A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.

Introduction: Modeling the blood-brain barrier has long been a challenge for pharmacological studies. Up to the present, numerous attempts have been devoted to recapitulating the endothelial barrier in vitro to assess drug delivery vehicles' efficiency for brain disorders. In the current work, we presented a new approach for analyzing the morphometric parameters of the cells of an insert co-culture blood-brain barrier model using rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes. This analytical approach could aid in getting further information on drug trafficking through the blood-brain barrier and its impact on the brain indirectly. Methods: In the current work, we cultured rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes and then used an insert well to culture the cells in contact with each other to model the blood-brain barrier. Then, the morphometric parameters of the porous membrane of the insert well, as well as each cell type were imaged by digital holographic microscopy before and after cell seeding. At last, we performed folate conjugation on the surface of the EVs we have previously tested for glioma therapy in our previous work called VEGF-A siDOX-EVs and checked how the trafficking of EVs improves after folate conjugation as a clathrin-mediated delivery setup. the trafficking and passage of EVs were assessed by flow cytometry and morphometric analysis of the digital holographic microscopy holograms. Results: Our results indicated that EVs successfully entered through the proposed endothelial barrier assessed by flow cytometry analysis and furthermore, folate conjugation significantly improved EV passage through the blood-brain barrier. Moreover, our results indicated that the VEGF-A siDOX-EVs insert cytotoxic impact on the cells of the bottom of the culture plate. Conclusion: folate-conjugation on the surface of EVs improves their trafficking through the blood-brain barrier and by using digital holographic microscopy analysis, we could directly assess the morphometric changes of the blood-brain barrier cells for pharmacological purposes as an easy, label-free, and real-time analysis.

A comprehensive review on methotrexate containing nanoparticles; an appropriate tool for cancer treatment.

For more than seven decades, methotrexate has been used all over the world for treatment of different diseases such as: cancer, autoimmune diseases, and rheumatoid arthritis. Several studies have addressed its formula, efficacy, and delivery methods in recent years. These studies have been focused on the effectiveness of different nanoparticles on drug delivery, delivery of the drug to the target cells, and attenuation of harm to the host cell. Whereas, the main usages of methotrexate are in cancer treatment field, this review provided a brief perspective into using different nanoparticles and their role in the treatment of different cancers.

Clinical theranostics applications of photo-acoustic imaging as a future prospect for cancer.

Photoacoustic imaging (PAI) of biological tissue has been a fast developing biomedical multi-wave imaging modality, after its introduction in the mid90s. PAI couples laser excitation to acoustic detection. Especially, in recent years its significant advantages in onco-surgery has attracted much attention due to its ability to detect malignant tissues. Monitoring cancer angiogenesis, assessment of blood oxygen saturation, functional brain imaging, evaluation of cortical blood volume, detection of skin/conjunctival melanoma depth, assessment of met-hemoglobin, investigating tumor hypoxia andcancer lymph node metastases are some of its promising applications. Moreover, as a real-time monitoring strategy, PAI allows intraoperative imaging of micro-metastases and residual islands in onco-surgery. Herein, we provide a brief introduction to biophysics and fundamentals of PAI, potential novel endogenous and exogenous contrast agents, and novel techniques to develop engineered and targeted contrast agents with theranostic applications. We also summarize the clinical trial pipelines for PAI. Furthermore, we discuss the potential obstacles and limitation of PAI theranostic agents for further clinical applications and strategies to overcome these hurdles.

Next-Generation Anti-Angiogenic Therapies as a Future Prospect for Glioma Immunotherapy; From Bench to Bedside.

Glioblastoma (grade IV glioma) is the most aggressive histopathological subtype of glial tumors with inordinate microvascular proliferation as one of its key pathological features. Extensive angiogenesis in the tumor microenvironment supplies oxygen and nutrients to tumoral cells; retains their survival under hypoxic conditions; and induces an immunosuppressive microenvironment. Anti-angiogenesis therapy for high-grade gliomas has long been studied as an adjuvant immunotherapy strategy to overcome tumor growth. In the current review, we discussed the underlying molecular mechanisms contributing to glioblastoma aberrant angiogenesis. Further, we discussed clinical applications of monoclonal antibodies, tyrosine kinase inhibitors, and aptamers as three major subgroups of anti-angiogenic immunotherapeutics and their limitations. Moreover, we reviewed clinical and preclinical applications of small interfering RNAs (siRNAs) as the next-generation anti-angiogenic therapeutics and summarized their potential advantages and limitations. siRNAs may serve as next-generation anti-angiogenic therapeutics for glioma. Additionally, application of nanoparticles as a delivery vehicle could increase their selectivity and lower their off-target effects.

