Confocal Laser Scanning Microscopy Studies Research Articles

Comparison of bacterial leakage resistance of various root canal filling materials and methods: Confocal laser-scanning microscope study.

This study evaluated the bacterial leakage resistance and root canal lining efficacy of various root canal filling materials and methods by using confocal laser-scanning microscope (CLSM). Sixty extracted human premolars with mature apex and single root canal were randomly divided into 2 control groups and 4 experimental groups. Group CW was filled with continuous wave technique using gutta-percha and AH Plus sealer. Group GC was coated with AH-Plus sealer and then obturated with soften GuttaCore. Group GF was obturated using GuttaFlow and gutta-percha. Group EM was filled with EndoSeal MTA and gutta-percha using ultrasonic vibration. The AH-Plus, GuttaFlow, and EndoSeal were labeled with Hoechst 33342 to facilitate fluorescence. The obturated root tip was incubated with Carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained E. faecalis for 14 days. CLSM was performed to evaluate the sealer distribution and bacterial leakage for the apical 1-, 2-, 3-mm specimens. Statistically significant differences were determined by 1-way ANOVA with Tukey's post-hoc test and Pearson's correlation analysis. Group EM showed the better sealer distribution score than the other groups (p < 0.05). Group CW and group GC exhibited the less bacterial leakage than the group GF, while group EM showed the similar bacterial leakage score with the groups CW and GC. There was no significant correlation between the sealer distribution and bacterial leakage (p > 0.05). Under the conditions of this study, different root canal filling materials and methods showed different efficacy for canal distribution and bacterial leakage resistance.

PEGylated Dendrimer-Doxorubicin Cojugates as pH-Sensitive Drug Delivery Systems: Synthesis and In Vitro Characterization.

To achieve liver-specific delivery of antitumor drug doxorubicin (DOX), PEGylated dendrimer-DOX conjugates were designed and synthesized, whereas DOX was conjugated to dendrimers via hydrazone bonds and the dendrimers were functionalized with galactose moieties. The release rates of DOX from the conjugates at pH 5.0 were much faster than those at pH 7.4 due to the pH-sensitive cleavage of the hydrazone bonds. The conjugates were shown to effectively kill HepG2 cells in vitro. Compared to other conjugates, the PEGylated dendrimer-DOX one with multiple galactose moieties (Dendrimer-DOX-PEG-Gal) demonstrated HepG2 cells specificity, higher efficacy and good biosafety due to the lower IC50 value and higher cellular uptake confirmed by in vitro cytotoxicity assays, confocal laser scanning microscopy and flow cytometric studies. These results suggest that Dendrimer-DOX-PEG-Gal is an efficient and biocompatible candidate for the specific delivery of antitumor drug to HepG2 cells and could be used as liver cancer specific drug delivery system.

Potential of a novel self nanoemulsifying carrier system to overcome P-glycoprotein mediated efflux of etoposide: In vitro and ex vivo investigations

The oral absorption of etoposide is mainly limited by its active efflux out of the cells by P-glycoprotein (P-gp) and metabolized by cytochrome P450. Therefore, an attempt was aimed to investigate the effect of etoposide-loaded self-nanoemulsifying drug delivery system (SNEDDS) on the absorption of etoposide. The transport of SNE3 formulation across rat intestine was significantly (p < 0.001) higher than that of drug solution alone as well as in presence of verapamil, used as P-gp inhibitor. Results of fluorescence activating cell sorting assay showed a 61.3 ± 2.56% intracellular uptake of fluorescence marker Rhodamine B loaded in SNEDDS while plain Rhodamine B solution showed only a 15.3 ± 1.62% uptake. Confocal laser scanning microscopy study showed significantly higher penetration i.e., 83.71 μm across the GI membrane as compared to plain Rhodamine B solution which showed penetration up to 21.67 μm. Mechanism of P-gp inhibition was confirmed by measuring ATPase activity in the DC-3F/ADX cell model. Moreover, histopathology revealed that epithelial membrane of rat intestine treated with SNE3 was free of abrasion and atrophy. The aforementioned results suggest that the inhibitory effect of SNEDDS on the activity of both P-gp and cytochrome P450 may enhance the oral absorption of etoposide.

