Confocal Scanning Microscopy Research Articles

Microstructural characterization of vacuum-fried matrices and their influence on starch digestion

Abstract During vacuum frying, starch gelatinization may be limited due to the water boiling point depression, which reduces starch digestibility. In addition, relevant structural changes are induced, which could affect the accessibility of amylolytic enzymes to the site of action. To differentiate the effect of structural changes in the food matrix over the effect of starch gelatinization on starch digestibility, we characterized the microstructure using x-ray micro-computed tomography (micro-CT), confocal laser scanning microscopy (CLSM) and environmental scanning electron microscopy (ESEM), and we also studied the starch in vitro digestibility in two different systems: vacuum (9.9 kPa, Twater-boiling-point = 45 °C) and traditional atmospherically-fried dough (170 °C) with a similar degree of starch gelatinization (60 %). Vacuum-fried matrices had a much higher oil content (∼51.3 % dry basis) than their atmospheric counterparts (∼20.3 % dry basis), as confirmed through micro-CT and CLSM. The quantitative analysis of micro-CT images showed that vacuum-fried samples had less air porosity (36.6 % air-filled pores) than atmospheric fried ones (49 % air-filled pores), whereas, no differences were found with respect to total porosity (p

Development of Lipid-Polymer Hybrid Nanoparticles for Improving Oral Absorption of Enoxaparin.

Enoxaparin, an anticoagulant that helps prevent the formation of blood clots, is administered parenterally. Here, we report the development and evaluation of lipid–polymer hybrid nanoparticles (LPHNs) for the oral delivery of enoxaparin. The polymer poloxamer 407 (P407) was incorporated into lipid nanoparticles to form gel cores and ensure high encapsulation efficiency and the controlled release of enoxaparin. In vitro results indicated that 30% of P407 incorporation offered higher encapsulation efficiency and sustained the release of enoxaparin. Laser confocal scanning microscopy (LCSM) images showed that LPHNs could not only significantly improve the accumulation of enoxaparin in intestinal villi but also facilitate enoxaparin transport into the underlayer of intestinal epithelial cells. In vivo pharmacokinetic study results indicated that the oral bioavailability of enoxaparin was markedly increased about 6.8-fold by LPHNs. In addition, its therapeutic efficacy against pulmonary thromboembolism was improved 2.99-fold by LPHNs. Moreover, LPHNs exhibited excellent biocompatibility in the intestine. Overall, the LPHN is a promising delivery carrier to boost the oral absorption of enoxaparin.

Diclofenac exhibits synergism with azoles against planktonic cells and biofilms of Candida tropicalis

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of planktonic cells was evaluated using the broth microdilution assay and checkerboard method. Biofilm formation by C. tropicalis in the presence of diclofenac, alone or in combination with antifungals, was also evaluated, and scanning electron microscope (SEM) and confocal microscope (CLSM) analyses were performed. Diclofenac showed an MIC of 1024 μg ml−1 against planktonic cells. The MICs of fluconazole and voriconazole against azole-resistant isolates were reduced 8- to 32-fold and 16- to 256-fold, respectively, when in combination with diclofenac. When in combination with fluconazole or voriconazole, diclofenac reduced the antifungal concentration necessary to inhibit C. tropicalis biofilm formation. In conclusion, diclofenac presents synergism with fluconazole and voriconazole against resistant C. tropicalis strains and improves the activity of these azole drugs against biofilm formation.

Fabrication of super-hydrophobic and highly oleophobic Ti-6Al-4 V surfaces by a hybrid method

In this study, the super-hydrophobic and highly oleophobic surfaces were fabricated on Ti-6Al-4V substrate by a hybrid method, which combined laser texturing followed by silanization treatment. To fabricate super-hydrophobic and oleophobic surfaces more accurately and effectively, three series of experiments have been carried out to determine the laser parameters for the fabrication of surfaces with the best wettability. Surface wettability was assessed by water contact angle, glycerin contact angle and the n-hexadecane contact angle. To investigate the laser texturing microstructure features and effects thereof on wettability, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and white light confocal scanning microscopy measurements were utilized to characterize the modifications of morphologic, chemical compositions and 3D profiles. The surface with best wettability presented a water contact angle of 153.3°, glycerin contact angle of 149.7°, and n-hexadecane contact angle of 136.6° was successfully achieved by setting the laser parameters to: distance between adjacent laser passes at 60 μm, output power at 12 W and scanning speed at 300 mm/s. The effect of corrosion and abrasion on wettability was also evaluated. Stability tests prove that the super-hydrophobic and oleophobic surfaces possess corrosion/abrasion resistance and good chemical and mechanical stability.

