Results Of Confocal Laser Scanning Microscopy Research Articles

Evaluation of Confocal Laser Scanning Microscopy in Histological Classification and Photodynamic Therapy of Low-risk Basal Cell Carcinoma.

Confocal laser scanning microscopy (CLSM) is noninvasive technique utilized for identification and analysis skin malignancies. Several studies have applied CLSM in monitoring the therapeutic effects of basal cell carcinoma (BCC). To investigate the diagnostic value of CLSM in low-risk BCC and the evaluation of photodynamic therapy (PDT). We have diagnosed 149 patients with BCC using CLSM and histopathological examination. Based on histopathology, we summarized the classification information of low-risk BCC along with imaging features observed through CLSM. Thirty-four low-risk BCC patients underwent PDT treatment, and we used CLSM to evaluate its efficacy. Out of 149 participants were diagnosed with BCC by CLSM, 52 were pigmented type, 87 were nodular type and 10 were superficial type. After histopathological examination, 44 out of 52 were pigmented type, five were nodular type and three were superficial type. The results of CLSM were consistent with those of 87 nodular type and 10 superficial type. The CLSM features of nodular were observed in the tissue fissures around the tumor, the pigment mass was the CLSM characteristic of pigmented type. The simultaneous occurrence of inflammation and increased vasculature were characteristics of superficial. The effective rate of PDT was 100%, and the cure rate was 67.6%. At 12 months follow up, the recurrence rate of PDT was 11.8%, 15.0% for nodule type, 10.0% for pigmented type and 0% for superficial type. The tissue classification of CLSM for low-risk BCC was consistent with histopathology. CLSM can be used to monitor the efficacy of PDT for low-risk BCC.

Effect of Pulsed Electric Field Treatment on the Protein, Digestibility, and Physicochemical Properties of Starch Granules in Wheat Flour.

The effect of pulsed electric field (PEF) treatment depends mainly on the electric field strength and treatment time. In this study, wheat flour-water suspensions were treated with PEF at an electric field strength of 3 kV/cm for 0 to 1400 pulses to obtain a specific energy input of 0 to 656 kJ/kg. The effect of PEF on the removal or unfolding of proteins from the starch surface, digestibility, starch granule structure, and physicochemical properties of wheat flour was studied. The removal of proteins from the surface and the damage to the internal structure of wheat starch granules after PEF treatment was detected by confocal laser scanning microscopy (CLSM) and FTIR. The damage of the PEF-treated wheat starch granules was observed by scanning electron microscopy (SEM). From CLSM results, penetration of dextran (Mw 10,000 Da) into starch granules of wheat flour was dependent on the energy input of PEF. The high the energy input showed the intense penetration of the biopolymer. The benefits of the accessibility of biopolymer in starch granules are to increase enzyme digestion, especially rapidly digestible starch (RDS). The RDS of wheat flour treated with PEF at 656 kJ/kg was 41.72%, whereas the RDS of wheat flour control was 27.59%.

Potential Use of Vivascope for Real-Time Histological Evaluation in Endoscopic Laryngeal Surgery.

We aimed to assess the feasibility of using confocal laser scanning microscopy (CLSM) for the real-time ex vivo examination of histological samples of laryngeal lesions and to evaluate the correlation between CLSM and definitive histological results. This preliminary study included eight consecutive patients with "suspected" laryngeal lesions who were candidates for endoscopic laryngeal surgery. The obtained samples were evaluated using CLSM and classified as "inadequate" or "adequate" (high- and low-grade dysplasia, in situ and invasive carcinoma, positive surgical margin, and inflammatory outbreaks). CLSM showed the macro image in all cases and generated a digital version. All the samples were defined as adequate during CLSM and confirmed at histopathology: low-grade dysplasia (n = 5), low- and high-grade dysplasia (n = 2), and high-grade dysplasia (n = 1). Four samples had an involved resection margin, and three samples revealed the presence of inflammatory outbreaks. CLSM can be applied to larynx pathology with excellent agreement with final histological results.

Analysis of Starch Structure and Pasting Characteristics of Millet Thick Wine during Fermentation.