Virulency of novel nanolarvicide from Trichoderma atroviride against Aedes aegypti (Linn.): a CLSM analysis

Aedes aegypti is the vector for transmitting dengue, chikungunya, and yellow fever. These diseases' transmission has increased predominantly in urban and semi-urban areas as a major public health concern. In present investigation, Trichoderma atroviride culture filtrates were used for the synthesis of silver nanoparticle. Moreover, T. atroviride is a free-living and rapidly growing fungi common in soil and root ecosystem. This fungi is an exceptionally good model for biocontrol and more significant as a bioagent. T. atroviride was grown in malt extract. T. atroviride culture filtrates were exposed to silver nitrates solution for 24 h at 25 °C for the synthesis of silver nanoparticles (AgNPs). These AgNPs were characterized to find their unique properties with UV-visible spectrophotometer and transmission electron microscope (TEM) analysis. The T. atroviride culture filtrates have formed hexagonal (diamond shape) AgNPs with the range of size of 14.01-21.02 nm. These AgNPs have shown significant efficacies against first, second, third, and fourth instar larvae of A. aegypti. The LC90 and LC99 values for the first instar were 1 and 3 ppm, second instar 2 and 3.18 ppm, third instar 3.12 and 4.12 ppm, and fourth instar 6.30 and 6.59 ppm, respectively, after an exposure of 7 h. The confocal laser scanning microscopy (CLSM) studies were verdict that these AgNPs embedded in the cuticle of larvae and cause instant lethality in 7 h. Present investigations have demonstrated that the AgNPs of T. atroviride culture filtrates synthesized can be used for larvae control of A. aegypti. T. atroviride is synthesized to silver nanoparticles to be a promising new candidate for application in mosquito control. We therefore suggested that the ability of T. atroviride culture filtrates in synthesis can also be explored for synthesizing silver nanoparticles for commercial exploitation.

Selective nuclei accumulation of ruthenium(II) complex enantiomers that target G-quadruplex DNA

Different enantiomers exhibit large differences in their biological activity and/or toxicity, but they rarely involve the relationship of the agents for molecular and cellular imaging with the chiral structure of ruthenium complexes. Here, we report that an enantiomer of a polypyridyl ruthenium complex can selectively accumulate in the nucleus of HepG2 cells. Confocal laser scanning microscopy studies show that this phenomenon occurs via a non-endocytotic, but temperature-dependent, mechanism of cellular uptake in HepG2 cells. DNA oligonucleotides with repetitive tracts of guanine bases that can form G-quadruplex structures have aroused interest as therapeutic agents and as targets for anticancer drug design. Various biophysical techniques show that the Λ-enantiomer of ruthenium complexes can selectively stabilize human telomeric G-quadruplex DNA and has a strong preference for G-quadruplex over duplex DNA. Judged from the NMR results, we speculate that at higher 4:1 ligand/G-quadruplex stoichiometry, complex Λ–Ru is likely to bind with each groove of the tetraplex in a dimeric form or intercalate with the G-tetrad in the 3′ terminal face and coexist with other modes. The molecular modeling analysis is in agreement with the NMR titrations performed in this investigation indicating that ruthenium complexes are actually characterized by a mixed binding mode. The results provide many opportunities for the development of novel agents for living cell-related studies.

Skin targeted lipid vesicles as novel nano-carrier of ketoconazole: characterization, in vitro and in vivo evaluation.

Liposomal carriers for topical drug delivery have been studied since the 1980s and have evoked a considerable interest. However, the conventional liposomes do not deeply penetrate into the skin and remain confined to the outer layer of SC. In order to increase skin targeting of ketoconazole (KCZ), a hydrophobic broad-spectrum antifungal agent, this study describes novel lipid vesicles as nano-carriers for topical delivery. In this paper, lipid vesicular systems including conventional liposomes (CL), ethosomes, deformable liposomes (DL) and ethanol-containing deformable liposomes (DEL) were prepared as nano-carriers for KCZ, respectively. Sodium dodecyl sulfate [SDS, 0.08 % (W/V)] was used as edge activator for DL and DEL preparation. Characterization of the vesicles was based on particle size, zeta potential, entrapment efficiency and transmission electron microscopy (TEM). In addition, in vitro permeation profile was obtained using vertical diffusion Franz cells by porcine skin. The in vivo accumulation of KCZ was also evaluated in rat skin. Confocal microscopy was performed to visualize the penetration of fluorescently labeled vesicles into skin. All of the lipid vesicles showed almost spherical structures with low polydispersity index (PDI < 0.3) and nano-metric size (no more than 160 nm). The results demonstrated that DEL dramatically improved both in vitro and in vivo skin deposition compared to the CLs (P < 0.05), which was further confirmed by confocal laser scanning microscopy study. In vivo pharmacodynamic studies showed DEL improved antifungal activity against Candida albicans in shorter duration of time. Therefore, based on present study, the novel nano-carrier DEL capable of enhancing skin target effect and forming a micro drug-depot could serve as an effective skin targeting delivery for KCZ as an anti-fungal agent in local therapy.