In Situ Intraepithelial Localizations of Opportunistic Pathogens, Porphyromonas gingivalis and Filifactor alocis, in Human Gingiva.

Porphyromonas gingivalis and Filifactor alocis are fastidious oral pathogens and etiological agents associated with chronic periodontitis. Although previous studies showed increased levels of the two obligate anaerobic species in periodontitis patients, methodologies for this knowledge were primarily limited to sampling subgingival plaque, saliva, or gingival crevicular fluid. To evaluate the extent to which P. gingivalis and F. alocis may invade the periodontal tissues, an in situ cross-sectional study was comparatively conducted on the gingival biopsy specimens of patients diagnosed with periodontal health or chronic periodontitis. Immunostained tissue sections for each organism were imaged by Super-Resolution Confocal Scanning Microscopy to determine the relative presence and localization of target bacterial species. Fluorescence-in-situ-hybridization (FISH) coupled with species specific 16S rRNA method was utilized to confirm whether detected bacteria were live within the tissue. In periodontitis, P. gingivalis and F. alocis revealed similarly concentrated localization near the basement membrane or external basal lamina of the gingival epithelium. The presence of both bacteria was significantly increased in periodontitis vs. healthy tissue. However, P. gingivalis was still detected to an extent in health tissue, while only minimal levels of F. alocis were spotted in health. Additionally, the micrographic analyses displayed heightened formation of epithelial microvasculature containing significantly co-localized and metabolically active dual species within periodontitis tissue. Thus, this study demonstrates, for the first-time, spatial arrangements of P. gingivalis and F. alocis in both single and co-localized forms within the complex fabric of human gingiva during health and disease. It also exhibits critical visualizations of co-invaded microvascularized epithelial layer of the tissue by metabolically active P. gingivalis and F. alocis from patients with severe periodontitis. These findings collectively uncover novel visual evidence of a potential starting point for systemic spread of opportunistic bacteria during their chronic colonization in gingival epithelium.

The use of the laser confocal scanning microscopy to measure resin remnants on customized lingual bracket

BackgroundThe study aimed to evaluate the permanence of resin and enamel remains on lingual brackets at the end of orthodontic treatment and after the debonding procedure. The evaluation of resin remnants on customized lingual brackets bases has never done before in other studies because they are curved, and traditional techniques are not applicable.MethodsThe sample consisted of 100 lingual brackets (25 incisors, 25 canines, 25 premolars, 25 molars) scanned with a confocal laser microscope (OLS4000). We measured the brackets' surface and the area of resin remnants with the software of the microscope. Median and quartiles were presented to describe the data. ARI calculation was indirect for each tooth, measuring the resin remnants to the total surface of the bracket. The Kruskal-Wallis test and Fisher test were applied respectively to compare the percentages of remnants and the frequencies of the ARI between the four groups.ResultsAfter the analyses, 13 brackets had no adhesive remnants (ARI 0), 29 brackets had less than 50% of resin remnants (ARI 1), 50 brackets had more than 50% of resin remnants (ARI 2), and 8 brackets had 100% of adhesive (ARI 3). Canines brackets presented the lower amount of resin followed by premolars, incisors, and molars.ConclusionLingual brackets showed a high frequency of ARI = 2. The median percentage of the bracket surface covered by resin was 41%. We observed a slight tendency of more resin remnants on molar brackets, due to half-pad configuration. The authors suggest paying attention during the debonding procedure of molar brackets since a stronger connection between the adhesive and the bracket mesh means a higher risk of enamel damage.

Systematic Investigation of Polyurethane Biomaterial Surface Roughness on Human Immune Responses in vitro.