Starch is the main substrate in millet thick wine (MTW). In order to control the fermentation process of MTW, it is critical to monitor changes in the starch structure and physicochemical characteristics during the fermentation of MTW. In the present study, the structural characteristics of MTW starch were analyzed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and rapid viscosity analysis (RVA). The results of SEM and CLSM showed that large starch granules in MTW swelled, developed cavities, and ruptured or even vanished with the prolongation of the fermentation time, whereas the size and shape of small starch granules barely changed, only falling off the pomegranate-seed-like aggregates. With the increase in fermentation time, the relative crystallinity of starch in MTW gradually increased. In addition, the short-range ordered structures underwent complex changes. Changes in the starch morphology and ordered structure led to an increase in the peak viscosity time and the initial gelatinization temperature. The present results reveal the beneficial effect of fermentation on MTW processing and suggest its potential applications in other millet-based fermented products.

Antimicrobial and cleaning effects of ultrasonic-mediated plasma-loaded microbubbles on Enterococcus faecalis biofilm: an in vitro study

BackgroundEnterococcus faecalis (E. faecalis) is the most frequently isolated bacteria from teeth with root canal treatment failure. This study aims to evaluate the disinfection effect of ultrasonic-mediated cold plasma-loaded microbubbles (PMBs) on 7d E. faecalis biofilm, the mechanical safety and the mechanisms.MethodsThe PMBs were fabricated by a modified emulsification process and the key reactive species, nitric oxide (NO) and hydrogen peroxide (H2O2) were evaluated. The 7d E. faecalis biofilm on human tooth disk was constructed and divided into the following groups: PBS, 2.5%NaOCl, 2%CHX, and different concentrations of PMBs (108 mL−1, 107 mL−1). The disinfection effects and elimination effects were verified with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Microhardness and roughness change of dentin after PMBs treatment were verified respectively.ResultsThe concentration of NO and H2O2 in PMBs increased by 39.99% and 50.97% after ultrasound treatment (p < 0.05) respectively. The CLSM and SEM results indicate that PMBs with ultrasound treatment could remove the bacteria and biofilm components effectively, especially those living in dentin tubules. The 2.5% NaOCl presented an excellent effect against biofilm on dishes, but the elimination effect on dentin tubules is limited. The 2% CHX group exhibits significant disinfection effect. The biosafety tests indicated that there is no significant changes on microhardness and roughness after PMBs with ultrasound treatment (p > 0.05).ConclusionPMBs combined with ultrasound treatment exhibited significant disinfection effect and biofilm removal effect, the mechanical safety is acceptable.

Synergistic antibacterial and anti-biofilm mechanisms of ultrasound combined with citral nanoemulsion against Staphylococcus aureus 29213

This study investigated the antibacterial and antibiofilm mechanism of ultrasound (US) combined with citral nanoemulsion (CLNE) against Staphylococcus aureus and mature biofilm. Combined treatments resulted in greater reductions in bacterial numbers compared to ultrasound or CLNE treatments alone. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein nucleic acid leakage, and N-phenyl-l-naphthylamine (NPN) uptake analysis showed that the combined treatment disrupted cell membrane integrity and permeability. Reactive oxygen species (ROS) and malondialdehyde (MDA) assays indicated that US+CLNE exacerbated cellular oxidative stress and membrane lipid peroxidation. Field emission scanning electron microscopy (FESEM) revealed that the synergistic processing of ultrasound and CLNE resulted in cell rupture and collapse. In addition, US+CLNE showed a more pronounced removal effect than both alone in the biofilm on the stainless steel sheet. US+CLNE reduced biomass, the number of viable cells in the biofilm, cell viability and EPS polysaccharide contents. The results of CLSM also showed that US+CLNE disrupted the structure of the biofilm. This research elucidates the synergistic antibacterial and anti-biofilm mechanism of ultrasound combined citral nanoemulsion, which provides a safe and efficient sterilization method for the food industry.