Co-processing of hydroxypropyl methylcellulose (HPMC) for improved aqueous dispersibility

Hydroxypropyl methylcellulose (HPMC), a widely employed film coating polymer, exhibits poor dispersibility in an aqueous medium. Rapid hydration leading to swelling and coherent gel formation is reported to be responsible for this problem. Present study focuses on the use of spray drying based approach for co-processing of HPMC to improve its dispersibility. Dispersion behavior of native HPMC showed formation of large lumps that did not dissolve completely for 40min. However, HPMC co-processed with lactose and sodium chloride exhibited improvement in dispersibility with complete dissolution attained within 20min. Mechanistic insights into improved dispersibility were obtained using contact angle studies, confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM) and scanning TEM (STEM) studies. Co-processed products exhibited higher immersional wetting as determined by sessile drop contact angle technique, which indicated spontaneous incursion of water. CLSM study revealed highly swollen and erodible gel in co-processed products. Novel application of TEM and STEM techniques was developed to understand the nature of mixing achieved during co-processing. Overall the improvement in dispersibility of co-processed products was predominantly due to the alteration in sub-particulate level properties during co-processing. The effect of excipients on the film properties of HPMC, like tensile strength and hygroscopicity, was also assessed. This study provides the comprehensive understanding of role of co-processing on improvement of dispersion behavior of HPMC and helps in the selection of suitable excipients for the same.

Combination of In Situ Microscopy and Calorimetry to Study Austenite Decomposition in Inclusion Engineered Steels

In situ high temperature confocal laser scanning microscopy and differential scanning calorimetry studies of ferrite formation in inclusion engineered (Ti2O3 and TiN) steels have been performed. The applied methodology allows distinction between intragranular ferrite, grain boundary ferrite, and pearlite. The effect of the inclusions and cooling rates on the initiation of phase transformation and the final microstructure is discussed. It is concluded that the applied hybrid methodology could provide vital details of solid‐state phase transformations within the field of inclusion engineering.

Biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo.

RNA-based delivery system for cancer therapy remains a challenge. In this study, a stearyl-peptide (SHR) was synthesized using arginine, histidine, cysteine, and stearyl moieties. Further, the stearyl-peptides were cross-linked by disulfide bonds to obtain cross-linked polypeptides (SHRss) with different molecular weight (SHRss1, SHRss2, SHRss3, SHRss4). The SHRss could effectively condense small interfering RNA (siRNA) into polyplexes with a hydrodynamic size of 100-300 nm and zeta potential of 20-40 mV. Flow cytometry and confocal laser scanning microscope studies revealed high cellular uptake and rapid dissociation behavior of SHRss2/siRNA complexes. Long-lasting high concentration of siRNA in cytoplasm was observed even at 24 h after SHRss2/Cy3-siRNA transfection. Compared with SHR, the SHRss showed much improved siRNA interference efficiency targeting luciferase on Luc-Hela cells. Moreover, SHRss2 exhibited higher interference efficiency and slower decay rate on Luc-Hela cells than Lipofectamine 2000 and SHR. In addition, much weaker expression of red fluorescence protein was also observed on SHRss2/simCh-treated mCherry-HEK293 cells than Lipofectamine 2000 and SHR. The SHRss did not induce cytotoxicity at siRNA concentrations of 25-200 nM under transfection. The in vivo studies demonstrated the gene interference efficiency of SHRss2/siRNA complexes. Our studies indicated that the SHRss are promising and efficient nonviral vectors for siRNA delivery.

