Application of calcium oxide as an efficient phase separation agent in temperature-induced counter-current homogenous liquid-liquid extraction of aflatoxins from dried fruit chips followed by high-performance liquid chromatography-tandem mass spectrometry determination.

A temperature-induced counter-current homogenous liquid-liquid extraction procedure performed in a burette has been proposed for the isolation of aflatoxins B1, B2, G1, and G2 from various fruit chip samples. In this method, a homogenous solution of deionized water and cyclohexylamine is added to the solid sample and the resulted mixture is vortexed. In the following, the liquid phase is taken and passed through the burette filled with a mixture of calcium oxide (as a phase separation agent) and sand (to avoid clumping the calcium oxide). By doing so, the temperature of the solution is increased by hydration of calcium oxide and consequently, the homogenous state is broken and the aflatoxins are migrated into the resulted tiny droplets of cyclohexylamine. This phase is collected on the top of the solution owing to its low density with respect to an aqueous solution. Numerous parameters which can affect the efficiency of the suggested approach were evaluated and under the best situations, great repeatability, low limits of determination and quantification, and high extraction recoveries were acquired. In the end, the suggested approach was employed for the quantification of the selected aflatoxins in various fruit chips samples marketed in Tabriz City, Iran.

The anti-biofilm activity of hydrogen peroxide against Escherichia coli strain FL-Tbz isolated from a pharmaceutical water system.

Biofilms are considered a significant reason for the failure of disinfection strategies in industrial water systems due to their resistance to antimicrobial agents. This study is designed to investigate the anti-biofilm activity of hydrogen peroxide (H2O2) at combinations of temperatures and contact times. For this purpose, an in vitro microtiter plate (MTP)-based model system was used for biofilm formation using Escherichia coli (E. coli) strain FL-Tbz isolated from the water system of a pharmaceutical plant. To investigate the anti-biofilm activity of H2O2, it was added at different concentrations (2-7% v/v) to biofilms and incubated at different temperatures (20-60 °C) for 10-40 min to find effective conditions to eradicate biofilms. Maximum biofilms were formed when bacterial suspensions were incubated at 37 °C for 96 h. The rate of biofilm formation using an environmental isolate was higher than that of standard strain. H2O2 at concentrations of ≥6.25% (v/v) at temperatures of ≥40 °C incubated for ≥25 min significantly eradicated the biofilms.

Direct quantitative detection of host cell residual DNA in recombinant Filgrastim by qPCR.

Host cell residual DNA is considered as an impurity in recombinant biopharmaceuticals. This study aimed to develop a direct qPCR method to quantify E. Coli residual DNA in recombinant Filgrastim. The specific primers were designed to amplify E. Coli's 16S-rDNA genomic region, which encodes the 16S-rRNA. The developed qPCR method showed that the designed primer has specifically amplified the target genome without any secondary reaction. The designed primer was also able to amplify the target gene as a representative of residual DNA in the drug matrix. Results show that the amount of residual DNA in Filgrastim is undetectable.

Combination of solvent extraction with deep eutectic solvent based dispersive liquid-liquid microextraction for the analysis of aflatoxin M1 in cheese samples using response surface methodology optimization.

A new extraction procedure based on combination of a solvent extraction and deep eutectic solvent based dispersive liquid-liquid microextraction has been introduced for the extraction of aflatoxin M1 from cheese samples. In this method, acetonitrile, deionized water, and n-hexane are added onto the sample and vortexed. Owning to different affinities of the substances in cheese toward the mentioned solvents, an efficient and selective extraction of the analyte is done in the acetonitrile phase. After centrifugation, the acetonitrile phase is removed and mixed with a new hydrophobic deep eutectic solvent prepared from N,N-diethanol ammonium chloride and carvacrol as an extraction solvent. The mixture is injected into deionized water, and a cloudy solution is obtained. Eventually, an aliquot of the organic phase is injected into high-performance liquid chromatography-fluorescence detection. After optimizing the effective parameters with the response surface methodology and a quadratic model, limits of detection and quantification were 0.74 and 2.56 ng/kg, respectively. The obtained extraction recovery and enrichment factor were 94% and 94, respectively. Also, intra- (n = 6) and interday (n = 4) precisions were less than or equal to 8.6% at a concentration of 5 ng/kg. The suggested method was applied to determine aflatoxin M1 in different cheese samples.

