques. GNRs were synthesized by using hexadecyltrimethyl ammonium bromide (CTAB) as stabilizer. A two-step detection strategy was applied: Firstly, the target bacteria were interacted with GNRs in suspensions, and then they were dropped onto silica substrates for detection. It was possible to obtain clear surface-enchanced Raman spectroscopy (SERS) peaks of the target bacteria, even without using phages. In the second step, the phage nanoemulsions were droped onto the bacterial-GNRs complexes on those surfaces and time-dependent changes in the Raman spectra were monitored at different time intervals upto 40 min. These results demonstrated that how one can apply phages with plasmonic nanoparticles for detection of pathogenic bacteria very effectively in a quite simple test.

Phages in modified alginate beads.

In order to increase stabilities and controlled/sustained released of T4 phages were encapsulated within alginate beads which were then coated with chitosan, polyethylene imine (PEI). Quite high loading capacities (over 90%) were achieved in these pH-sensitive microbeads. Coating with those polycations increased significantly stability both in "simulated gastric fluid" and bile salts especially in the case of PEI coating. The tests conducted in "simulated intestinal fluid" demonstrated that phages were released from the beads which were active at basic pH in which the release rates were smaller in case of chitosan. PEI concluded to be a better coating then chitosan.

Cranial bone regeneration via BMP-2 encoding mesenchymal stem cells.

Cranial bone repair and regeneration via tissue engineering principles has attracted a great deal of interest from researchers during last decade. Here, within this study, 6 mm critical-sized bone defect regeneration via genetically modified mesenchymal stem cells (MSC) were monitored up to 4 months. Cranial bone repair and new bone formations were evaluated by histological staining and real time PCR analysis in five different groups including autograft and bone morphogenetic protein-2 (BMP-2) transfected MSC groups. Results presented here indicate a proper cranial regeneration in autograft groups and a prospering regeneration for hBMP-2 encoding mesenchymal stem cells.

Evaluation of the effectiveness of biodegradable electrospun caprolactoneand poly(lactic acid-ε-caprolactone) nerve conduits for peripheral nerveregenerations in a rat sciatic nerve defect model.

BACKGROUND/AIM: The aim of this study was to compare electrospun caprolactone (EC) and poly(lactic acid-ε-caprolactone) (PLCL) nerve conduits with nerve graft in a rat sciatic nerve defect model. MATERIALS AND METHODS: A total of 32 male Wistar albino rats were divided into 4 groups, with 8 rats in each group. A nerve defect of 1 cm was constructed in the left sciatic nerve of the rats. These defects were left denuded in the sham group, and reconstructed with nerve grafts, PLCL, and EC nerve conduits in the other groups. After 3 months, nerve regenerations were evaluated macroscopically, microscopically, and electrophysiologically. The numbers of myelinated axons in the cross-sections of the nerves were compared between the groups. RESULTS: Macroscopically, all nerve coaptations were intact and biodegradation was detected in nerve conduits. Electromyographic assessment and count of myelinated axons in the cross-sections of the nerves displayed the best regeneration in the nerve graft group (P < 0.001) and similar results were obtained in the PLCL and EC nerve conduit groups (P = 0.79). Light and electron microscopy studies demonstrated nerve regeneration in both nerve conduit groups. CONCLUSION: EC nerve conduits and PLCL nerve conduits yielded similar results and may be alternatives to nerve grafts as they biodegrade.

Separation by nanoparticles plasmonic resonance with low stress in microfluidics channel (analytical and design).

In this study, nanoparticles near-field plasmonic resonance is used to improve the traditional cell separation main outputs such as viability and efficiency. The live cells viability is severely depend on stresses, which are applied on cells in the microfluidics channel. Hence, for improving the cell viability, the enforced stresses inside of the structure should be declined. The major factors of the enforced stresses are related to the electric field non-uniformity, which are attributed to the hurdles and applied voltage magnitude. Therefore, in this study, a new structure is presented and thereby, the magnitude of the applied stresses on live cells is minimised which is contributed to the decreasing the non-uniformity strength of channel. It should be noted that in the new structure two arrays of nanoparticles were used to produce a short range and localised non-uniform electrical field because of their near-field plasmonic resonance. Hence, the enforced stress on the live cell severely decreased at the far-field and confined at the small section of the channel. It is due to, the near-field plasmonic amplitude is dramatically disappeared by increasing distance, hence, the cells far from the nanoparticles will be endured the low level but effective amount of the optical force.

Ti implants with nanostructured and HA-coated surfaces for improved osseointegration.