Effect of brief cetylpyridinium chloride treatments during early and mature cariogenic biofilm formation.

The aim of this study was to investigate the antibiofilm activity of brief cetylpyridinium chloride (CPC) treatments during early and mature Streptococcus mutans biofilm formation. Streptococcus mutans biofilms were formed on saliva-coated hydroxyapatite disks. The biofilms were treated with CPC twice daily (1min/treatment) from 0 to 50h or from 48 to 98h. Acidogenicity, dry weight, viability, and water-insoluble extracellular polysaccharides of the biofilms were analyzed. Confocal laser scanning microscopy (CLSM) images were obtained to confirm the antibiofilm activity during mature biofilm formation and to evaluate the relationship between treatment time and the antibiofilm activity. CPC showed complete antibiofilm activity during early biofilm formation at 0.025% to 0.1%. During mature biofilm formation, CPC inhibited dry weight, viability, and acidogenicity at 0.075% and 0.1%. CLSM images showed an increase in dead cells at 0.075% and 0.1% CPC. The antibiofilm activity during mature biofilm formation increased as the concentration of CPC increased. Images from the CLSM study also showed that antibiofilm activity increased as treatment time increased. Our findings suggest that brief CPC treatments have strong anti-S.mutans biofilm activity. The antibiofilm activity was dependent on the stage of biofilm formation, CPC concentration, and treatment time.

Bioactive hydrogel-nanosilica hybrid materials: a potential injectable scaffold for bone tissue engineering

Novel bioactive organic–inorganic hybrid materials that can serve as injectable hydrogel systems for bone tissue regeneration were obtained. The silica nanoparticles (SiNP) prepared in situ by the Stöber method were dispersed in collagen, collagen-chitosan or chitosan sols, which were then subsequently crosslinked. Laser scanning confocal microscopy studies, in which fluorescent SiNP were applied, and SEM images indicated that the nanosilica particles were distributed in the whole volume of the hydrogel matrix. In vitro studies on fibroblast cell viability indicated that the hybrid materials are biocompatible. The silica nanoparticles dispersed in the biopolymer matrix had a positive effect on cell viability. Studies on the mineralization process under simulated body fluid (SBF) conditions confirmed the bioactivity of prepared materials. SEM images revealed mineral phase formation in the majority of the hybrid materials developed. EDS analysis indicated that these mineral phases are mainly composed of calcium and phosphorus. The XRD studies confirmed that mineral phases formed during SBF incubation of hybrid materials based on collagen are bone-like apatite minerals. The silica nanoparticles added to the hydrogel at the stage of synthesis induced the occurrence of mineralization. This process occurs not only at the surface of the material but in its entire volume, which is important for the preparation of scaffolds for bone tissue engineering. The ability of these materials to undergo in situ gelation under physiological temperature and their bioactivity as well as biocompatibility make them interesting candidates for bioactive injectable systems.

Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonumaviculare L. extract

In this study, Polygonum aviculare L. extract, which has superior antioxidative and cellular membrane protective activity, was loaded onto cell penetrating peptide (CPP) conjugated liposomes to enhance transdermal delivery. The physical characteristics of typical liposomes and CPP-conjugated liposomes containing P. aviculare extract were evaluated. The particle sizes of both liposomes were approximately 150nm. Whereas the zeta potential of typical liposomes was −45mV, that of CPP-conjugated liposomes was +42mV. The loading efficiency of P. aviculare extract in both liposomes was calculated to be about 83%. Fluorescent-labeled liposomes were prepared to evaluate cellular uptake and skin permeation efficiency. Using flow cytometry, we found that CPP-conjugated liposomes improved cellular uptake of the fluorescent dye as compared with the typical liposomes. In addition, the skin permeation of CPP-conjugated liposomes was proved higher than that of typical liposomes by confocal laser scanning microscopy studies and Franz diffusion cell experiments. The improved cellular uptake and skin permeation of the CPP-conjugated liposomes were due to the cationic arginine-rich peptide. In vivo studies also determined that the CPP-conjugated liposomes were more effective in depigmentation and anti-wrinkle studies than typical liposomes. These results indicate that the CPP-conjugated liposomes could be effective for transdermal drug delivery of antioxidant and anti-aging therapeutics.

