Andrographolide-loaded ethosomal gel for transdermal application: Formulation and in vitro penetration study

Background: Andrographolide is a phytoconstituent with anti-inflammatory activity, however, the compound’s poor oral bioavailability has hindered its effective formulation for oral administration. This study, therefore, aims to develop an ethosome for improving andrographolide penetration through the transdermal delivery system. Methods: This study developed 3 ethosome formulas with different andrographolide-phospholipid weight ratios (1:8, 1:9; 1:10), using the thin-layer dispersion-sonication method. Subsequently, the ethosomes were evaluated for particle size, polydispersity index, zeta potential, morphology, as well as entrapment efficiency, and incorporated into a gel dosage form. Subsequently, an in vitro penetration study was performed using Franz diffusion cells for 24 hours and the stability of the gels at 5 ± 2°C, 30 ± 2°C, and 40 ± 2°C, were studied for 3 months. Results: The results showed the optimal formula was E2, a 1:9 weight ratio formula of andrographolide and phospholipid. Based on the transmission electron micrograph, E2 possessed unilamellar, as well as spherical-shaped vesicles, and exhibited superior characteristics for transdermal delivery, with a particle size of 89.95 ± 0.75 nm, polydispersity index of 0.254 ± 0.020, a zeta potential of -39.3 ± 0.82 mV, and entrapment efficiency of 97.89 ± 0.02%. Furthermore, the cumulative andrographolide penetration and transdermal flux for the ethosomal gel of E2 (EG2) were 129.25 ± 4.66 µg/cm2 and 5.16 ± 0.10 µg/cm2/hours, respectively. All the ethosomal gel formulations exhibited improved penetration enhancement of andrographolide, compared to the nonethosomal formulations. Also, the andrographolide levels in the ethosomal and nonethosomal gels after 3 months ranged from 98.13 to 104.19%, 97.93 to 104.01%, and 97.23 to 102.26% at storage temperatures of 5 ± 2°C, 30 ± 2°C/RH 65% ± 5%, and 40 ± 2°C/RH 75% ± 5%, respectively. Conclusions: This study concluded that encapsulation into ethosome enhances andrographolide delivery through the skin.

Isolation and Identification of a Siphoviridae Bacteriophage Targeting Multidrug-resistant Klebsiella pneumoniae

Background: Based on the WHO, multidrug-resistant Klebsiella pneumoniae is a priority pathogen that causes opportunistic infections and is widely spread in the environment. Phage therapy is considered a natural, safe, and very efficient alternative to treat difficult-to-treat infections. Objectives: This study aimed to isolate highly virulent, lytic bacteriophages and evaluate their efficacy for lysing multidrug-resistant K. pneumoniae. Methods: Municipal wastewater samples were collected and filtered using 0.22 µm syringe filters and cultivated with log-phase cultures of K. pneumoniae using enrichment media. After 48 h of incubation, the cultures were centrifuged, and the resultant supernatant was filtered (0.22 µm). The detection of the phage was done using the spot assay with K. pneumoniae as the host. One-step growth kinetics and bacterial reduction tests were conducted to assess the growth kinetics of the isolated phage. The stability of the isolated phage was characterized by subjecting it to various temperature and pH conditions. The chemical stability of the K. pneumoniae phage was determined by exposing it to various organic compounds. A panel of 20 bacterial strains was tested using the spot assay, as well as double agar overlying assay, to determine the host range of the isolated phage. Results: Out of 40 wastewater samples tested, only one sample was tested positive for the K. pneumoniae phage (2.5%) that was lytic against the host strain. The K. pneumoniae phage had a latent period of 15 min and a burst size of 100 virions per infected cell. It was most stable at 37°C and pH range of 6.0 to 10.0. Chemically, the K. pneumoniae phage was resistant to 10% chloroform treatment. Transmission electron micrograph indicated that the K. pneumoniae phage belonged to the order Caudovirales, family Siphoviridae, morphotype B1. Conclusions: Most of the characteristic features of the K. pneumoniae phage indicated the potential of this phage to be used in phage therapy. Hence, a comprehensive study is highly recommended to characterize the K. pneumoniae phage genome, detect its molecular interactions with the host cell, and determine its lytic activity in combination with other phages, which may lead to the efficient utilization of this phage in phage therapy against K. pneumoniae infections.