It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how different topographies contribute to pro-inflammatory or anti-inflammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with defined roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 μm to 18 μm in average roughness (Ra), which was confirmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inflammatory and anti-inflammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no influence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inflammatory immune response. Therefore, we propose that such modifications do not affect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material.

Microbial biofilm community structure and composition on the lithic substrates of Herculaneum Suburban Baths

In this work, we want to investigate the impact of different substrates and different environmental condition on the biofilm communities growing on plaster, marble, and mortar substrates inside the Herculaneum Suburban Baths. To do so, we measured environmental conditions and sampled biofilm communities along the walls of the baths and used culture-dependent and -independent molecular techniques (DGGE) to identify the species at each sampling sites. We used the species pool to infer structure and richness of communities within each site in each substrate, and confocal light scanning microscopy to assess the three-dimensional structure of the sampled biofilms. To gather further insights, we built a meta-community network and used its local realizations to analyze co-occurrence patterns of species. We found that light is a limiting factor in the baths environment, that moving along sites equals moving along an irradiation gradient, and that such gradient shapes the community structure, de facto separating a dark community, rich in Bacteria, Fungi and cyanobacteria, from two dim communities, rich in Chlorophyta. Almost all sites are dominated by photoautotrophs, with Fungi and Bacteria relegated to the role of rare species., and structural properties of biofilms are not consistent within the same substrate. We conclude that the Herculaneum suburban baths are an environment-shaped community, where one dark community (plaster) and one dim community (mortar) provides species to a “midway” community (marble).

New Factors for Protoplast-Callose-Fiber Formation in Salt-Tolerant Mangrove Plants, Avicennia alba and Bruguiera sexangula and Analysis of Fiber Substructures

Elongated and spiral β-1,3-glucan (callose) fibers were obtained by new factors from protoplasts cultured in liquid medium from suspension cultured cells of two salt-tolerant mangrove species; Avicennia alba and Bruguiera sexangula. Differences in salt factor for protoplast-fiber formation were compared with those of the callose fibers developed from protoplasts of non-mangrove tree plants, Larix leptolepis and Betula platyphylla, which high concentrations of divalent cations, Mg2+ (50 mM) or Ca2+ (100 mM), were stimulatory. In the halophilic A. alba protoplasts, whose cell division was stimulated by up to 400 mM NaCl, addition of Mg2+, Ca2+, K+ ions inhibited protoplast-fiber formation. In B. sexangula, protoplast-fibers were rapidly and efficiently formed only by another new factor, electric cell fusion treatment of protoplasts. Spiral fibers developed from mangrove protoplasts were detected under an inverted microscope, and their specific blue-green color for callose after staining with Aniline Blue dye was detected under a fluorescence microscope. Enzymatic certification of callose was further performed with laminarinase, specific for callose, in comparison with cellulase CBH1, specific for cellulose. Differences in sub-structures, fibrils and sub-fibrils of two mangrove protoplast-fibers were analyzed using laser confocal scanning microscopy, atomic force microscopy and image J analysis. Tube-like fine structure was observed using transmission electron microscopy in single protoplast-fiber of B. sexangula selected with a micromanipulator.

The Development of Pemetrexed Diacid-Loaded Gelatin-Cloisite 30B (MMT) Nanocomposite for Improved Oral Efficacy Against Cancer: Characterization, In-Vitro and Ex-Vivo Assessment.

The intention of this investigation was to develop Pemetrexed Diacid (PTX)-loaded gelatine-cloisite 30B (MMT) nanocomposite for the potential oral delivery of PTX and the in vitro, and ex vivo assessment. Gelatin/Cloisite 30 B (MMT) nanocomposites were prepared by blending gelatin with MMT in aqueous solution. PTX was incorporated into the nanocomposite preparation. The nanocomposites were investigated by Fourier Transmission Infra Red Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscope (SEM) X-Ray Diffraction (XRD) and Confocal Laser Microscopy (CLSM). FT-IR of nanocomposite showed the disappearance of all major peaks which corroborated the formation of nanocomposites. The nanocomposites were found to have a particle size of 121.9 ± 1.85 nm and zeta potential -12.1 ± 0.63 mV. DSC thermogram of drug loaded nanocomposites indicated peak at 117.165 oC and 205.816 oC, which clearly revealed that the drug has been incorporated into the nanocomposite because of cross-linking of cloisite 30 B and gelatin in the presence of glutaraldehyde. SEM images of gelatin show a network like structure which disappears in the nanocomposite. The kinetics of the drug release was studied in order to ascertain the type of release mechanism. The drug release from nanocomposites was in a controlled manner, followed by first-order kinetics and the drug release mechanism was found to be of Fickian type. Ex vivo gut permeation studies revealed 4 times enhancement in the permeation of drug present in the nanocomposite as compared to plain drug solution and were further affirmed by CLSM. Thus, gelatin/(MMT) nanocomposite could be promising for the oral delivery of PTX in cancer therapy and future prospects for the industrial pharmacy.

