Confocal Laser Scanning Microscopy Studies Research Articles

α,α-disubstituted β-amino amides eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal

Bacterial biofilms account for up to 80% of all infections and complicate successful therapies due to their intrinsic tolerance to antibiotics. Biofilms also cause serious problems in the industrial sectors, for instance due to the deterioration of metals or microbial contamination of products. Efforts are put in finding novel strategies in both avoiding and fighting biofilms. Biofilm control is achieved by killing and/or removing biofilm or preventing transition to the biofilm lifestyle. Previous research reported on the anti-biofilm potency of α,α-disubstituted β-amino amides A1, A2 and A3, which are small antimicrobial peptidomimetics with a molecular weight below 500 Da. In the current study it was investigated if these derivatives cause a fast disintegration of biofilm bacteria and removal of Staphylococcus aureus biofilms. One hour incubation of biofilms with all three derivatives resulted in reduced metabolic activity and membrane permeabilization in S. aureus (ATCC 25923) biofilms. Bactericidal properties of these derivatives were attributed to a direct effect on membranes of biofilm bacteria. The green fluorescence protein expressing Staphylococcus aureus strain AH2547 was cultivated in a CDC biofilm reactor and utilized for disinfectant efficacy testing of A3, following the single tube method (American Society for Testing and Materials designation number E2871). A3 at a concentration of 90 μM acted as fast as 100 μM chlorhexidine and was equally effective. Confocal laser scanning microscopy studies showed that chlorhexidine treatment lead to fluorescence fading indicating membrane permeabilization but did not cause biomass removal. In contrast, A3 treatment caused a simultaneous biofilm fluorescence loss and biomass removal. These dual anti-biofilm properties make α,α-disubstituted β-amino amides promising scaffolds in finding new control strategies against recalcitrant biofilms.

Antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin on Enterococcus faecalis: A confocal laser scanning microscopic study

Background: Sealers with antimicrobial properties play an important role in endodontic therapy success especially against Enterococcus faecalis infection found in failed root canal therapy. Addition of antibiotic agents to endodontic sealers may show significant increase in their antibacterial properties both against anaerobic and aerobic microbes. The purpose of the present study was to evaluate antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin against E. faecalis and post-root canal treatment viability of Enterococcus faecalis on the first and seventh day. Methods: A total of 60 extracted human mandibular premolar teeth were decoronated after initial decontamination with 1% NaOCl. Root length standardized to 12 mm. Canal instrumentation was done using ProTaper Universal file system till size F2 using 5.25% NaOCl. It was then infected with a pure strain of E. faecalis for a period of four days. Obturation was done using plain sealer, (n=30) and sealer-antibiotic combination, (n=30). Half of the teeth were sectioned at 24 hours (S, SA) and other half were sectioned seven days after obturation (S7, SA7). All samples were stained with SYTO9 and propidium iodide for imaging under Confocal Laser Scanning microscope. Statistical analysis was performed with the statistical software SPSS v. 17.0 (SPSS for Windows; SPSS Inc, Chicago, IL). Data was analysed using One Way ANOVA and post hoc Tukey test to determine statistical significance with p value < 0.01 considered significant. Results: Statistically significant differences were observed in green to red ratio between group S (9.561976) and S7 (0.435418) (p < 0.01). There was no difference found between SA (mean of green to red ratio, (0.70431) and SA7 (mean of green to red ratio, 0.85184). Conclusions: Antibiotics added to the sealer effectively eradicated of E. faecalis 24 hours post-obturation. However, after seven days, plain sealer was as effective as sealer-antibiotic combination.

Small Molecule AIEgens for Illuminating Sub-Cellular Endoplasmic Reticulum, Mitochondria, and Lysosomes.