Atomic Layer Deposition of Nanolayered Carbon Films

In this paper, carbon thin films were grown using the plasma-enhanced atomic layer deposition (PE-ALD). Methane (CH4) was used as the carbon precursor to grow the carbon thin film. The grown film was analyzed by the high-resolution transmission electron micrograph (TEM), X-ray photoelectron spectroscopy (XPS) analysis, and Raman spectrum analysis. The analyses show that the PE-ALD-grown carbon film has an amorphous structure. It was found that the existence of defective sites (nanoscale holes or cracks) on the substrate of copper foil could facilitate the formation of nanolayered carbon films. The mechanism for the formation of nanolayered carbon film in the nanoscale holes was discussed. This finding could be used for the controlled growth of nanolayered carbon films or other two-dimensional nanomaterials while combining with modern nanopatterning techniques.

One Pot Synthesis, Surface and Magnetic Properties of Cu2O/Cu and Cu2O/CuO Nanocomposites

A series of copper-based systems containing two different nanocomposites (Cu2O/CuO and Cu2O/Cu) was synthesized by the egg white assisted auto-combustion route. This method was distinguished by the simplicity of its steps, low cost, one-pot synthesis process at low temperature and, short time. The characterization of the as prepared nanocomposites was carried out by using X-ray diffraction (XRD), Fourier-transform infrared (FTIR), Scanning electron microscope (SEM) and transmission electron micrograph (TEM), Energy dispersive spectrometry (EDS) techniques. Surface and magnetic properties of the obtained systems were determined by using N2 adsorption/desorption isotherms at 77 K and the vibrating sample magnetometer (VSM) technique. XRD results confirmed the formation of Cu2O/CuO and Cu2O/Cu nanocomposites with different ratios of well crystalline CuO, Cu2O, and Cu phases. FTIR results of the combusted product displays the presence of both CuO and Cu2O, respectively. SEM/EDS and TEM results confirm the formation of a porous nanocomposite containing Cu, O, and C elements. The change in concentration of the oxygen vacancies at the surface or interface of both Cu2O/CuO and Cu2O/Cu nanoparticles resulted in different changes in their magnetization. Based on this study, it is possible to obtain nanocomposite-based copper with multiple valances by a simple and inexpensive route which can be suitable for the fabrication of different transition metal composites.

Process Parameters Study on Structure and Properties of Zr-Based Bulk Metallic Glasses

The effects of different process parameters on the microstructure and mechanical properties of Zr-based bulk metallic glass were studied by using four factors and four levels of orthogonal experiment. Through the observation and analysis of the X-ray diffraction pattern, room temperature compressibility, microhardness distribution and transmission electron micrograph of the [Zr0.72-x(Cu0.59Ni0.41)0.28+x]88-yAl12+y (atomic percent) amorphous alloy with different process parameters, the results show that the compressive strength of the amorphous alloys with composition of [Zr0.73(Cu0.59Ni0.41)0.27]88Al12 and [Zr0.73(Cu0.59Ni0.41)0.27]87Al13 amorphous alloy under the corresponding processing parameters has reached 2411MPa and 1993MPa, and the plastic strain of the alloys is 33.8% and 19.3%. At the same time, through orthogonal analysis, in the four process parameters, the superheat temperature of the melt during the preparation of the alloy mainly affects the compressive strength of the alloy, while the cooling rate of the melt during the preparation of the alloy has a great influence on the plastic properties of the alloy.