Study on the effect of slightly acid hypochlorous water on dental unit waterlines biofilm model

Pseudomonas aeruginosa biofilm was established on PU and PVC's surface by Center of Disease Control (CDC) Biofilm Reactor, to evaluate the effectiveness of Slightly Acidic Hypochlorous water (SAHW) for removing Pseudomonas aeruginosa biofilm on Polyurethane (PU) or Polyvinyl Chloride (PVC), which are commonly used in dental unit waterlines (DUWL). The biofilms were treated with 10 mg/L SAHW, 40 mg/L SAHW and sterile distilled water (control group) by the continuous immersion method for 3, 7, and 10 days. The total viable count (TVC) and Confocal laser scanning microscopy (CLSM) were selected to evaluate the biofilm removal effect and calculate the biofilm reduction rate. The results of TVC showed that 99%-100% biofilm reduction could be achieved in 10 mg/L SAHW group and 40 mg/L SAHW group. The results of CLSM showed that 10 mg/L SAHW group and 40 mg/L SAHW group could reach 89%-100% biofilm reduction after 10 days of treatment.

Preparation and Characterization of Sodium Alginate and Polyquaternium‐10 Hollow Microcapsules by a Layer‐by‐Layer Self‐Assembly Technique

AbstractNovel micro‐scaled hollow capsules were obtained by consecutively adsorbing oppositely‐charged polyelectrolytes of sodium alginate (ALG) and polyquaternium‐10 (PQ‐10) on biocompatible porous calcium carbonate (CaCO3) microspheres, followed by the subsequent removal of the CaCO3 template using acetic acid. The properties of the microcapsules were characterized at various stages of the preparation procedure by using a zeta potential analyzer, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TG) and confocal laser scanning microscopy (CLSM). The results demonstrated that ALG and PQ‐10 were successfully assembled on the surface of the CaCO3 microspheres, and the resulting hollow microcapsules exhibited spherical morphology with typical folds by SEM observations. Furthermore, TEM observations indicated that the average thickness of the monolayer film was ∼1–2 nm, and CLSM results revealed that the microcapsules possessed selective permeability for different charged molecules.

Genotype, biofilm formation ability and specific gene transcripts characteristics of endodontic Enterococcus faecalis under glucose deprivation condition

ObjectiveTo study the relationship between the specific gene and biofilm formation ability of seven wild type Enterococcus faecalis (E. faecalis) under glucose deprivation conditions. DesignWild type E. faecalis (3RC, 5RC, 25RC, 31RC, 33RC, 37RC, 58RC) extracted from the teeth with persistent apical periodontitis were cultured under glucose deprivation conditions and then resequenced. The biofilm formation ability was compared using primary adherence assay, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The transcriptional level of biofilm formation–related genes (ace, gelE, efa, esp and fsrB) were detected. ResultsGenomic resequencing showed that 3RC and 58RC (Class B) were similar, while 5RC, 25RC, 31RC, 33RC and 37RC (Class A) were similar. Based on primary adherence assay, CLSM and SEM results, biofilm formation ability of Class B strains was lower, while Class A strains were higher when compared with control group (0.25 % glucose). Furthermore, compared with control group (0.25 % glucose), the transcriptional levels of ace, efa and fsrB genes were upregulated in all strains; the transcriptional levels of gelE were downregulated in Class B strains, upregulated in Class A strains; the transcriptional levels of esp of Class B strains were downregulated, while upregulated in 25RC, 31RC and 37RC (Class A), and not observed in 5RC and 33RC. ConclusionThe genotypes of wild type E. faecalis of different persistent periapical periodontitis teeth are different. The genotype differences and the transcription levels of related virulence genes (ace, gelE, efa, esp and fsrB) are related to the biological phenotype.