This study was aimed at comparing the osseointegration of titanium (Ti)-based Küntscher nails (K-nails) and plates with modified nanostructured and hydroxyapatite-coated surfaces in a rat femur model. Material surfaces were first modified via a simple anodization protocol in which the materials were treated in hydrogen fluoride (1% w/w) at 20 V. This modification resulted in tubular titanium oxide nanostructures of 40-65 nm in diameter. Then, hydroxyapatite-deposited layers, formed of particles (1-5) µm, were produced via incubation in a simulated body fluid, followed by annealing at 500°C. Both surface modifications significantly improved cell proliferation and alkaline phosphatase (ALP) activity as compared to the control (non-modified Ti implants). The controls and modified nails and plates were implanted in the femur of 21 male Sprague-Dawley rats. The implants, with surrounding tissues, were removed after 10 weeks, and then mechanical tests (torque and pull-out) were performed, which showed that the modified K-nails exhibited significantly better osseointegration than the controls. Histologic examinations of the explants containing plates showed similar results, and the modified plates exhibited significantly better osseointegration than the controls. Surface nanostructuring of commercially available titanium-based implants by a very simple method - anodization - seems to be a viable method for increasing osseointegration without the use of bioactive surface coatings such as hydroxyapatite.

Fabrication and characterization of novel starch-grafted poly l-lactic acid/montmorillonite organoclay nanocomposites.

In this work, poly(L-lactic acid)-g-starch layered silicate nanocomposites (NCs) (PLLA-g-starch/MMT) were fabricated by intercalative bulk graft copolymerization of LA with starch, in the presence of either stannous octoate acting as a catalyst or LA-MMT organoclay acting as a cocatalyst-nanofiller. This procedure was performed inside a custom vacuum micro-reactor. To better understand the graft copolymerization mechanism, in situ processing types, interfacial interactions and nanostructure formation of PLLA-g-starch/MMT NCs, methods such as FT-IR, XRD, (1)H NMR, (13)C CP/MAS-NMR, DSC/TGA, TEM and SEM were utilized. The morphology and thermal behaviors of nanocomposites were found to be strongly dependent on the loading mass fraction of LA-MMT within the nanocomposite structure and the type of in situ processing such as interfacial, physical and chemical interactions. Preintercalated LA-MMT organoclay exhibited dual functions. It demonstrated the ability to act as a catalyst, essentially accelerating in situ graft copolymerization via esterification of LA carboxyl groups with hydroxyl groups of starch macromolecules, whilst also acting as a nananofiller-compatibilizer.

Silver or gold deposition onto magnetite nanoparticles by using plant extracts as reducing and stabilizing agents.

In this paper, we describe an environmentally friendly procedure to produce silver (Ag) or gold (Au)-deposited magnetite nanoparticles by using plant extracts (Ligustrum vulgare) as reducing and stabilizing agents. Firstly, magnetite nanoparticles (∼6 nm) with superparamagnetic properties - SPIONs - were synthesized by co-precipitation of Fe(+ 2) and Fe(+ 3) ions. Color changes indicated the differing amounts of Au and Ag ions reduced and deposited on to the SPIONs when the plant extracts were used. UV-vis and transmission electron microscope (TEM) with energy dispersive X-ray (EDX) apparatus confirmed the metallic deposition. Magnetic saturation decreased when the amount of the metallic deposition increased, which was measured by vibrating sample magnetometry (VSM). Due to the molecules coming into contact with - and even remaining on - the surface of the nanoparticles after aggressive washing procedures, the Ag/Au-deposited SPIONs were stable, and almost no agglomeration was observed for months. Fourier Transform Infrared (FTIR) spectra depicted that functional groups such as carboxylic and ketone groups, which are most probably responsible for the reduction and stabilization of Ag/Au- carrying magnetite nanoparticles, originated from the plant extract. The proposed route was facile, viable, and reproducible, and it should be stressed that nanoparticles do contain only safe biomolecules as stabilizing agents on their surfaces.

Anti-biofilm effect of nanometer scale silver (NmSAg) coatings on glass and polystyrene surfaces against P. mirabilis, C. glabrata and C. tropicalis strains.

PURPOSE: Nowadays, in order to terminate biofilm associated infections, coating of particular biomaterial surfaces with particular substances, via some nanotechnological tools, is being applied. Therefore, in the present study, investigation of anti-biofilm effects of nanometer scale silver (NmSAg) coatings on glass and polystyrene surfaces against clinical strains of Proteus mirabilis, Candida glabrata and Candida tropicalis was aimed. METHODS: In this study, glass and polystyrene slabs with 1.5 cm × 1.5 cm × 0.3 mm dimensions were cleaned by using surface plasma technology, covered with NmSAg by using a physical vapor deposition machine, and biofilm inhibition was determined by crystal violet binding assay. RESULTS: According to our results, 32 nm of silver layer on a glass slab decreased biofilm formation of P. mirabilis strain to a maximum amount of 88.1% and caused 20.9% inhibition in biofilm formation of C. glabrata strain. On the other hand, NmS coating of Ag on a polystyrene slab caused 34.4% and 20% inhibitions, respectively, in biofilm formations of C. glabrata and C. tropicalis strains. Although biofilm inhibition of NmSAg layer on polystyrene slab was more (34.4%) than biofilm inhibition caused by NmSAg layer on glass slab (20.9%), C. glabrata strain's biofilm formation on uncoated glass slab was lower than both uncoated and NmSAg-coated polystyrene slabs. CONCLUSIONS: Our results show that glass surfaces with NmSAg coatings can be used as a new surface material of various indwelling devices on which P. mirabilis colonizations frequently occur and in order to avoid C. glabrata-associated biofilm infections, it is more useful to choose a surface material of glass rather than choosing a surface material of polystyrene.