The bactericidal effect of lysostaphin coupled with liposomal vancomycin as a dual combating system applied directly on methicillin-resistant Staphylococcus aureus infected skin wounds in mice.

BACKGROUND AND AIM: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of surgical infection, and its resistance to numerous conventional antibiotics makes treatment difficult. Although vancomycin is often an effective agent for the initial therapy of MRSA, clinical failure sometimes occurs. Therefore, there is an urgent need to develop better therapies. Here, we prepared some vancomycin-loaded nanoliposomes coupled with anti-staphylococcal protein (lysostaphin) and evaluated their in vitro and in vivo efficacy as a topical MRSA therapy. METHODS: Vancomycin was encapsulated in liposomes, and the coupling of lysostaphin with the surface of liposomes was carried out through cyanuric functional groups. The bactericidal efficacies and a full characterization were evaluated. To define different nanoliposomal-bacterium interactions and their bactericidal effect, flow cytometry was employed. Finally, in vivo, the topical antibacterial activity of each formulation was measured against surgical wound MRSA infection in a mouse model. RESULTS: High encapsulation and conjugation efficiency were achieved for all formulations. All the formulations showed a significant reduction in bacterial counts (p<0.05). The targeted liposomes more effectively suppress bacterial infection in vitro and in vivo relative to equivalent doses of untargeted vancomycin liposome. The flow cytometry results confirmed liposome-bacterium interactions, which increased during the incubation time. The maximum binding rate and the bactericidal effect were significantly higher in targeted liposomes (p<0.05) compared with control liposomes. CONCLUSION: Our data suggest a novel nano-vehicle (lysostaphin-conjugated coupled liposomal vancomycin) which could be used as a great topical antimicrobial construct for treatment of MRSA skin infections.

The bactericidal effect of liposomal vancomycin as a topical combating system against Methicillin-resistant Staphylococcus aureus skin wound infection in mice.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of skin infections and treatment is difficult due to its resistance to the most of antibiotics. Although vancomycin is often considered as an antibacterial agent of choice for the treatment of MRSA, its use is limited because of the high side effects. One solution is using liposomal formulation for local drug delivery. The aim of this study was to determine in vitro and in vivo efficacies of liposomal vancomycin as topical use. Methods: To prepare liposomal vancomycin, the ammonium sulfate gradient using remote loading and freeze-thaw methods was applied. Then, synthesized nanoliposomes were evaluated in terms of particle size, morphology, stability, and encapsulation efficiency. Minimum inhibitory concentration (MIC) of synthesized nanoliposome against MRSA was detected. The cytotoxicity of synthesized nanoliposome was evaluated using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Finally, the topical antibacterial activity of each formulation was tested against MRSA-infected skin wound model in mice. Results: High encapsulation efficiency was achieved for all synthesized nanoliposomes. The results of in vitro and in vivo showed that liposomal vancomycin was more effective than free vancomycin. Also, synthesized nanoliposome showed no cytotoxicity on human epidermoid cell line. Conclusion: The results showed that synthesized nanoliposome could be applied as a great topical antimicrobial construct for treatment of MRSA skin infections.

Detection of Integrons and Staphylococcal Cassette Chromosome mec Types in Clinical Methicillin-resistant Coagulase Negative Staphylococci Strains.

OBJECTIVES: Integrons are thought to play an important role in the spread of antibiotic resistance. This study investigates class 1 and 2 integron-positive methicillin-resistant coagulase-negative staphylococci strains isolated in Iran and characterizes their patterns of antimicrobial resistance. METHODS: Hundred clinical isolates of coagulase-negative staphylococci were characterized for integron content and staphylococcal cassette chromosome mec (SCCmec) type. RESULTS: Sixteen isolates carried class 1 (intI1) integrons and four isolates carried class 2 (intI2) integrons. One resistance gene array was identified among the class 1 integrons (aadA1 cassette). The distribution of SCCmec types in 50 methicillin-resistant coagulase-negative staphylococci strains showed that SCCmec types III and V dominated among the tested strains. CONCLUSION: This is the first report of methicillin-resistant coagulase-negative staphylococci strains that carry two mobile genetic elements, including class 1 and 2 integrons and SCCmec, in Iran.