Organelles are the working hubs of the cells. Hence, visualizing these organelles inside the cells is highly important for understanding their roles in pathological states and development of therapeutic strategies. Herein, we report the development of a novel highly substituted oxazoles with modular scaffolds (AIE-ER, AIE-Mito, and AIE-Lyso), which can home into endoplasmic reticulum (ER), mitochondria, and lysosomes inside the cells. These oxazoles showed remarkable aggregation-induced emission (AIE) property in water and in the solid state due to dual intramolecular H-bonding, which was confirmed by pH- and temperature-dependent fluorescence studies followed by molecular dynamics (MD) simulations and density functional theory (DFT) calculations. Confocal laser scanning microscopy studies revealed that AIE-ER, AIE-Mito, and AIE-Lyso efficiently homed into ER, mitochondria and lysosomes, respectively, in the HeLa cervical cancer cells and non-cancerous human retinal pigment epithelial RPE-1 cells within 3 h without showing any toxicity to the cells with high sub-cellular photostability. To the best of our knowledge, this is the first report of highly substituted oxazole-based small molecule AIEgens for organelle imaging. We anticipate these novel AIEgens have promise to image sub-cellular organelles in different diseased states as well as understanding the inter-organelle interactions towards the development of novel therapeutics.

Beta Caryophyllene-Loaded Nanostructured Lipid Carriers for Topical Management of Skin Disorders: Statistical Optimization, In Vitro and Dermatokinetic Evaluation.

This work aimed to overcome the disadvantages of the oral administration of beta-caryophyllene and boost efficiency by developing a nanostructured lipid carrier for topical administration of the drug in skin disorders. The heat emulsification method was utilized to produce beta-caryophyllene-loaded nanostructured lipid carriers. The newly created formulation was examined for its particle size, entrapment efficiency, and zeta potential after being improved using the Box-Behnken Design. The chosen formulation underwent tests to determine its ex vivo skin retention, dermatokinetic, in vitro release, antioxidant, and confocal laser scanning microscopy study. The findings of the characterization of the nanostructured lipid carriers demonstrated that the particles had a spherical form and a size of 210.86 nm (0.263 polydispersity index). The entrapment efficiency was determined to be 86.74%, and the zeta potential was measured to be -26.97 mV. The in vitro release investigation showed that nanostructure lipid carriers were capable of releasing regulated amounts of beta-caryophyllene for up to 24 hrs. In comparison to the traditional gel formulation, the ex vivo investigation demonstrated a 1.94-fold increase in the skin's capacity to retain the substance. According to the findings of the study, nanostructure lipid carriers loaded with beta-caryophyllene have the potential to be investigated for use as a topical administration method in skin disorders with enhanced skin retention and effectiveness.

Comparative evaluation of the effectiveness of XP-endo Finisher and passive ultrasonic activation on dentinal tubule penetration of irrigation solution: A confocal laser scanning microscopy study

ABSTRACT Aim: The aim of this in vitro study was to comparatively evaluate the effectiveness of XP-endo Finisher and passive ultrasonic (PU) activation on dentinal tubule penetration of irrigation solution: A confocal laser scanning microscopy study. Materials and Methods: Forty extracted single-rooted human mandibular canines and premolars were instrumented up to size 40/06 taper and randomly divided into 2 groups (n = 20) based on the activation technique of final irrigation solution into Group A: PU activation and Group B: XP-endo Finisher activation. In each group, 5 ml of 5% NaOCl labeled with fluorescent dye was used during activation as the final irrigation solution. Specimens were sectioned at 2, 5, and 8 mm from the apex and examined under confocal microscope to calculate the dentinal tubule penetration area. Data were analyzed using Tukey’s post hoc tests (P = 0.05). Results: PU activation exhibited a significantly higher penetration area than XP-endo Finisher activation (P < 0.05). In addition, at coronal, middle, and apical thirds, all regions of PU activation had significantly higher penetration than XP-endo Finisher activation. Statistically significant differences were found between each root canal third (coronal > middle > apical) (P < 0.001). Conclusion: The PU activation is more effective than XP-endo Finisher in terms of irrigant penetration into the dentinal tubules.

Micromorphology and analysis of dentin surfaces after preparation with Er:YAG laser and application of self-etch adhesive system with 10-MDP. A confocal laser scanning microscope study.