Thermoresponsive biodegradable HEMA-lactate-Dextran-co-NIPA cryogels for controlled release of simvastatin.

Abstract NIPA and HEMA-lactate-Dextran-based biodegradable and thermoresponsive cryogels were synthesized at different compositions by cryogelation. Chemical and morphological properties of the HEMA-lactate-Dextran-co-NIPA cryogel matrices were demonstrated by FTIR, SEM, and ESEM. Thermoresponsivity of the prepared cryogels was investigated by DSC, imaging NMR, and swelling studies. For possible use of the cryogels in potential bone tissue engineering applications, either hydrophobic simvastatin was embedded, or hydrophilic simvastatin was incorporated in the cryogels. Release profiles of simvastatin delivering cryogel scaffolds depending on their composition, hydrophobicity or hydrophilicity of loaded simvastatin and the medium temperature were demonstrated.

Controlled graft copolymerization of lactic acid onto starch in a supercritical carbon dioxide medium.

This work presents a new approach for the synthesis of a starch-g-poly L-lactic acid (St-g-PLA) copolymer via the graft copolymerization of LA onto starch using stannous 2-ethyl hexanoate (Sn(Oct)2) as a catalyst in a supercritical carbon dioxide (scCO2) medium. The effects of several process parameters, including the pressure, temperature, scCO2 flow rate and reaction time, on the polymerization yield and grafting degree were studied. Amorphous graft St-g-PLA copolymers with increased thermal stability and processability were produced with a high efficiency. The maximum grafting degree (i.e., 52% PLA) was achieved with the following reaction conditions: 6h, 100°C, 200 bar and a 1:3 (w/w) ratio of St/LA. It was concluded that these low cost biobased graft biopolymers are potential candidates for several environment-friendly applications.

Stem cell suspension injected HEMA-lactate-dextran cryogels for regeneration of critical sized bone defects.

HEMA-Lactate-Dextran cryogel scaffolds were produced by cryogelation. Mesencyhmal stem cells (MSC) were isolated from rat bone marrow. Critical sized cranial bone defects were created in rat cranium. Stem cells were injected inside the macropores of the cryogel scaffolds prepared from HEMA-Lactate-Dextran possessing the same dimensions with the defect and placed in the cranial bone. The cryogels placed in the defect without stem cells served as control. After selected time intervals the experimental sites were removed from the animals and new bone formation and tissue integration were investigated by histological analysis. The in vivo results exhibited osseous tissue integration within the implant and mineralized functionally stable bone restoration of the cranial defects. Tissue formation started in the macrospores of the scaffold starting from periphery to the center. A significant ingrowth of connective tissue cells and new blood vessels allowed new bone formation. Histological data demonstrated that new bone per total defect area ratio, were not significantly different in "scaffold-stem cells" group compared to that of "scaffold only" group on all time points. However, the blood vessel density was significantly higher in "scaffold-stem cells" group comparing to that of the "scaffold only" group on day 30. "Scaffold-stem cells" given group gave better tissue response score when compared to "scaffold only" group on day 180.

Nanoemulsions and nonwoven fabrics carrying AgNPs: antibacterial but may be cytotoxic.

Abstract The aim of this study is to prepare nonwoven fabrics carrying silver nanoparticles (AgNPs), and to investigate their antibacterial activities and cytotoxicities in parallel. AgNPs were impregnated from their nanoemulsions onto two commercially available nonwoven fabrics: pure-cotton fabrics (PCF) and polyester/viscous fabrics (PVF), by a simple adsorption (dipping) and were then heat stabilized. PCF exhibited stronger antibacterial effects on both Staphylococcus aureus and Escherichia coli. In-vitro cell culture studies demonstrated that AgNPs nanoemulsions and also fabrics carrying them were cytotoxic on L929-fibroblasts in all concentrations used here (6.25-400 ppm) in different extends. Only the fabrics loaded with AgNPs using nanoemulsion with the lowest concentration of 6.25 ppm exhibited low cytotoxicity but were still antibacterial.

Voltammetric and impedimetric DNA detection at single-use graphite electrodes modified with gold nanorods.

In this study, single-use pencil graphite electrodes (PGEs) were modified by gold nanorods (AuNRs) and then single stranded oligonucleotide probe molecules (ssODNs) were immobilized onto the surface of AuNRs modified PGE. The electrochemical characterizations using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were firstly performed to present the successfull modification of AuNR onto PGE surface. Under the optimum experimental conditions based on enhanced sensor response, single stranded thiol linked DNA probe ssODNs was immobilized onto AuNR modified PGE and accordingly, a sequence-selective DNA hybridization related to Hepatitis B virus (HBV) DNA was performed at the surface of AuNR modified electrodes. The impedimetric sequence-selective HBV DNA hybridization was then explored in the case of complementary (target), or noncomplementary (NC) DNA sequences.