Antibacterial effect of a new bioactive glass on cariogenic bacteria

ObjectivesTo investigate the antibacterial effect of a new bioactive glasses on cariogenic bacteria. Methods and materialsA newly developed bioactive glass named Huaxi bioactive glass-ceramic (HX-BGC) and its antibacterial effects were investigated. 3 M™ Specialty Glass 19933 (SG) was used as the positive control while deionized water was used as a negative control. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of HX-BGC and SG against four cariogenic bacteria (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilius, Lactobacillus rhamnosus) were determined by a microdilution method. Subsequently, bacterial growth and acid production were assessed by co-culturing the four cariogenic bacteria species with HX-BGC or SG at different concentrations anaerobically at 37 °C. Optical density (at 660 nm) was assessed by a microplate reader and the acidity of the supernatant was measured by a pH electrode at 6, 12, 24, and 48 h. Microbial kinetics, morphology and viability were evaluated by counting colony forming units (CFU), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), respectively. ResultsThe MBC of HX-BGC againstS.mutans, S.sobrinus, L.acidophilus and L.rhamnosus were 15.63 mg/mL, 15.63 mg/mL, 7.81 mg/mL and 62.5 mg/mL, respectively. The optical density (7.8 mg/mL) value of the bacterial suspensions did not change during the 48 h of culture. The acidity of bacterial suspension kept steady at around pH 8.8. Results from counting the CFU indicated that HX-BGC had stronger antibacterial effect when compare to SG, which is consistent with the SEM and CLSM results. ConclusionsHX-BGC inhibits acid production and growth of cariogenic bacteria.

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.

In Vitro Antibacterial Activity and Mechanism of Vanillic Acid against Carbapenem-Resistant Enterobacter cloacae.

Vanillic acid (VA) is a flavoring agent found in edible plants and fruits. Few recent studies exhibited robust antibacterial activity of VA against several pathogen microorganisms. However, little was reported about the effect of VA on carbapenem-resistant Enterobacter cloacae (CREC). The purpose of the current study was to assess in vitro antimicrobial and antibiofilm activities of VA against CREC. Here, minimum inhibitory concentrations (MIC) of VA against CREC was determined via gradient diffusion method. Furthermore, the antibacterial mode of VA against CREC was elucidated by measuring changes in intracellular adenosine triphosphate (ATP) concentration, intracellular pH (pHin), cell membrane potential and membrane integrity. In addition, antibiofilm formation of VA was measured by crystal violet assay and visualized with field emission scanning electron microscopy (FESEM) and confocal laser scanning microscopy (CLSM). The results showed that MIC of VA against E. cloacae was 600 μg/mL. VA was capable of inhibiting the growth of CREC and destroying the cell membrane integrity of CREC, as confirmed by the decrease of intracellular ATP concentration, pHin and membrane potential as well as distinctive variation in cellular morphology. Moreover, crystal violet staining, FESEM and CLSM results indicated that VA displayed robust inhibitory effects on biofilm formation of CREC and inactivated biofilm-related CREC cells. These findings revealed that VA exhibits potent antibacterial activity against CREC, and thus has potential to be exploited as a natural preservative to control the CREC associated infections.

MODERN APPROACH TO DIAGNOSTICS, PREVENTION AND CORRECTION OF ACNE SCARRING ON THE BASIS OF STUDY OF SKIN MORPHOLOGY BY NONINVASIVE METHODS

The efficacy of the long-acting drug of hyaluronidase (bovhyaluronidase azoximer, BHA) in acne and postacne patients has been studied. The main criteria for the efficacy of acne and post-acne therapy in the assessment by confocal laser scanning microscopy and laser Doppler flowmetry are proposed.Materials and methods. Randomized, double-blind, placebo-controlled study, comparative in parallel groups. 90 patients aged 18 to 35 years (23.76 ± 4.59 years) with medium and severe acne and postacne were divided into 2 groups of 45 each by randomization (the first group received BHA 3000ME, the second — a placebo). There were two methods of administration of drug or placebo: intramuscular injection or ultraphoonophoresis. Evaluation of the skin was performed by confocal laser scanning microscopy (CLSM) in vivo (VivaScope®, NY, USA), microcirculation — by the laser Doppler flowmetry (LDF) with the laser capillary blood flow analyzer (LAKK-01, Russia). The effectiveness was evaluated by the Qualitative scaring grading system and Goodman’s quantitative global acne scarring system.Results and conclusions: With the use of BHA 80 % of patients had an improvement, 8.89 % — a significant improvement, postoperative index was reduced from 14.27 ± 5.61 to 10.18 ± 4.63 points (p 0.05). In the placebo group the reduction of indices is insignificant. The effectiveness of the use of BHA by the ultraphonophoresis was lower than by intramuscular injection. A significant change in the microcirculation index (flux) was established from 20.09 ± 1.74 PU (Perfusion Units) up to 15.2 ± 2,1 PU (p = 0,01). The normalization of the microcirculation was noted. According to the results of CLSM, among BHA patients were observed a significant decrease of severe fibrosis and inflammatory infiltration, the number of dilated vessels. In the placebo group signs persisted. Clinical and morphological monitoring showed the expediency, high efficiency and safety of BHA in the therapy of patients with medium and severe acne vulgaris and postacne.