The purpose of this study was to analyze and compare the micromorphology of the hybrid layer and the dentinal surface when the dentin was prepared with an Er:YAG laser or burs, and to find if there was any difference if the time of application of the self-etching primer on the dentin prepared with an Er:YAG laser was doubled.

Antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin on Enterococcus faecalis: A confocal laser scanning microscopic study.

Background: Sealers with antimicrobial properties play an important role in endodontic therapy success especially against Enterococcus faecalis infection found in failed root canal therapy. Addition of antibiotic agents to endodontic sealers may show significant increase in their antibacterial properties both against anaerobic and aerobic microbes. The purpose of the present study was to evaluate antimicrobial efficacy of Kerr pulp canal sealer (EWT) in combination with 10% amoxicillin against E. faecalis and post-root canal treatment viability of Enterococcus faecalis on the first and seventh day. Methods: A total of 60 extracted human mandibular premolar teeth were decoronated after initial decontamination with 1% NaOCl. Root length standardized to 12 mm. Canal instrumentation was done using ProTaper Universal file system till size F2 using 5.25% NaOCl. It was then infected with a pure strain of E. faecalis for a period of four days. Obturation was done using plain sealer, (n=30) and sealer-antibiotic combination, (n=30). Half of the teeth were sectioned at 24 hours (S, SA) and other half were sectioned seven days after obturation (S7, SA7). All samples were stained with SYTO9 and propidium iodide for imaging under Confocal Laser Scanning microscope. Statistical analysis was performed with the statistical software SPSS v. 17.0 (SPSS for Windows; SPSS Inc, Chicago, IL). Data was analysed using One Way ANOVA and post hoc Tukey test to determine statistical significance with p value < 0.01 considered significant. Results: Statistically significant differences were observed in green to red ratio between group S (9.561976) and S7 (0.435418) (p < 0.01). There was no difference found between SA (mean of green to red ratio, (0.70431) and SA7 (mean of green to red ratio, 0.85184). Conclusions: Antibiotics added to the sealer effectively eradicated of E. faecalis 24 hours post-obturation. However, after seven days, plain sealer was as effective as sealer-antibiotic combination.

Tools of the Trade: Image Analysis Programs for Confocal Laser-Scanning Microscopy Studies of Biofilms and Considerations for Their Use by Experimental Researchers.

Confocal laser-scanning microscopy (CLSM) is the bedrock of the microscopic visualization of biofilms. Previous applications of CLSM in biofilm studies have largely focused on observations of bacterial or fungal elements of biofilms, often seen as aggregates or mats of cells. However, the field of biofilm research is moving beyond qualitative observations alone, toward the quantitative analysis of the structural and functional features of biofilms, across clinical, environmental, and laboratory conditions. In recent times, several image analysis programs have been developed to extract and quantify biofilm properties from confocal micrographs. These tools not only vary in their scope and relevance to the specific biofilm features under study but also with respect to the user interface, compatibility with operating systems, and raw image requirements. Understanding these considerations is important when selecting tools for quantitative biofilm analysis, including at the initial experimental stages of image acquisition. In this review, we provide an overview of image analysis programs for confocal micrographs of biofilms, with a focus on tool selection and image acquisition parameters that are relevant for experimental researchers to ensure reliability and compatibility with downstream image processing.

Investigating the effectiveness of Difluprednate-Loaded core-shell lipid-polymeric hybrid nanoparticles for ocular delivery