Effect of NH4Cl on the microstructure, wettability and corrosion behavior of electrodeposited Ni–Zn coatings with hierarchical nano/microstructure

In this study, nickel‐zinc (Ni–Zn) superhydrophobic coatings with a hierarchical nano/microstructure were deposited through a two-step electrodeposition process on the copper substrate. The effect of adding NH4Cl crystal modifier concentration on the composition, surface morphology, roughness and wettability of coatings were evaluated by energy dispersive spectroscopy (EDS), X-ray diffraction (XRD), scanning electron microscopy (SEM), laser confocal scanning microscopy (LCSM) and contact angle measurements. Additionally, the corrosion behavior of selected coatings was investigated using potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) techniques. The results showed that adding NH4Cl could affect the composition and surface morphology of the Ni–Zn coatings. A transition from a Zn-rich Ni–Zn coatings with polyhedral shape to the Ni-rich Ni–Zn coatings with hierarchical nano/mico-conical structure was taken place through adding NH4Cl as a crystal modifier. The Ni–Zn coating prepared with 400 g·L−1 NH4Cl showed superhydrophobic behavior with the highest water droplet contact angle of ~155°. The confocal microscopy results confirmed this coating has the highest roughness and power spectral density (PSD) values at entire spatial frequencies. In addition, the superhydrophobic coatings exhibited higher corrosion resistance compared to the other coatings.

Use of 3D Distance Mapping to examine Neuro‐Glia‐Vascular Plasticity in Rodent Models of Cardiovascular Disease

Regulating cerebral blood flow to supply oxygen and nutrients and remove waste products is of utmost importance for neuronal health. The brain’s capillary network responds to acute local increases of neuronal activity with vasodilation of upstream arterioles (neurovascular coupling, NVC). NVC mechanisms are impaired in a number of neurodegenerative and cardiovascular diseases. A compounding factor in these diseases is the remodelling of capillary networks. A reduced density of capillaries and/or changes in the arrangement of capillaries so that the parenchyma is not adequately supplied can exacerbate these conditions. We set out to develop novel tools to analyse the architecture of cerebral microvascular networks and assess neuronal‐glial‐vascular plasticity in rodent models of hypertension, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and impaired endothelial Ca2+ signalling (TRPV4‐KO).Animals (mice or rats) were transcardially perfused with a liposome‐encapsulated fluorescent dye (DiI) and brains were harvested and fixed in 4 % paraformaldehyde. Transparency of the tissue was increased using the SeeDB protocol and the microvasculature was imaged in 2 mm thick coronal brain slabs using 2‐photon microscopy. Subsequently, slabs were cryopreserved, re‐sectioned at 80 μm thickness and processed for immunofluorescence (glial fibrillary astrocytic protein, Gfap). Slices were imaged using scanning confocal microscopy. Imaging volumes were taken in the rostro‐caudal axis to a depth of ~1 mm. They were deconvolved, attenuation‐corrected and scaled to equal length voxels. Then, fluorescent structures were converted into 3D objects (Volumetry G9 ‐ GWH) and non‐vessel particulates were filtered. Then, expanding boundary shells were used at every voxel in parenchymal space. Once the expanding boundary shells collided with a vessel, the distance (radius) was converted to a color and a new imaging volume containing the distance mapping results was created. The proportion of parenchymal voxels located at different distances from their closest blood vessel was calculated.We found that in both mice and rats, overall vessel density was the highest in the paraventricular nucleus of the hypothalamus (10–20 % of voxels), followed by cortex (~ 10 %) and hippocampus (~ 5 %). 90 % of the parenchymal volume in the cortex was within 26 μm of a blood vessel compared to > 34 μm in the hippocampus. Interestingly, Gfap staining was intense in the hippocampus but not in the cortex. In disease models, we found that aged mice (CADASIL, CADASIL control and TRPV4‐KO) had a lower vessel density than younger animals especially in cortex and PVN. In contrast, it was hippocampal vessel density in the dentate gyrus that was lower in spontaneously hypertensive rats than in controls.In summary, we describe a simple, yet powerful method to assess the relationship of capillary networks and parenchymal volume and show that rodent models of cardiovascular diseases are associated with plasticity of the neuro‐glia‐vascular unit.Support or Funding InformationR01 HL133211