Multifunctional layered magnetic composites.

A fabrication method of a multifunctional hybrid material is achieved by using the insoluble organic nacre matrix of the Haliotis laevigata shell infiltrated with gelatin as a confined reaction environment. Inside this organic scaffold magnetite nanoparticles (MNPs) are synthesized. The amount of MNPs can be controlled through the synthesis protocol therefore mineral loadings starting from 15 wt % up to 65 wt % can be realized. The demineralized organic nacre matrix is characterized by small-angle and very-small-angle neutron scattering (SANS and VSANS) showing an unchanged organic matrix structure after demineralization compared to the original mineralized nacre reference. Light microscopy and confocal laser scanning microscopy studies of stained samples show the presence of insoluble proteins at the chitin surface but not between the chitin layers. Successful and homogeneous gelatin infiltration in between the chitin layers can be shown. The hybrid material is characterized by TEM and shows a layered structure filled with MNPs with a size of around 10 nm. Magnetic analysis of the material demonstrates superparamagnetic behavior as characteristic for the particle size. Simulation studies show the potential of collagen and chitin to act as nucleators, where there is a slight preference of chitin over collagen as a nucleator for magnetite. Colloidal-probe AFM measurements demonstrate that introduction of a ferrogel into the chitin matrix leads to a certain increase in the stiffness of the composite material.

The Effect of Addition of an EPS Degrading Enzyme with and without Detergent to 2% Chlorhexidine on Disruption of Enterococcus faecalis Biofilm: A Confocal Laser Scanning Microscopic Study.

Enterococcus faecalis is one of the most commonly occurring organisms retrieved from root canal treated teeth that show refractory apical periodontitis. Though it is well known that the ability of E. faecalis to form a matrix-encased biofilm contributes to its pathogenicity, the role of extracellular dextran and DNA in biofilm formation and its effect on the susceptibility of the biofilm to chlorhexidine remains poorly understood. It was hypothesized that the addition of an Extracellular Polymeric Substance (EPS) degrading enzyme along with a detergent to chlorhexidine may increase the susceptibility of the E. faecalis biofilm. To evaluate the sensitivity of Enterococcus faecalis biofilms treated with DNase enzyme and their susceptibility to 2% chlorhexidine used alone or in conjunction with a detergent in a dentin disinfection model and examine under confocal laser scanning microscopy (CLSM). Semi cylindrical shaped dentin specimens were infected with E. faecalis and incubated for 24 hours. Following incubation, the infected dentin specimens were exposed for 3 minutes to the four disinfecting solutions and grouped accordingly. {Group I- Sterile saline, Group II- 2% Chlorhexidine (CHX), Group III- Dnase1 Enzyme + 2% CHX, Group IV- DNase1 Enzyme + 2% CHX & Tween 80. Bacterial viability was then assessed by staining the specimens and examining under CLSM to analyse the proportion of dead and live bacteria within the dentinal tubules. The Groups II, III and IV showed statistically significant (p<0.05) percentage of dead bacteria compared to the control (Group I). However there was no significant difference in the killing effectiveness within the experimental groups (II-IV) at (p<0.05). EPS degrading enzyme (DNase I) disrupts the biofilm and increases the susceptibility of E.faecalis when exposed to 2% Chlorhexidine and the use of a surfactant with this combination significantly contributes to improving the antibacterial efficacy.

Self-assembled zein-sodium carboxymethyl cellulose nanoparticles as an effective drug carrier and transporter.

In this work, biodegradable nanoparticles (NPs) were assembled with sodium carboxymethyl cellulose (CMC) and zein to produce zein-CMC NPs. Paclitaxel (PTX) was 95.5% encapsulated at a zein-CMC weight ratio of 1 : 3 and the NPs were spherical with an average particle size of approximately 159.4 nm, with the PTX concentration maintained at 80 μg mL-1. The NPs demonstrated good stability over a broad range of pH ranging from 3.7 to 11.0. The zein-CMC NPs were seen to provide a sustained release of PTX for up to 72 h, which led to an 80% release of the total loaded PTX in vitro. Confocal laser scanning microscopy (CLSM) and flow cytometry studies showed that the zein-CMC NPs could effectively transport encapsulated molecules into both drug-sensitive (HepG2 cells) and drug-resistant cancer cells (MCF-7 cells). Moreover, in vitro viability studies revealed that the PTX-loaded zein-CMC NPs had greater potency than free PTX in the PTX resistant MCF-7 cells at higher concentration. Furthermore, PTX-loaded NPs displayed obvious efficiency in the apoptosis of HepG2 cells. Zein-CMC NPs have shown significant potential as a highly versatile and potent platform for cancer therapy.