Multi-drug-resistant enterotoxigenic and enterohemorrhagic Escherichia coli isolated from children with diarrhea.

Multi-drug-resistant (MDR) diarrheagenic Escherichia coli (DEC) has rapidly spread worldwide and represents the most serious threat to the management of diarrhea in developing countries. During the period from March 2011 to January 2012, a total of 450 stool samples of diarrheal children aged 0-60 months were studied. In order to detect enterotoxigenic E. coli (ETEC) and enterohemorrhagic E. coli (EHEC) simultaneously, a mixture of four primer pairs specific for eltB, estA, vt1, and vt2 genes was used in a multiplex PCR. Antimicrobial susceptibility testing was performed as the Clinical and Laboratory Standards Institute (CLSI) guidelines. A total of 140 (31·1%) DEC were isolated from 450 stool samples. Diarrheagenic E. coli exhibited high-level resistance to aztreonam (80·7%), amoxicillin (74·4%), and tetracycline (69·3%). Also, 86·4% of E. coli isolates were resistant to at least three different classes of antimicrobial agents and considered as MDR. The frequency of ETEC and EHEC pathotypes was 46·4 and 12·1%, respectively and all of these isolates were MDR. In conclusion, MDR ETEC continues to be an important agent associated with diarrhea in children from Tabriz, Iran.

Molecular characterization of integrons in clinical isolates of betalactamase-producing Escherichia coli and Klebsiella pneumoniae in Iran.

Integrons are considered to play a significant role in the evolution and spread of antibiotic resistance genes. A total of 349 clinical isolates of Escherichia coli and Klebsiella pneumoniae were investigated for molecular characterization of integrons and betalactamases. Antimicrobial susceptibility testing was also performed as the Clinical and Laboratory Standards Institute (CLSI) guidelines. The frequency of extended spectrum betalactamases (ESBL) or metallo-betalactamases (MBL)-producing isolates, patient demographics, and the susceptibility to various antimicrobial agents were described. BlaCTX-M was the most frequently detected betalactamase in all isolates. Moreover, MBL producing K. pneumoniae carried blaIMP and blaVIM at 100 and 41·6%, respectively but no MBL-positive E. coli was detected. Class 1 integrons were more frequent among E. coli and K. pneumoniae isolates in comparison with class 2 integrons and the frequency of intI2 in K. pneumoniae was significantly higher than E. coli isolates. Five different resistance gene arrays were identified among class 1 integrons. Dihydrofolate reductase (dfrA) and aminoglycoside adenyltransferase (aad) gene cassettes were found to be predominant in the class 1 integrons. These results indicate that class 1 integrons are widespread among ESBL-producing isolates of K. pneumoniae and E. coli and appropriate surveillance and control measures are essential to prevent further dissemination of these elements among Enterobacteriaceae in our country.

Frequency and antimicrobial resistance of diarrhoeagenic Escherichia coli from young children in Iran.

Diarrhoea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. Diarrhoeagenic Escherichia coli (DEC) is an emerging agent among pathogens that cause diarrhoea. Between March 2011 and January 2012, a total of 600 stool specimens from children younger than 5 years of age (450 with and 150 without diarrhoea) were investigated for enteroaggregative E. coli (EAEC), enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli (ETEC) using PCR. Antimicrobial susceptibility testing was performed following Clinical and Laboratory Standards Institute guidelines. The prevalence of DEC pathotypes was 30.4% (137 patients) and 12% (18 patients) in the diarrhoea group and the control group, respectively. The most frequently isolated pathotype in diarrhoeal children was ETEC. This pathotype was detected significantly more often in children with diarrhoea (14.4%) than in children without diarrhoea (5.3%). EAEC and EPEC were detected with slightly higher frequencies in children with (8 and 4.2%, respectively) than in children without (4.6 and 2%, respectively) (P>0.05) diarrhoea. EHEC was only detected in children with diarrhoea (3.8%). Of the children from the diarrhoea group, 10% were colonized with more than one DEC pathotype. The DEC isolates exhibited high-level resistance to erythromycin (100%), azteronam (80.7%), amoxicillin (74.4%) and tetracycline (69.3%), and 86.4% of isolates were multidrug resistant. In conclusion, ETEC continues to be an important agent associated with diarrhoea in children from Tabriz, Iran.