Mitochondrial Fragmentation in Skeletal Muscle Derived Cells from an Old Male Donor May Relate to Decreased Oxygen Consumption Rates

With age, skeletal muscle mitochondria lose their ability to fuse and regenerate following stress and exercise leading to fragmentation. These phenomena can lead to reduced oxygen, muscle atrophy, reduced maximal O2 consumption, loss of metabolic flexibility and risk of developing age‐related diseases such as sarcopenia. Skeletal muscle cells derived from human donors can be used to investigate these physiological capacities in primary culture. Our objective is to investigate mitochondrial morphology and cellular maximal oxygen consumption rates (OCR) of skeletal muscle cells derived from healthy young and old male donors. Primary skeletal muscle cells derived from the Rectus abdominis muscle of healthy active eighteen and sixty‐nine year old men (SKM18M and SKM69M, respectively) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Cells were stained with MitoTracker Red (Cell Signaling; Danvers, MA) and mitochondrial morphology was observed using a Zeiss LSM 710 AxioObserver confocal inverted scanning microscope (Carl Zeiss; White Plains, NY). The mitochondrial network was analyzed using the Mitochondrial Network Analysis tool in ImageJ (MiNA, FIJI) to estimate mitochondrial footprint from a binarized image. Oxygen consumption rates were measured in intact cells using Seahorse Cell Mito Stress Tests on a XFp extracellular flux analyzer (Agilent Technologies; Santa Clara, CA). Primary cells derived from the young donor (SKM18M) had a larger mitochondrial footprint, longer branch length, and a greater number of network branches compared to the old donor (SKM69M) (Footprint: 34.65 ± 25.30 vs. 11.64 ± 9.53 μm2; Branch Length: 20.59 ± 7.23 vs. 12.10 ± 6.84 μm; Network: 17.25 ± 0.16 vs. 7.67 ± 4.97 counts). SKM18M also showed higher Basal and Maximal OCR compared to SKM69M (Basal: 38.78 ± 8.34 vs. 12.82 ± 2.07; Maximal: 60.09 ±10.84 vs. 20.52 ± 2.36 pmol/min/protein). We observed differences morphologically and metabolically between the primary skeletal muscle cells derived from young and old donors. These preliminary results give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial fragmentation in vitro will help us elucidate the effects of aging, illness and injury on skeletal muscle mitochondrial fragmentation, dysfunction, and loss of metabolic flexibility.Support or Funding InformationFunded by the National Institute of Aging of the National Institute of Health; Grant Number R01‐AG059715.

Combined quantitative microscopy on the microstructure and phase evolution in Li1.3Al0.3Ti1.7(PO4)3 ceramics

Lithium aluminum titanium phosphate (LATP) is one of the materials under consideration as an electrolyte in future all-solid-state lithium-ion batteries. In ceramic processing, the presence of secondary phases and porosity play an important role. In a presence of more than one secondary phase and pores, image analysis must tackle the difficulties about distinguishing between these microstructural features. In this study, we study the phase evolution of LATP ceramics sintered at temperatures between 950 and 1100 °C by image segmentation based on energy-dispersive X-ray spectroscopy (EDS) elemental maps combined with quantitative analysis of LATP grains. We found aluminum phosphate (AlPO4) and another phosphate phase ((Lix)PyOz). The amount of these phases changes with sintering temperature. First, since the grains act as an aluminum source for AlPO4 formation, the aluminum content in the LATP grains decreases. Second, the amount of secondary phase changes from more (Lix)PyOz at 950 °C to mainly AlPO4 at 1100 °C sintering temperature. We also used scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to study the evolution of the LATP grains and AlPO4, and LATP grain size increases with sintering temperature. In addition, transmission electron microscopy (TEM) was used for the determination of grain boundary width and to identify the amorphous structure of AlPO4.