Preparation and in vivo evaluation of in situ gel system as dual thermo-/pH-responsive nanocarriers for sustained ocular drug delivery.

OBJECTIVE: Ciprofloxacin (CIP) was effective in treating bacterial keratitis. The purpose of this study was to prepare an effective prolonged-release of CIP by both temperature and pH-triggered in situ nanogels for the treatment of keratitis. MATERIALS AND METHODS: Poly(N-isopropylacrylamide-methacrylicacide-vinylpyrrolidone) [P (NIPAAm-MAA-VP)] nanoparticles was synthesised and used for preparation of CIP-loaded nanogels. Antimicrobial and in vivo animal studies of the CIP-loaded nanoformulation were performed. RESULTS: Nanoformulation with a mean particle size between 10 and 50 nm and higher than 95% encapsulation efficiency was obtained. Ciprofloxacin released from the nanoparticles showed an enhanced antibacterial effect as determined by minimal inhibitory concentrations. In vivo studies demonstrated reasonable efficacy in severe keratitis using the developed nanoformulation. CONCLUSIONS: Nanoformulation had acceptable efficacy in treating bacterial keratitis in an animal model. Therefore, the developed system has the potential to be used in localised application for the treatment of keratitis.

Intraperitoneal delivery of nanoparticles for cancer gene therapy.

Nanoparticle-based gene delivery systems may be more efficient for administration of therapeutic genes to solid tumors and cancer metastases, owing to the numerous advantages in terms of enhanced tissue penetrability, improved cellular uptake and targeted gene delivery to the cells of interest compared with other gene delivery systems. Intraperitoneal (IP) delivery of therapeutic agents offers special merits because of the anatomical situation of peritoneum for local cancer therapy. Via the IP administration route, it is possible to target the therapeutic agents exactly to the target cells and protect healthy tissues outside the peritoneal cavity from side effects. IP delivery could be applicable for the treatment of disorders of organs in the peritoneal cavity covered with peritoneum and subperitoneal connective tissue, including cancers such as ovarian and gastric. The goal of this article is to review the current state of IP delivery of nanoparticles for cancer gene therapy.

Preparation and characterization of gelatin nanoparticles containing pDNA encoding IL-12 and their expression in CT-26 carcinoma cells.

AIM: Gelatin as a biodegradable, nontoxic and biocompatible natural protein is a good candidate for gene delivery. In this study, pDNA-loaded gelatin nanoparticles were prepared and characterized for the expression of the cytokine IL-12 and anti-tumor effects. MATERIALS & METHODS: Gelatin-pUMVC3-hIL-12 nanoparticles were prepared by the ethanol precipitation technique and evaluated for physicochemical characteristics, cytotoxiciy and transfection efficiency. RESULTS: The prepared particles were spherical in shape with sizes varying from 344.27 to 826.23 nm, ζ-potentials between -944 and -165 mV, and greater than 97% encapsulation efficiency. The particles were nontoxic to CT-26 carcinoma cells. The nanoparticles prepared using 0.5% gelatin solution (G14) with a mean particle size of 816.87 nm (polydispersity index = 0.56 ± 0.01) demonstrated maximum transfection efficiency with 2.5-times higher expression compared with the naked plasmid. CONCLUSION: Gelatin-DNA nanoparticles using 0.5% gelatin solution had minimal cytotoxicity and can be used as a suitable candidate for further gene delivery studies and applications.

Prevalence of bacteriocin activities and bacteriocin-encoding genes in enterococcal clinical isolates in Iran.