Optimization of protocols for generation of insulin-producing cells from human embryonic stem cells using small molecules

The differentiation of human embryonic stem cells (hESCs) into β-cells has potential uses for cell therapy in diabetes. There were massive reported protocols which can be used to generate insulin-producing cells (IPCs) from hESCs, and most of these methods were using recombinant growth factors. However, small molecules can coax ES cells towards pancreatic fate, and could substitute recombinant proteins. To obtain an unbiased characterization, we performed a comparative study with the recombinant protein induction protocol and the small molecule induction protocol and found that the small molecule induction protocol could better inhibit the differentiation of cells into hepatic lineage cells than the recombinant protein induction protocol. On this basis, we further optimized and established an efficient small molecule differentiation system, and the optimized protocol could better inhibit the differentiation of pancreatic exocrine glands. The differentiated hESCs obtained by our optimized protocol comprised approximately 32% IPCs as assayed by flow cytometry analysis. Transmission electron micrograph demonstrated that IPCs obtained in optimized small molecule induction protocol had more and larger secretory granules than those in induction protocol before optimization.

Polyaniline modified silica gel coupled with green solvent as eco favourable mobile phase in thin layer chromatographic analysis of organic dyes

This article studies a new green eco-friendly TLC (thin layer chromatography) using silica gel and polyaniline modified silica gel as stationary phase in combination with ethyl acetate (EA), n-butyl acetate (BA) and butane-1-ol (BO) solutions as mobile phase for the comparative study of migration behaviour of organic dyes to identify the most suitable thin layer chromatographic system for the resolution of co-existing dyes. Better separation efficiency was observed by modifying silica gel with polyaniline as compared to pure silica stationary phase. Densitogrpahic presentation of separations achieved on polyaniline modified silica gel Pani@SG-EB1 was also presented. The thin layer chromatographic system comprising of polyaniline modified silica gel Pani@SG-EB1 as stationary phase and n-butyl acetate:DDW, 5:5 as green mobile phase was observed to be the most favourable for the separation of various combinations of three or four-component mixtures of organic dyes viz. methyl thymol blue, tartrazine, carmoisine, rose bengal, amidoblack 10B, bromopyrogallol red and 4-nitrobenzene dizonium tetrafluoroborate. The effect of presence of cations and anions on separation trend was also examined and the limits of detection of the separated organic dyes were estimated. Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron micrograph (TEM) studies were undertaken to characterize silica gel and modified silica gel (stationary phase). The developed method has been successfully applied for the identification of carmoisine in Solvin cold DS syrup and tartrazine in MefastTM syrup.

Impaired Hepcidin Metabolism Promotes Hemolysis Induced Hepatobiliary Injury in Sickle Cell Disease

Sickle cell disease (SCD) is an autosomal-recessive-genetic disorder that affects millions of people worldwide. Although hepatic crisis affects 10-40% of hospitalized SCD patients and can progress to fatal liver failure, the current treatment is primarily supportive and the molecular pathophysiology remains largely unknown. We found that transgenic, humanized SCD mice developed liver injury with age, manifested by increased inflammation, necrosis and hepatic iron accumulation. The presence of iron particles in SCD liver was confirmed by transmission electron micrograph (TEM) analysis and prussian blue staining which revealed increased iron accumulation in the central and midzonal region of the SCD liver tissue. An increase in aggregates of iron pigment reminiscent of hemosiderin-laden macrophages was also observed in SCD liver tissue. Interestingly, the SCD mice also showed significant enrichment of both hepatic (p=0.02) and serum iron (p=0.04) compared to control AS mice. We determined the expression level of genes commonly involved in iron homeostasis by RT-PCR. Interestingly, a significantly lower expression level of hepcidin transcripts was observed in the hepatocytes of SCD mice compared to control mice (AS) (p=0.01). In order to define the pathways controllinghepcidintranscription in SCD, we performed an RNA-seq analysis in the liver of SCD mice. Remarkably, our data showed significant misexpression of BMP signaling pathways. Further analysis revealed a significant misexpression in BMP2 and 6 levels in the liver of SCD mice by western blot. Reduced levels of hepcidin were also confirmed in serum samples from SCD patients compared to controls. Work is currently underway to understand how BMP2/6 might hypothetically regulate hepcidin expression in the liver. In summary, our results reveal a significant defect in iron homeostasis in the liver of SCD mice, suggesting that impaired iron homeostasis may contribute to hepatobiliary injury in SCD independent of blood transfusions. Our study also highlights the importance of hepcidin as potential therapeutic target in regulation of hepatic injury in SCD. Disclosures No relevant conflicts of interest to declare.