Biofilm Inhibition by Novel Natural Product- and Biocide-Containing Coatings Using High-Throughput Screening.

The use of natural products (NPs) as possible alternative biocidal compounds for use in antifouling coatings has been the focus of research over the past decades. Despite the importance of this field, the efficacy of a given NP against biofilm (mainly bacteria and diatoms) formation is tested with the NP being in solution, while almost no studies test the effect of an NP once incorporated into a coating system. The development of a novel bioassay to assess the activity of NP-containing and biocide-containing coatings against marine biofilm formation has been achieved using a high-throughput microplate reader and highly sensitive confocal laser scanning microscopy (CLSM), as well as nucleic acid staining. Juglone, an isolated NP that has previously shown efficacy against bacterial attachment, was incorporated into a simple coating matrix. Biofilm formation over 48 h was assessed and compared against coatings containing the NP and the commonly used booster biocide, cuprous oxide. Leaching of the NP from the coating was quantified at two time points, 24 h and 48 h, showing evidence of both juglone and cuprous oxide being released. Results from the microplate reader showed that the NP coatings exhibited antifouling efficacy, significantly inhibiting biofilm formation when compared to the control coatings, while NP coatings and the cuprous oxide coatings performed equally well. CLSM results and COMSTAT analysis on biofilm 3D morphology showed comparable results when the NP coatings were tested against the controls, with higher biofilm biovolume and maximum thickness being found on the controls. This new method proved to be repeatable and insightful and we believe it is applicable in antifouling and other numerous applications where interactions between biofilm formation and surfaces is of interest.

In vitro photodynamic inactivation effects of benzylidene cyclopentanone photosensitizers on clinical fluconazole-resistant Candida albicans.

The incidence of Candida infections has increased for various reasons, including, the more frequent use of immunosuppresants or broad-spectrum antibiotics. Photodynamic inactivation (PDI) is a promising approach for treating localized Candida infections. The PDI efficacies of three benzylidene cyclopentanone-based (BCB) photosensitizers (PSs: P1, P2 and Y1) against three fluconazole-resistant C. albicans (cal-1, cal-2, and cal-3) and one control C. albicans (ATCC 90028), respectively, were evaluated using an established plate dilution method. The binding of PSs to C. albicans was determined by fluorescence spectroscopy. The mechanism of antifungal PDI was investigated using confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). Three BCB PSs all bound rapidly to C. albicans. After incubation with PSs for 30 min and irradiation with a 532 nm laser for 10 min (40 mW cm-2, 24 J cm-2), the fungicidal activity was achieved as 7.5 μM for P1 and P2, and 25 μM for Y1. CLSM confirmed that P1 and Y1 were located in intracellular components, including mitochondria, while P2 bound to the protoplast exterior and failed to enter the cells. TEM revealed the damage of mitochondria ultrastructures after P1- or Y1-mediated PDI, consistenting with the CLSM results. However, most cells became edematous, enlarged or deformation after P2-mediated PDI. The three BCB PSs all have remarkable PDI effects on C. albicans. The best effect is obtained by P1, which has one cationic charge with a proper lipophilicity. The respective subcellular localization of the three PSs led to different PDI mechanisms.