The aim of the project was to isolate and characterize bacteriocin-producing enterococci, as well as determine the prevalence of enterocin structural genes in 187 enterococcal clinical isolates from the northwest of Iran. The isolates were screened for antibacterial activity against 15 different indicator strains. The proteinaceous nature of the antimicrobial substances was confirmed by sensitivity to proteinase K; their stability to heat treatment was tested at 60 °C and 100 °C for 20 and 10 min, respectively. The PCR method was applied to detect previously identified enterocin genes. Our results showed that 38 (20.3%) of the enterococcal isolates were considered to be potential bacteriocinogenic strains. Furthermore, genes encoding diverse bacteriocin are highly distributed among clinical enterococci, and the strains with multi-bacteriocin genes displayed high antimicrobial activity. Enterocin A, enterolysin A, and enterocin L50A/B were the most abundant structural genes detected in bacteriocinogenic strains. This work is the first survey on the prevalence of bacteriocin genes among clinical enterococci in Iran that has isolated a strain with high antimicrobial activity and sensitivity to clinically relevant antibiotics.

Preparation and characterization of alginate and psyllium beads containing Lactobacillus acidophilus.

This paper describes preparation and characterization of beads of alginate and psyllium containing probiotic bacteria of Lactobacillus acidophilus DMSZ20079. Twelve different formulations containing alginate (ALG) and alginate-psyllium (ALG-PSL) were prepared using extrusion technique. The prepared beads were characterized in terms of size, morphology and surface properties, encapsulation efficiency, viabilities in acid (pH 1.8, 2 hours) and bile (0.5% w/v, 2 hours) conditions, and release in simulated colon pH conditions. The results showed that spherical beads with narrow size distribution ranging from 1.59 ± 0.04 to 1.67 ± 0.09 mm for ALG and from 1.61 ± 0.06 to 1.80 ± 0.07 mm for ALG-PSL with encapsulation efficiency higher than 98% were achieved. Furthermore, addition of PSL into ALG enhanced the integrity of prepared beads in comparison with ALG formulations. The results indicated that incorporation of PSL into alginate beads improved viability of the bacteria in acidic conditions as well as bile conditions. Also, stimulating effect of PSL on the probiotic bacteria was observed through 20-hour incubation in simulated colonic pH solution. According to our in vitro studies, PSL can be a suitable polymer candidate for partial substitution with ALG for probiotic coating.

Safety issues of nanomaterials for dermal pharmaceutical products.

Nanomaterials (NMs) have favorable application in the medicine area, specifically in regard to the carry of pharmaceutical ingredients to provide targeted drug delivery systems. The skin is an excellent route for the delivery of pharmaceutical nano-transporters for skin-related applications. The physicochemical properties of nanomaterials such as size, hydrophobicity, loading capacity, charge and weight are vital for a skin penetrating system. Many nanocarriers such as polymeric nanoparticles, inorganic nanomaterials and, lipid nanostructures have been utilized for dermal delivery of active ingredients and others such as carbon nanotubes and fullerenes require more examination for future application in the skin-related area. Some negative side effects and nano-cytotoxicity of nanomaterials require special attention while investigating different nanomaterials for medicinal applications. Then, in the current review, we had a view on the safety issues of nanomaterials for dermal pharmaceutical products.

Targeting Multidrug Resistance With Antimicrobial Peptide-Decorated Nanoparticles and Polymers.

As a category of small peptides frequently found in nature, antimicrobial peptides (AMPs) constitute a major part of the innate immune system of various organisms. Antimicrobial peptides feature various inhibitory effects against fungi, bacteria, viruses, and parasites. Due to the increasing concerns of antibiotic resistance among microorganisms, development of antimicrobial peptides is an emerging tool as a favorable applicability prospect in food, medicine, aquaculture, animal husbandry, and agriculture. This review presents the latest research progress made in the field of antimicrobial peptides, such as their mechanism of action, classification, application status, design techniques, and a review on decoration of nanoparticles and polymers with AMPs that are used in treating multidrug resistance. Lastly, we will highlight recent progress in antiviral peptides to treat emerging viral diseases (e.g., anti-coronavirus peptides) and discuss the outlook of AMP applications.

Dispersive micro-solid-phase extraction of aflatoxins from commercial soy milk samples using a green vitamin-based metal-organic framework as an efficient sorbent followed by high performance liquid chromatography-tandem mass spectrometry determination.