Clinical, Functional and Structural Studies of Humans and Merino Sheep Corneas from Two Different Regions of Argentina

Objective: The purpose of our work was to study and compare the cornea and tear film of people and sheep inhabiting in a region of Argentinean Patagonia (PATG), and in a geographically and climatically different region in the Argentinean Pampa (CAPT). Methods: Using cross sectional study, corneal examinations were performed in people and Merino sheep inhabiting PATG and CAPT regions, respectively. All people completed a questionnaire related to work activity, diet, and the use of hats or sunglasses during their life. Eyes from all participants were examined with a portable handheld slitlamp biomicroscope (BM) to evaluate corneal appearance, epithelium integrity, and transparency. Later on, in a subgroup of participants we studied eyeblinking frequency (EBF), ocular surface staining (FS), breakup time (BUT), Schirmer Tear Test (STT), corneal structure using confocal laser scanning microscopy (CLSM) and light microscopy (LM), and concentrations of serum ascorbate (sAA). Results: BM studies revealed numerous cases of Climatic Droplets Keratopathy (CDK) only in people inhabiting the PATG region. CLSM studies confirmed typical punctiform deposits at the Bowman´s layer in CDK patients living in that region. CLSM images from sheep did not show any abnormalities at the Bowman´s layer but exhibited small hyper reflective dots at the epithelium only in animals pasturing in the PATG region. FS and EBF mean values were significant higher in sheep grazing in the PATG region (p <0.05). No differences were found in surface eye tests when people from both regions were analyzed. Low sAA levels were found only in CDK people living in PATG region. Conclusions: CDK is a multi-factorial disease not only associated to harsh climate. We provide data that low sAA levels can play a role in its genesis in Patagonia patients’... Sheep pasturing in this region with a high prevalence of CDK have high concentration of sAA and did not present any sub epithelial corneal abnormalities.

Nanoethosomal formulation for skin targeting of amphotericin B: an in vitro and in vivo assessment

The present study is envisaged to develop nanoethosomal formulation for enhanced topical delivery of amphotericin B (AmB) for the treatment of cutaneous fungal infections. AmB encapsulated nanoethosomes were prepared using mechanical dispersion method in a strength of 0.1% w/w similar to the strength of marketed topical formulation. Vesicle size of nanoethosomal formulations was found to be in the range of 186 ± 2 to 298 ± 4 nm. The optimized nanoethosomal formulation was further incorporated in gel base to form AmB nanoethogel formulation. Rheological characterization study of nanoethogel demonstrated its viscoelastic nature with high elasticity and resistance to deformation at 37 °C. The yield stress value was found to be 108.05 ± 2.4 and 52.15 ± 0.9 Pa for nanoethogel and marketed gel formulation, respectively. The nanoethogel formulation exhibited 2.7- and 3.5-fold higher steady state transdermal flux and skin deposition of AmB, respectively, in comparison to marketed formulation. Confocal laser scanning microscopy (CLSM) study also revealed enhanced skin permeation and deposition with nanoethogel formulation. In vivo study showed that topical application of nanoethogel does not exhibit any skin irritation as tested by Draize test. The developed formulation, in comparison to the marketed gel, demonstrated a remarkable increase in the antifungal activity against Candida albicans. It is thus corroborated from the above results that nanoethosomal formulation represents an efficacious carrier for effective topical delivery of AmB.