Effects of ethanol on gelation of iota-carrageenan

The gelation of iota carrageenan (IC) with different ethanol concentrations (0–40 mL/100 mL) was investigated in terms of rheology, chemical structure, and microstructure. Ethanol facilitated IC gel formation with predictable increasing gelling temperature (52.6–62.6 °C) and critical gelling points (50.8–61.3 °C). The ethanol –OH moiety was responsible for Kirkwood-Buff theory through exclusion effects and direct binding with IC. Fourier transform infrared (FTIR) confirmed the binding with the chemical shifts on both sulphated groups (G4S from 850 to 854 cm−1 and DA2S from 805 to 808 cm−1). The gel strength decreased at < 10 mL/100 mL ethanol and increased up to 40 mL/100 mL ethanol. From Winter-Chambon equation, 40 mL/100 mL ethanol provided a significantly lower fractal dimension df (2.09). Confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) also showed a less complex network. These results indicated that a small amount of ethanol stabilised IC gelation and that the increased gel strength was more significant at high ethanol concentrations (>10 mL/100 mL), which improved the design of IC-ethanol systems.

Development of food-grade Pickering emulsions stabilized by a biological macromolecule (xanthan gum) and zein

In this work, food-grade sunflower oil/W Pickering emulsions stabilized by xanthan gum-zein complex were developed. For this purpose, laser diffraction, rheological, multiple light scattering, confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) measurements were carried out. A response surface methodology was used to determine the optimized zein and oil concentration of the emulsion by using D4,3 and Turbiscan Stability Index (TSI) as objective functions to minimize. Subsequently, the optimized formulation with minimum D4,3 was selected and the biological macromolecule, advanced performance xanthan gum (APXG), was added. CLSM results of emulsions without gum showed the location of zein in the oil-water interface protecting droplets against coalescence as Pickering stabilizer. They also demonstrated that zein did not present important aggregation at the working pH. The addition of APXG changed the flow behaviour from Newtonian to shear thinning which fitted to the Cross model. This fact provoked the occurrence of viscoelastic properties and an increase in stability. FESEM results suggested the formation of a zein-gum complex, which forms a layer covering the droplets, protecting them against oxidation and physical destabilization. Therefore, this research supports the role of zein-APXG complex as a stabilizer of future emulsions.

Structural comparison, physicochemical properties, and in vitro release profile of curcumin-loaded lyotropic liquid crystalline nanoparticle: Influence of hydrotrope as interface stabilizers

Abstract Lyotropic liquid crystal (LLC) is a nano-biomimetic lipid-based system, which has thermodynamic property that is peculiar to it, and suitable for its structural ordering, which facilitates more intimate contact with the tissues. The LLC as a carrier of curcumin has been much studied, however, this is the first time that hydrotrope method approach has been used in the lamellar system precursor of the LLC nanoparticles (LLC-NPs). Curcumin was used due to its pharmacological properties. However, bioavailability is limited by poor water solubility, high chemical instability and metabolic susceptibility. The aim of this study was developed and lipid-based LLC systems prepared by the hydrotrope method were evaluated. Unlike most studies, sodium lauryl sulphate and Poloxamer 407® were used as hydrotropes and the stability, dissolution rate, and physicochemical properties of LLC-NPs were evaluated. The analysis of results showed that hydrotropes increase the stability of LLC-NPs and modify the curcumin release profile. The structural ordering of the lamellar mesophase and LLC-NPs was revealed by microscopy of polarized light and laser confocal scanning microscopy, the results showed a structure with the maltese cross. The LLC-NPs arising from lamellar mesophase with hydrotropes into water-lipid matrices hosted curcumin in the hilum of the maltese cross, and controlled release of curcumin.