Uveitis is a sight-threatening disease that causes inflammation in the uvea; difluprednate (DFB) is the first approved drug molecule for postoperative pain, inflammation, and endogenous uveitis. Complex ocular physiology and structure make it difficult to deliver drugs to the eye. Increased permeation and retention in the layer of the eye are required to improve the bioavailability of ocular drugs. In the current research investigation, DFB-loaded lipid polymer hybrid nanoparticles (LPHNPs) were designed and fabricated to enhance the corneal permeation and sustained release of DFB. A well-established two-step approach was used to fabricate the DFB-LPHNPs, comprising of Poly-Lactic-co-Glycolic Acid (PLGA) core that entrapped the DFB and DFB loaded PLGA NPs covered by lipid shell. The manufacturing parameters were optimized for the preparation of DFB-LPHNPs; the optimal DFB-LPHNPs showed a mean particle size of 117.3 ± 2.9 nm, suitable for ocular administration and high entrapment efficiency of 92.45 ± 2.17 % with neutral pH (7.18 ± 0.02) and isotonic Osmolality (301 ± 3 mOsm/kg). Microscopic examination confirms the core–shell morphological structure of DFB-LPHNPs. The prepared DFB-LPHNPs were extensively characterized using spectroscopic techniques and physicochemical characterization, which confirms the entrapment of the drug and the formation of the DFB-LPHNPs. The confocal laser scanning microscopy studies revealed that Rhodamine B-loaded LPHNPs were penetrated into stromal layers of the cornea in ex-vivo conditions. The DFB-LPHNPs showed a sustained release pattern in simulated tear fluid and 4- folds enhanced permeation of DFB as compared to pure DFB solution. The ex-vivo histopathological studies revealed that DFB-LPHNPs didn't cause any damage or no alteration in the cellular structure of the cornea. Additionally, the results of the HET-CAM assay confirmed that the DFB-LPHNPs were not toxic for ophthalmic administration.

Internalization of PLGA nanoparticles coated with poloxamer 188 in glioma cells: A confocal laser scanning microscopy study

Internalization of poloxamer 188-coated PLGA nanoparticles (NPs) in GL261 murine glioma cells was studied using confocal laser scanning microscopy. For visualization, both poloxamer 188 (P188) and PLGA were labeled covalently with fluorescent dyes Rhodamine B and Cyanine5, respectively. The results indicated that the PLGA NPs coated with poloxamer 188 enter a cell as an integral core–shell structure, which can be helpful for gaining further insight into the in vivo performance of surfactant-coated polymeric NPs as core–shell delivery systems

Evaluation of interfacial adaptation and penetration of bioceramic-based sealers in oval root canals: A confocal laser scanning microscope study.

To assess the interfacial adaptation and penetration depth of three different bioceramic-based sealers (CeraSeal, EndoSeal MTA, Nishika Canal Sealer BG) compared to an epoxy resin-based sealer (AH Plus) in oval root canals. Fourty extracted single-rooted mandibular premolar with oval canal were prepared and randomly allocated according to the obturation into; CeraSeal, EndoSeal MTA, Nishika Canal Sealer BG and AH Plus. The roots were sectioned at 3, 6 and 9 mm from the apex. The sealer adaptation and the penetration depth were evaluated under confocal laser scanning microscope. One-way ANOVA and Repeated measure ANOVA were used to statistically analyze the data. Nishika Canal Sealer BG showed significantly higher sealer adaptation than EndoSeal MTA (P < .001) at apical and middle thirds. Meanwhile, AH Plus showed significantly higher sealer adaptation than EndoSeal MTA (P = 0.011) at middle third. For sealer penetration, Nishika Canal Sealer BG showed the longest sealer penetration that was significant compared to AH Plus (P < .001) and EndoSeal MTA (P < .001) whereas CeraSeal was significantly higher than EndoSeal MTA (P = 0.029) at coronal third. For AH Plus, there was a significant less sealer penetration at coronal third compared to apical and middle thirds (P < .05). Whereas for EndoSeal MTA, the coronal third has significant less penetration compared to the middle third (P = 0.032). Endoseal has the lowest adaptation and penetration depth. Nishika Canal Sealer BG has better adaptation and penetration depth using single cone obturation technique in oval canal. RESEARCH HIGHLIGHTS: All the tested root canal sealers have some percentage of gaps and vary in their penetration capability into dentinal tubules. Nishika Canal Sealer BG has significantly better sealer adaptation to root dentinal walls than that of EndoSeal MTA at the apical and middle third but not significantly different from other type of sealers used. Nishika Canal Sealer BG has significantly better penetration depth than AH Plus and EndoSeal MTA at the coronal third of radicular dentin.