In the current work, for the first time, a vitamin-based metal-organic framework constructed from cobalt ions and vitamin B3 has been used as a sorbent in dispersive micro-solid-phase extraction. The proposed method was used to extract and enrich aflatoxins (B1, B2, G1, and G2) from soy milk samples before their quantification by high performance liquid chromatography-tandem mass spectrometry. In this work, first the metal-organic framework was synthesized and characterized using techniques such as X-ray diffraction, Fourier transform infrared spectrophotometry, scanning electron microscopy, nitrogen adsorption/desorption, and energy-dispersive X-ray spectroscopy. Then it was used as an efficient sorbent in the proposed dispersive micro-solid-phase extraction. For this purpose, after precipitating the proteins of soy milk sample with the aid of trichloroacetic acid, the supernatant phase was taken, mixed with the synthesized sorbent, and vortexed. After centrifuging, the analytes loaded on the adsorbent were eluted with methanol to transfer them into an organic phase which was compatible with the subsequently employed separation system. The adsorption capacity of the synthesized MOF for aflatoxins B1, B2, G1, and G2 were 0.77, 0.83, 0.70, and 0.54 mg g-1, respectively. Under the best experimental situations, satisfactory outcomes including acceptable extraction recoveries (64-75%), low limits of detection (11.3-48.2 ng L-1) and quantification (42.8-161.6 ng L-1), and good repeatability (relative standard deviations equal or less than 4.0 and 4.7% for intra- and inter- day precisions, respectively) were obtained. In addition, green synthesis of the metal-organic framework (using vitamin B3 as a linker, water as the reaction solvent, and mild conditions) and usage low amount or volume of the adsorbent and organic solvents during the extraction process were the other beneficial aspects of this work which caused the suggested analytical method to be environmentally friendly.

A novel method for the development of plasmid DNA-loaded nanoliposomes for cancer gene therapy.

We aimed to develop a simple yet novel method to prepare plasmid DNA-loaded nanoliposomes for cancer gene therapy. Murine interleukin-12 (mIL-12) pDNA-loaded nanoliposomes were prepared via novel freeze-drying of a monophase solution method. The physicochemical characteristics, cytotoxicity, and transfection efficiency of the prepared nanoliposomes in murine CT-26 colon carcinoma cells were evaluated. Furthermore, tumor progression and survival rate in CT-26 colon carcinoma-bearing BALB/c mice subsequent to direct intratumoral injections were investigated over a period of 40 days. Using this preparation method, nanoliposomes with particle size of around 300 nm and zeta potential of 96.5 mV were obtained. The transmission electron microscope results showed that the liposomes were nano-sized and almost spherical. The agarose gel retardation assay revealed the pDNA encapsulation in the nanoliposomes. The nanoliposomes with 72.4% encapsulation efficiency and low cell toxicity could significantly improve mIL-12 expression by approximately 25-fold relative to the naked mIL-12 pDNA. There was a significant tumor growth inhibition after repeated injections of mIL-12 pDNA-loaded nanoliposomes. This is the first study on the freeze-drying of a monophase solution method as a simple yet novel technique for the preparation of pDNA-loaded nanoliposomes. Given the ease of preparation method and promising in vitro and in vivo characteristics, this investigation demonstrates advances in pDNA lipid formulation for cancer gene therapy.

Preparation of chitosan-plasmid DNA nanoparticles encoding interleukin-12 and their expression in CT-26 colon carcinoma cells.