A novel approach to the pacemaker infection with non-thermal atmospheric pressure plasma

Although the pacemaker (PM) is a key cardiac implantable electrical device for life-threatening arrhythmias treatment, the related infection is a challenge. Thus, the aim of this study is to validate cold plasma as a potential technology for the disinfection of infected pacemakers. Fifty donated PMs were cleaned and sterilized before use and then infected with Staphylococcus aureus (S. aureus). Then, each experimental group was treated with cold plasma treatment for 1 min, 3 min, 5 min and 7 min, while the control group was immersed with sterilized water. Effectiveness of disinfection was evaluated by using CFU counting method and confocal laser scanning microscopy (CLSM). The physicochemical properties of water treated with cold plasma at different time were evaluated, including water temperature change and oxidation reduction potential (ORP). The major reactive species generated by the cold plasma equipment during cold plasma were analyzed with optical emission spectroscopy (OES). No live bacteria were detected with CFU counting method after 7 min of cold plasma treatment, which matches with the CLSM results. The ORP value of water and H2O2 concentration changed significantly after treating with cold plasma. Furthermore, reactive oxygen species (ROS) and reactive nitrogen species (RNS), especially NO, O (777 nm) and O (844 nm) were probably key inactivation agents in cold plasma treatment. These results indicate that cold plasma could be an effective technology for the disinfection of implantable devices.

Preparation of doxorubicin-loaded nanoparticles based on modified popaccharide and its targeting effect on hepatocellular carcinoma cells

Objective To prepare a redox-responsive doxorubicin-loaded nanoparticle, and to study its in vitro realease behavior and targeting effect on heptoma cells. Methods Cystamine was grafted on the side chains of hyaluronic acid with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide catalyst, and then β-cyclodextrin (β-CD) was conjugated on the amine groups of the cystamine by Schiff's base reaction to prepare β-CD modified hyaluronic acid (HACD). The HACD/DOX nanoparticles were prepared by encapsulating DOX into HACD using dialysis method. The drug loading, encapsulation efficiency, particle size and distribution, zeta potential and other physical and chemical properties, as well as in vitro drug release behavior of the HACD/DOX nanoparticles were characterized. The cytotoxicity of HACD/DOX nanoparticles to HepG2 cells was studied by cell counting kit-8 (CCK-8) method. The targeting effect of HACD/DOX nanoparticles on HepG2 cells was studied using flow cytometry and confocal laser scanning microscopy (CLSM). Results HACD were successfully synthesized, which could carry DOX to form uniform homogeneous nanoparticles. The drug loading of DOX in the nanoparticles was (16.1±0.2)% and the encapsulation efficiency was (64.2±0.9)%. The transmission electron microscope images indicated that the shape of the HACD/DOX nanoparticles was homogeneous sphere. The results of granularity analysis showed that the average size of the HACD/DOX nanoparticles was (203.1±2.5) nm with a narrow size distribution (PDI= 0.202). The zeta potential of the HACD/DOX nanoparticles was (-29.1±0.8) mV. The in vitro release behavior of the nanoparticles exhibited obvious redox-sensitivity. The results of in vitro cytotoxicity showed that the blank carrier material HACD had no obvious toxicity to hepatoma cells, and the HACD/DOX nanoparticles could effectively kill hepatoma cells with the 0.38 μg/ml half maximal inhibitory concentration (IC50) value at 48 h. Flow cytometry and CLSM results demonstrated that the HACD/DOX nanoparticles could target hepatoma cells through the mediating effect of hyaluronic acid. Conclusions The prepared HACD/DOX nanoparticles have suitable particle size, high drug loading and encapsulation efficiency, and can release DOX under the stimulation of reducing agent. These nanoparticles have obvious targeting effect on hepatoma cells, which is expected to be applied as the drug delivery system of hepatocellular carcinoma (HCC) therapy. Key words: Hyaluronic acid; β-cyclodextrin; Redox-sensitive; Drug delivery; Hepatocellular carcinoma

Time dependence of the enhancement effect of chemical enhancers: Molecular mechanisms of enhancing kinetics