Bacterial Colonization and Proliferation in Primary Molars following the Use of the Hall Technique: A Confocal Laser Scanning Microscopy Study.

Restorative dentistry aims to create a favorable environment to arrest caries with minimal operative intervention. The Hall technique (HT) involves the seating and cementation of stainless steel crowns (SSC) on primary molars without any tooth preparation, caries removal, or local anesthesia. In this manner, it entombs bacteria and arrests caries' progress. We compared bacterial distribution and quantity among primary molars affected with caries and restored with SSC using the HT (n = 10), the conventional technique (CT; n = 10), or not restored at all (control; n = 10). The teeth were contaminated with Enterococcus faecalis to mimic the clinical situation in the oral cavity and then incubated for 21 days. They were then cut mesiodistally and evaluated with confocal laser scanning microscopy. Total bacterial load (live + dead) in the mesial and distal areas of the crown showed no significant difference between the groups (p = 0.711), but there were significantly more dead than live bacteria in the CT and control groups versus the HT group (p = 0.0274 and p = 0.0483, respectively). Inside the pulp chamber and the crown area, the total bacterial load was significantly higher in the HT compared to the CT group (p < 0.001). Significantly more dead than live bacteria were observed in all tooth areas treated with the HT (p = 0.0169). Bacterial penetration depth was significantly correlated with bacterial load (p = 0.0167). In conclusion, although more bacteria were present in teeth that had undergone the HT versus those treated with the CT, they were mainly unviable. Additionally, the CT and the HT showed a similar performance in terms of marginal leakage, indicating that complete caries removal is not essential to achieve good sealing.

Assessment of dentinal tubule penetration of ah plus, mta fillapex and sealapex after various disinfection procedures: A confocal laser scanning microscopic study

Assessment of dentinal tubule penetration of ah plus, mta fillapex and sealapex after various disinfection procedures: A confocal laser scanning microscopic study

PH-Responsive Drug Delivery Nanoplatforms as Smart Carriers of Unsymmetrical Bisacridines for Targeted Cancer Therapy.

Selective therapy and controlled drug release at an intracellular level remain key challenges for effective cancer treatment. Here, we employed folic acid (FA) as a self-navigating molecule in nanoconjugates containing quantum dots (QDs) and β-cyclodextrin (β-CD) for the delivery of antitumor unsymmetrical bisacridine compound (C-2028) to lung and prostate cancers as well as normal cells. The bisacridine derivative can form the inclusion complex with β-cyclodextrin molecule, due to the presence of a planar fragment in its structure. The stability of such a complex is pH-dependent. The drug release profile at different pH values and the mechanism of C-2028 release from QDs-β-CD-FA nanoconjugates were investigated. Next, the intracellular fate of compounds and their influence on lysosomal content in the cells were also studied. Confocal Laser Scanning Microscopy studies proved that all investigated compounds were delivered to acidic organelles, the pH of which promoted an increased release of C-2028 from its nanoconjugates. Since the pH in normal cells is higher than in cancer cells, the release of C-2028 from its nanoconjugates is decreased in these cells. Additionally, we obtained the concentration profiles of C-2028 in the selected cells treated with unbound C-2028 or nanoconjugate by the HPLC analysis.

Smear Layer and Debris Removal from Root Canals Comparing Traditional Syringe Irrigation and 3D Cleaning: An Ex Vivo Study.