PURPOSE: Interleukin-12 (Il-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Non-viral gene delivery systems exhibit low toxicity and are easier to prepare compared to their viral counterparts. In this study, we aimed to prepare plasmid DNA loaded chitosan nanoparticles for expression of Il-12 and to evaluate their physicochemical characteristics, cytotoxicity and transfection efficiency in Murine CT-26 colon carcinoma cells. METHODS: Nanoparticles were prepared using a complex coacervation process at different N/P ratios and characterized in terms of size, zeta potential, polydispersity index, morphology, encapsulation efficiency and polyplex formation. The cytotoxicities and transfection efficiencies of the prepared polyplexes were evaluated by MTT assay and ELISA (for hIL-12, p70), respectively. RESULTS: Size and zeta potential varied from 76.73 to 867.03 nm and between 5.68 and 16.77 mV, respectively. Strong attachment of the DNA to chitosan was observed after polyplex preparation. Encapsulation efficiencies were high (72.97-94.87%). The transfection efficiencies of the prepared complexes were obviously higher than those of naked pDNA when N/P ratios were between 16 and 60. Maximum level of phIL-12 expression was obtained at (N/P = 16) with mean particle size of 381.83±82.77 nm (polydispersity index=0.44) indicating the improved transfection of pUMVC3-hIL12 about 2.80 times compared to that of the naked pUMVC3-hIL12. Prepared polyplexes were nontoxic to CT-26 cells. CONCLUSIONS: Chitosan-DNA nanoparticles at N/P = 16 with minimal cytotoxicity, can be used as suitable candidate for Il-12 delivery. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Effect of two prophylaxis methods on adherence of Streptococcus mutans to microfilled composite resin and giomer surfaces.

OBJECTIVES: Surface attributes of a restoration play an important role in adherence of plaque bacteria. Prophylaxis methods may be involved in modification of or damaging the restoration surface. The aim of the present study was to evaluate the effect of two prophylaxis methods on adherence of Streptococcus mutans to the surface of two restorative materials. STUDY DESIGN: A total of 60 specimens were prepared from each material; a microfilled composite resin (HelioProgress) and a giomer (Beautifil II). For each material, the specimens were randomly divided into three groups (n=20). Group 1: no prophylaxis treatment (control); Group 2: prophylaxis with pumice and rubber cup; Group 3: prophylaxis with air-powder polishing device (APD). The surfaces of selected specimens from each group were evaluated under a scanning electron microscope (SEM), and the surface topography formed by the two prophylaxis methods was determined by atomic force microscopy (AFM). Adherence of Streptococcus mutans to the surface of specimens was determined by the plate counting method following immersion in a bacterial innoculum for 4 hours, rinsing and sonication. Data were analyzed by two-way ANOVA and post hoc Tukey test for multiple comparisons. Statistical significance was set at P<0.05. RESULTS: Bacterial adherence was significantly affected by both factors: restorative material type and prophylaxis method (P<0.0005). Mean bacterial adhesion was significantly higher in composite groups compared to corresponding giomer groups. Within each material, bacterial adherence was significantly lower in the control group compared to prophylaxis groups. Prophylaxis with pumice and rubber cup resulted in a significantly lower bacterial adherence compared to prophylaxis with APD. CONCLUSIONS: Based on the results of the present study, giomer specimens demonstrated lower bacterial adherence compared to composite resin specimens. In both materials, the highest bacterial adherence was observed with prophylaxis with APD, pumice and rubber cup and the control group, respectively.

A minireview on nanoparticle-based sensors for the detection of coronaviruses.

Coronaviruses (CoVs) are a class of viruses that cause respiratory tract infections in birds and mammals. Severe acute respiratory syndrome and Middle East respiratory syndrome are pathogenic human viruses. The ongoing coronavirus causing a pandemic of COVID-19 is a recently identified virus from this group. The first step in the control of spreading the disease is to detect and quarantine infected subjects. Consequently, the introduction of rapid and reliable detection methods for CoVs is crucial. To date, several methods were reported for the detection of coronaviruses. Nanoparticles play an important role in detection tools, thanks to their high surface-to-volume ratio and exclusive optical property enables the development of susceptible analytical nanoparticle-based sensors. The studies performed on using nanoparticles-based (mainly gold) sensors to detect CoVs in two categories of optical and electrochemical were reviewed here. Details of each reported sensor and its relevant analytical parameters are carefully provided and explained.

Safety and Toxicity Issues of Therapeutically Used Nanoparticles from the Oral Route.