Chemical enhancers are widely used for facilitating drug penetration in transdermal drug delivery system (TDDS). However, there is a lack of knowledge about how the enhancement effect changes over time. In this study, on the basis of kinetic parameters of enhancement effect, molecular details of the dynamic enhancement process was described and a new hypothesis of the recovery mechanism of the skin barrier function was proposed. Using pretreated skin and flurbiprofen patch, the effects of Azone (AZ) and menthyl decanoate (MT-10) were evaluated with in vitro permeation experiment and further confirmed by confocal laser scanning microscopy (CLSM) and TEWL. The results showed that the enhancement ratio (ER) increased firstly, then reached a plateau and finally decreased. The enhancement effect of MT-10 was slower (Tonset, MT-10>Tonset, AZ), weaker (ERmax, MT-10<ERmax, AZ) and shorter (Teff, MT-10<Teff, AZ) than that of AZ. According to the results of CLSM, ATR-FTIR and molecular dynamic simulation, the dynamic enhancement effect was caused by the variation of the diffusion coefficient of intercellular lipid in the stratum corneum (SC), which was dependent on the affinity between enhancers and lipid. Moreover, the skin barrier function recovered although a large amount of enhancers still existed in the SC. Additionally, according to the results of ATR-FTIR, molecular docking and skin retention study, the dynamic effect of AZ on the skin protein only induced skin irritation but showed no influence on drug penetration, so did the amount of the enhancer. In conclusion, dynamic enhancement effect was caused by the dynamic effect of the enhancer on the SC intercellular lipid, and the skin barrier function recovered by accepting the enhancer as a new component of the lipid bilayer.

Comparative analysis of the value of information provided by anterior segment optical coherence tomography and confocal laser scanning microscopy for identifying the palisades of Vogt in normal limbus

to conduct a comparative analysis of the value of information provided by anterior segment optical coherence tomography (AS-OCT) and in vivo confocal laser scanning microscopy (CLSM) for identifying the palisades of Vogt (POV) in normal limbus. POV were studied in 15 healthy participants (30 eyes) without any anterior segment disease. Two examination methods were used: AS-OCT En Face imaging (RTVue XR Avanti, 3D Cornea protocol) and CLSM (HRT3). Concordance of the results was then analyzed. Structural features of POV were distinguishable by both methods. CLSM was able to visualize POV in both the superior and inferior limbi of all the 15 patients (30 eyes). Within the inferior corneal hemisphere, POV had the appearance of well-differentiated hyperreflective double-contoured structures organized in parallel lines at the level of basal epithelium. The structure of the superior limbus, as judged from CLSM data, varied significantly: «classic» palisades were seen in only 13 eyes (43%), while in the other 17 cases (57%) POV were atypical. On OCT, POV structure was seen in detail in 29 eyes. By comparing the abilities of CLSM and OCT to visualize POV, a strong positive correlation (rS=0.99, p<0.05) was revealed between the results obtained in both the superior and inferior limbi. OCT data provide a detailed understanding of POV structure and strongly correlate with CLSM results. Taking into consideration the noninvasiveness, demonstrativeness, and repeatability of AS-OCT En Face imaging as well as the high value of provided information, the method cay be recommended as an alternative in POV diagnostics.

Enhancement of the topical tolnaftate delivery for the treatment of tinea pedis via provesicular gel systems

Tolnaftate is a thiocarbamate antifungal drug which is therapeutically active against dermatophytes that cause various forms of tinea. Due to the small amount of tolnaftate released from ordinary ointment bases and insufficient penetration through the infected skin layers the need to incorporate the drug in a more suitable pharmaceutical form has evolved. A provesicular system is one such form that can solve these problems. Once in contact with the skin, dilution with moisture occurs and the provesicular system rapidly transforms into a vesicular one. Provesicular systems were prepared according to full-factorial experimental design. Plain provesicular systems were compared with systems containing Phospholipon 80 H and Lipoid S45 as penetration enhancers. Design expert software was used to analyze the effect of formulation variables (type of Span used as well as the presence or the absence of the penetration enhancer and its type) on the dependent variables: percent encapsulation efficiency (EE%), vesicle size and percent in vitro drug released). Three formulations were chosen; a plain provesicular system (PV-2), one containing Phospholipon 80H (PV-6) and another containing Lipoid S45 (PV-10) with the goal to reveal the effect of penetration enhancer on morphology, rheological properties and ex vivo permeation using confocal laser scanning microscopy (CLSM). Analysis of CLSM results showed that the penetration enhancing effect for the tested formulations followed the order PV-10 > PV-6 > PV-2. Promising clinically active treatment for tinea patients could be expected as shown by the in vivo permeation results for the provesicular systems as suggested by the CLSM results.