Background: Endodontic treatment objectives comprise eliminating or decreasing bacterial load inside the complex endodontic space. Removing the smear layer and debris becomes mandatory to achieve good three-dimensional (3D) cleaning. Aim: This study assesses the difference in smear layer removal using the 3D cleaning technique and traditional syringe needle irrigation. The 3D cleaning technique includes the ultrasonic activation of intracanal-heated NaOCl. Materials and Methods: Our current study used single-rooted human mandibular premolar teeth to test the earlier-mentioned technique (n = 30). Initially, an endodontic access cavity was performed. Consequently, specimens were randomly distributed into three study groups according to irrigation protocol. The groups were Group 1, where the traditional syringe needle irrigation system was applied; Group 2, where the 3D cleaning technique was performed; and Group 3, in which teeth remained uncleaned as it was regarded as the control group. Once the experiment was completed, the teeth were decoronated at the cementoenamel junction (CEJ) and examined using scanning electron microscopy (SEM). Debris and smear layers were viewed in 1000× magnification and scored. Results: Statistical analysis was performed with a standard statistical software package (SPSS, version 28.0; SPSS IBM, Armonk, NY, USA). Data were analyzed with a nonparametric analysis of variance (Kruskal−Wallis ANOVA) among the groups tested and among the thirds of the canals. The level of significance was set at p < 0.05. A statistically significant (p < 0.05) lower mean smear layer and debris score was observed in both study groups compared to the control group. Group 2 showed better results compared to Group 1. Conclusions: The present study concluded that the 3D cleaning technique is an effective irrigation method for removing debris and smear layers. Future research, such as CLSM (Confocal Laser Scanning Microscopy) and Histological study, should be employed to confirm this study’s conclusion.

Comparison of bacterial removal from dentinal tubules with two different bacterial biosurfactants and a herbal extract: An in vitro confocal laser scanning microscopic study

Comparison of bacterial removal from dentinal tubules with two different bacterial biosurfactants and a herbal extract: An in vitro confocal laser scanning microscopic study

Influence of access cavity design on calcium hydroxide removal using different cleaning protocols: a confocal laser scanning microscopy study.

The purpose of this study was to evaluate the influence of endodontic access cavities design on the removal of calcium hydroxide medication of the apical third of mandibular incisor root canal walls and dentinal tubules with different cleaning protocols: EDDY sonic activation, Er,Cr:YSGG laser-activated irrigation, or conventional irrigation with IrriFlex. Seventy-eight extracted human mandibular incisors were assigned to 6 experimental groups (n = 13) according to the endodontic access cavity and cleaning protocol for calcium hydroxide removal: traditional access cavity (TradAC)/EDDY; ultraconservative access cavity performed in the incisal edge (UltraAC.Inc)/EDDY; TradAC/Er,Cr:YSGG; UltraAC.Inc/Er,Cr:YSGG; TradAC/IrriFlex; or UltraAC.Inc/IrriFlex. Confocal laser scanning microscopy images were used to measure the non-penetration percentage, maximum residual calcium hydroxide penetration depth, and penetration area at 2 and 4 mm from the apex. Data were statistically analyzed using Shapiro-Wilk and WRS2 package for 2-way comparison of non-normally distributed parameters (depth of penetration, area of penetration, and percentage of non-penetration) according to cavity and cleaning protocol with the significance level set at 5%. The effect of cavity and cleaning protocol interactions on penetration depth, penetration area and non-penetration percentage was not found statistically significant at 2 and 4 mm levels (p > 0.05). The present study demonstrated that TradAC or UltraAC.Inc preparations with different cleaning protocols in extracted mandibular incisors did not influence the remaining calcium hydroxide at 2 and 4 mm from the apex.

Comparison of sealer penetration of sonic activation versus conventional needle irrigation: a systematic review and meta-analysis of randomized controlled trials