The emerging science of nanotechnology sparked a research attention in its potential benefits in comparison to the conventional materials used. Oral products prepared via nanoparticles (NPs) have garnered great interest worldwide. They are used commonly to incorporate nutrients and provide antimicrobial activity. Formulation into NPs can offer opportunities for targeted drug delivery, improve drug stability in the harsh environment of the gastrointestinal (GI) tract, increase drug solubility and bioavailability, and provide sustained release in the GI tract. However, some issues like the management of toxicity and safe handling of NPs are still debated and should be well concerned before their application in oral preparations. This article will help the reader to understand safety issues of NPs in oral drug delivery and provides some recommendations to the use of NPs in the drug industry.

Application of new N- and S-doped amorphous carbon in D-µSPE and its combination with deep eutectic solvent-based DLLME for the extraction of some mycotoxins from soymilk.

A new amorphous carbon-based dispersive micro solid-phase extraction method was developed for the extraction and preconcentration of several mycotoxins from soymilk samples. The extracted analytes were concentrated by a deep eutectic solvent-based dispersive liquid-liquid microextraction method, and then, quantified by a high-performance liquid chromatography-fluorescence detector. The sorbent was prepared from ß-cyclodextrin and methionine under mild conditions. The sorbent was doped by N and S, which improved its physicochemical properties. The optimization and validation of the method were performed using the "one-variable-at-a-time" method and International Council Harmonization guideline, respectively. Under the optimal conditions, low limits of detection and quantifications in the ranges of 0.08-0.56 and 0.27-1.9 ng L-1 were obtained, respectively. Also, intra- (n = 6) and inter-day (n = 6) precisions showed an acceptable repeatability of the present work as they were in the ranges of 3.9-6.2 and 4.6-8.9% at a concentration of 3 ng L-1 of each analyte, respectively. Finally, the proposed method was performed on different soymilk samples marketed in Tabriz city, and aflatoxin B1 was found in all samples. One soymilk was contaminated by ochratoxin A.

Air-assisted liquid-liquid microextraction of total 3-monochloropropane-1,2-diol from refined edible oils based on a natural deep eutectic solvent and its determination by gas chromatography-mass spectrometry.

In this paper, a fast, sensitive, and selective sample preparation procedure was presented for the determination of 3-monochloropropane-1,2-diol (3-MCPD) in refined edible oils using gas chromatography-mass spectrometry. In this method, firstly, the sample lipids and analyte fatty esters are saponified by sodium hydroxide under sonication. After that the analyte was derivatized using phenylboronic acid (as the derivatization agent) and the obtained derivative was extracted during an air-assisted liquid-liquid microextraction procedure (AALLME). Six different deep eutectic solvents (DESs) were prepared as the extraction solvents and the most effective extraction for 3-MCPD was obtained in the presence of a natural DES (NDES) consisting of choline chloride (ChCl)-acetic acid (AcOH). Important variables such as sodium hydroxide concentration and volume, sonication time, temperature, extraction solvent type and volume, and phenylboronic acid concentration and volume have been optimized. Using the optimum conditions, broad linear range (0.88-1000 ng g-1), suitable coefficient of determination (0.995), and low limits of detection (0.26 ng g-1) and quantification (0.88 ng g-1) were obtained. Relative standard deviations for intra- (n=8) and inter-day (n=6) precisions at a concentration of 5 ng g-1 were 2.6 and 3.2%, respectively. The developed method has been successfully applied to 3-MCPD determination in refined edible oil samples including sunflower, corn, and canola oils.

Osteogenic Differentiation of Mesenchymal Stem Cells via Curcumin-Containing Nanoscaffolds.

The diverse pleiotropic pharmacological effects of curcumin nanoformulations have turned it into an attractive natural compound in different health-related problems. A great body of evidence has shown the impact of curcumin and its nanoformulations on the differentiation of stem cells. The current review highlights cellular and molecular mechanisms connected with the osteogenic differentiation of mesenchymal stem cells (MSCs) in the scaffolds benefiting from the presence of nanocurcumin pointing toward the role of inhibitory or stimulant signal transduction pathways in detail. Moreover, the effects of different concentrations as well as the structural modifications of curcumin on the differentiation of MSCs have been addressed.