Mechanistic profiling of the siRNA delivery dynamics of lipid–polymer hybrid nanoparticles

Understanding the delivery dynamics of nucleic acid nanocarriers is fundamental to improve their design for therapeutic applications. We investigated the carrier structure–function relationship of lipid–polymer hybrid nanoparticles (LPNs) consisting of poly(dl-lactic-co-glycolic acid) (PLGA) nanocarriers modified with the cationic lipid dioleoyltrimethyl-ammoniumpropane (DOTAP). A library of siRNA-loaded LPNs was prepared by systematically varying the nitrogen-to-phosphate (N/P) ratio. Atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM) combined with small angle X-ray scattering (SAXS) and confocal laser scanning microscopy (CLSM) studies suggested that the siRNA-loaded LPNs are characterized by a core–shell structure consisting of a PLGA matrix core coated with lamellar DOTAP structures with siRNA localized both in the core and in the shell. Release studies in buffer and serum-containing medium combined with in vitro gene silencing and quantification of intracellular siRNA suggested that this self-assembling core–shell structure influences the siRNA release kinetics and the delivery dynamics. A main delivery mechanism appears to be mediated via the release of transfection-competent siRNA–DOTAP lipoplexes from the LPNs. Based on these results, we suggest a model for the nanostructural characteristics of the LPNs, in which the siRNA is organized in lamellar superficial assemblies and/or as complexes entrapped in the polymeric matrix.

Study of Pickering emulsion stabilized by sulfonated cellulose nanowhiskers extracted from sisal fiber

Sulfonated cellulose nanowhiskers (sulfonated CNWs), a kind of needle-like colloidal particles, could be obtained through sulfuric acid hydrolysis of microcrystal cellulose extracted from sisal fibers. Here, stable Pickering emulsions were prepared using the sulfonated CNWs as a stabilizer. The effects of the sulfonated CNW concentration in aqueous phase, volume ratio of water to oil, oil type, and the pH value on the stability, type, and morphology of these emulsions were investigated. The average droplet size decreases with increasing sulfonated CNW concentration and volume ratio of water to oil. Many types of oil phase were stabilized by sulfonated CNWs to form stable oil-in-water (o/w) Pickering emulsion only by adjusting the pH value of water phase. In addition, poly(methyl methacrylate)PMMA/sulfnated CNW composite microspheres were fabricated by solvent volatilization of the as-prepared Pickering emulsion. Confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) studies confirmed that sulfonated CNWs adsorbed on the surface of the PMMA microparticles. With the interesting renewability and biodegradability of the sulfonated cellulose nanowhisker, these PMMA@sulfonated CNW composite microsphere could find great potential application in food, cosmetics, drug delivery, and biological tissue engineering, etc.

Elastic liposome-based gel for topical delivery of 5-fluorouracil: in vitro and in vivo investigation

Objective: This investigation has focused to characterize the elastic liposome containing 5-fluorouracil (5-FU) and to enhance drug permeation across stratum corneum (SC) of the skin (rat) using various surfactants and in vivo dermal toxicity evaluation.Methodology: 5-FU-loaded elastic liposomes were developed, prepared and characterized for their entrapment efficiency, vesicle size, number of vesicles, morphological characteristics, surface charge and turbidity. In vitro drug release profile, in vitro skin permeation potential and in vitro hemolytic ability of the formulation have been evaluated to compare with drug solution for 24 h. In vitro skin permeation potential was also compared with marketed cream. Furthermore, in vivo skin irritation potential, drug penetration into the skin using confocal laser scanning microscopy (CLSM) and in vivo toxicity studies were performed.Results and conclusions: The optimized elastic liposomes demonstrated maximum drug entrapment efficiency, optimum vesicular size and considerable elasticity. In vitro skin permeation studies showed the highest drug permeation flux like 77.07 ± 6.34, 89.74 ± 8.5 and 70.90 ± 9.6 µg/cm2/h for EL3-S60, EL3-S80 and EL3-T80, respectively, as compared to drug solution (8.958 ± 6.9 µg/cm2/h) and liposome (36.80 ± 6.4 µg/cm2/h). Drug deposition of optimized elastic liposome EL3-S80 was about three fold higher than drug solution. Skin irritation and CLSM studies suggested that optimized gel was free from skin irritation and capable to deliver 5-FU into the epidermal area for enhanced topical delivery than drug solution. The in vitro study showed minimum hemolysis in the optimized formulation. Finally, in vivo toxicity studies followed with hisptopathological assessment showed that elastic liposome was able to extract SC to improve drug permeation without changing general anatomy of the skin.