DEVELOPMENT AND CHARACTERIZATION OF DOXORUBICIN AND siRNA ENCAPSULATED CHITOSAN NANOPARTICLES

Objective: Chitosan nanoparticles (ChNP’s) have been widely studied for drug and gene delivery. In this study, we prepared ChNP’s for co-delivery of doxorubicin (DOX) and siRNA for cancer treatment. Methods: The ionic gelation method was used to develop ChNP’s. The positively charged DOX and negatively charged siRNA encapsulated into ChNP’s. The particle size and zeta potential of the developed ChNP’s were studied by particle size analyzer and morphology was examined by TEM. Encapsulation of DOX in ChNP’s was confirmed by FTIR spectroscopy. The encapsulation efficiency and in vitro release of DOX were studied by UV-Vis spectrophotometry. The siRNA loading into ChNP’s was confirmed by gel retardation assay. Results: The developed ChNP’s showed particle size ranged from 127±6.5 to 215±8.5 nm with zeta potential ranged from 16.5±0.3 to 25.8±0.3. Transmission Electron Micrograph showed DOX and siRNA encapsulated ChNP’s are polydisperse and spherical in nature. FTIR study confirmed the binding of DOX with ChNP’s with absorption peaks at 1016 cm-1,1316 cm-1, 1412 cm-1, 1645 cm-1 and 3370 cm-1. The TPP:Ch ratio 0.1:0.5 showed the highest encapsulation efficiency 69±3.24%, with initial burst release and then sustained or slow release of DOX. Agarose gel retardation study confirmed the encapsulation of siRNA in ChNP’s by retarded migration of siRNA-ChNP’s in comparison with naked siRNA. Conclusion: The developed ChNP’s successfully encapsulated the DOX and siRNA and showed the sustain release of DOX. In conclusion, our study shown that ChNP’s is having a potential of co-loading of DOX-siRNA as an efficient drug delivery system for the treatment of various cancers such as colorectal cancer, breast cancer etc.

Antibacterial mechanism of rhodomyrtone involves the disruption of nucleoid segregation checkpoint in Streptococcus suis

Rhodomyrtone has been recently demonstrated to possess a novel antibiotic mechanism of action against Gram-positive bacteria which involves multiple targets, resulting in the interference of several bacterial biological processes including cell division. The present study aims to closely look at the downstream effect of rhodomyrtone treatment on nucleoid segregation in Streptococcus suis, an important zoonotic pathogen. Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) values of rhodomyrtone against recombinant S. suis ParB-GFP, a nucleoid segregation reporter strain, were 0.5 and 1 µg/ml, respectively, equivalent to the potency of vancomycin. Using fluorescence live-cell imaging, we demonstrated that rhodomyrtone at 2 × MIC caused incomplete nucleoid segregation and septum misplacement, leading to the generation of anucleated cells. FtsZ immune-staining of rhodomyrtone-treated S. suis for 30 min revealed that although large amount of FtsZ was trapped in the region of high fluidity membrane, it appeared to be able to polymerize to form a complete Z-ring. However, the Z-ring was shifted away from midcell. Transmission electron micrograph further confirmed disruption of nucleoid segregation and septum misplacement at 120 min following rhodomyrtone treatment. Asymmetric septum formation resulted in either generation of minicells without nucleoid, septum formed over incomplete segregated nucleoid (guillotine effect), or formation of multi-constriction of Z-ring within a single cell. This finding spotlights on antibacterial mechanism of rhodomyrtone involves the early stage in bacterial cell division process.