BackgroundMost existing studies comparing the efficiency of sonic irrigation (SI) and conventional needle irrigation (CNI) in increasing the penetration of sealers into dentine tubules are controversial; and this study aimed to determine whether the use of SI can lead to greater sealing ability than CNI, during the root canal treatment.MethodsThe EMBASE, PubMed, and Cochrane Library databases were used to find confocal laser scanning microscopy studies evaluating percentage and maximum depth of sealer penetration following the use of SI or CNI in mature permanent teeth until October 2022. The critical estimative checklist of randomized controlled trials of the standardized Joanna Briggs Institute was adopted to independently score the quality of each study. The random-effect model for meta-analysis was used to analyse for each canal segment (apical, middle, coronal). The results are shown in the forest plots as weighted mean differences (WMDs) with 95% confidence intervals (95% CIs).ResultsNinety-seven articles were included in the preliminary screening, and nine of them were included in this study. Eight studies were included in the meta-analysis.The meta-analysis exhibited great increases in the coronal (WMD: 8.09, 95% CI 2.78–13.40/WMD: 165.32, 95% CI 128.85–201.80), and middle segments (WMD: 8.81, 95% CI 5.76–11.87/WMD: 132.98, 95% CI 68.71–197.25) for the percentage and maximum depth of sealer penetration, respectively. The percentage of sealer penetration in the apical thirds region was nonsignificant (WMD: 4.73, 95% CI − 2.34–11.80). However, the maximum depth of sealer penetration in the apical thirds region was significant (WMD: 121.46, 95% CI 86.55–156.38). Chi-squared analysis revealed heterogeneity scores of 0.0–70.0% and 44.0–90.0% for the percentage and maximum depth of sealer penetration, respectively.DiscussionThis review verified that SI significantly improves tubular dentin sealer penetration in most areas of the root canal; thus, SI may lead to better filling efficiency and anti-reinfection effects than CNI during and after the root canal therapy. Nevertheless, a large heterogeneity in the current data comparing the irrigation efficiency of SI versus CNI in the apical third of the root canal was found, implying the necessity to standardize root canal irrigation procedures and obtain more accurate results in this area.Trial Registration: INPLASY database (INPLASY202270116).

Reducible amino acid based cationic lipids with a naphthalimide moiety as non-viral gene vehicles

Three basic amino acid–based cationic lipids bearing a fluorescent naphthalimide moiety and a reducible disulfide linkage are synthesized and applied as non-viral gene vehicles. Their DNA interactions are investigated by agarose-gel retardant and ethidium bromide replacement assays. The sizes and zeta potentials of the liposome/DNA complexes are measured by dynamic light scattering. The cytotoxicities of the liposome/DNA complexes are examined using HeLa and 7702 cell lines by MTT assays. The glutathione-responsive DNA release process is studied through time-dependent fluorescence assays. Luciferase gene expression showed the transfection efficiency of the liposome is dramatically increased in the presence of 10% serum. Confocal laser scanning microscopy studies corroborated that the liposome/DNA complexes are successfully uptaken into HeLa cells. These results demonstrate the promising use of amino acids and naphthalimide-containing lipids for safe and efficient gene delivery.

Central Composite Design Implemented Azilsartan Medoxomil Loaded Nanoemulsion to Improve Its Aqueous Solubility and Intestinal Permeability: In Vitro and Ex Vivo Evaluation.

The present research attempted to design and develop a nanoemulsion formulation of azilsartan medoxomil to improve its aqueous solubility and intestinal permeability. Based on the solubility profile, ethyl oleate, tween 80, and Transcutol P were selected as the oil phase, surfactant, and co-surfactant, respectively. Central composite design (CCD) suggested an optimized azilsartan medoxomil- nanoemulsion formulation (optimized AZL-NE formulation) with 1.25% oil, 15.73% Smix, and 90 s ultrasonication time; it was found to have the droplet size, percentage transmittance, and % cumulative drug release (%CDR) of 71.5 nm, 93.46 ± 1.13%, and 90.14 ± 0.94%, respectively. Furthermore, it exhibited a 0.141 polydispersity index, 34.05 mV zeta potential, a 1.413 ± 0.03 refractive index, 6.68 ± 0.22 pH, 28.17 ± 0.52 cps viscosity, and a 96.98 ± 0.94% percentage drug content. Transmission electron microscopy (TEM) assessed the nano-sized spherical shape, and a differential scanning calorimeter (DSC) assessed the solubilization of the drug in the optimized formulation. The %CDR was 1.71 times higher and the % cumulative drug permeation was 2.1 times higher for the optimized AZL-NE formulation than for the drug suspension through an intestinal segment of a rat, which was also supported by confocal laser scanning microscopy (CLSM) studies. Thus, the nanoemulsion formulation of azilsartan medoxomil ensured the enhancement of the drug availability in the body.