A Sensitive Electrochemical Ascorbic Acid Sensor Using Glassy Carbon Electrode Modified by Molybdenite with Electrodeposited Methylene Blue.

A non-enzymatic amperometric sensor using natural molybdenite (MLN) electrodeposited with methylene blue (MB) has been fabricated and characterized and its analytical performances were investigated for the determination of ascorbic acid (AA). The surface morphology of the electrode modified by electrodeposited MB was studied by use of the Advanced Mineral Identification and Characterization System (AMICS) and laser confocal high-temperature scanning microscope (LCSM). The poly(MB) and MLN immobilized sensor showed good stability, reproducibility, sensitivity, and selectivity. It exhibited a linear performance range from 3 to 1000μM, with a lower detection limit of 0.083μM (signal/noise = 3) and short response time (< 5s). No obvious decrease in the current was observed after 20days storage. The methodology reproducibility of this sensor was 2.6%. It showed good anti-interference ability for the potential interfering compounds. The poly(MB) film not only can enhance the electron-transfer rate but also increase the lifetime of the sensor. This study demonstrated the applicability of natural molybdenite for the fabrication of non-enzymatic electrochemical AA sensor.

Preparation and optimization of fisetin loaded glycerol based soft nanovesicles by Box-Behnken design.

The present study focused on the development and optimization of glycerosomes for dermal delivery of fisetin. The fisetin loaded glycerosomes formulation was optimized by Box-Behnken design. The independent variables were the lipoid S 100, glycerol, and sonication time, whereas the dependent variables were the vesicles size, entrapment efficiency, and flux. The mechanism of skin penetration of fisetin loaded glycerosomes formulation was determined by the DSC and FTIR studies. Confocal scanning microscopy was used to detect the penetration ability of glycerosomes. The optimized fisetin loaded glycerosomes formulation was converted into a Carbopol® gel matrix, and the latter was analyzed for various parameters. The optimized formulation of glycerosomes presented vesicles size, entrapment efficiency and flux of 138.8±4.09nm, 86.41±2.95% and 5.04±0.17µg/cm2/h, respectively. The transmission electron microscopy of optimized fisetin loaded formulation revealed the spherical and sealed structure of glycerosomes vesicles. The confocal study confirmed that the Rhodamine B incorporated glycerosomes penetrated up to deeper layers of skin. The DSC and FTIR studies revealed that the hydration of skin layers and skin lipid fluidization could be the penetration mechanism of fisetin glycerosomes formulation. The optimized fisetin loaded glycerosomes gel formulation presented a flux of 4.24±0.14μg/cm2/h, and exhibited zero-order release kinetics. The texture analysis of fisetin glycerosomes gel displayed a sufficient hardness, consistency, cohesiveness, and index of viscosity. It was concluded that the prepared fisetin loaded glycerosomes gel was suitable for the dermal application.

Stokes-vector Resolved Multiphoton/Fluorescence Confocal Scanning Microscopy

Microscopy techniques based on the measurements of polarimetric contrasts as proved to be an important tool to extract additional information about the organization and orientation of any anisotropic sample. More particularly, it has been proven their efficiency for quantitative methods for materials science and biomedical diagnosis. Among these techniques, we have developed a complete Stokes-vector and Mueller-matrix scanning microscope allowing the acquisition of all the physical effects induced by the interaction of the polarized light with a medium. This interaction can be summarized in a single 4x4 elements Mueller-matrix by comparing the independent coding and decoding polarization states, giving access to physical parameters such as dichroism, birefringence, and scattering, from the macro- to the microscopic scale. In this work, we proposed to image the anisotropic emission of molecules under illumination from multiphoton and fluorescence microscopy available on the same scanning microscope. Using Stokes-Mueller formalism, we demonstrate the potentiality to acquire intensity images combined with the anisotropy factor orientation and the scattering (Degree of Polarization) mapping for both modalities. As proof of principle, we have imaged particular biological structures of interests (collagen fibers and muscles) for thin and thick samples. Combining all this information, we propose to quantify locally the cellular and molecular organization at a different scale, from the microscopic to the nanoscopic level.