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Rajashekhar Kanchanapally,1 Sachin Kumar Deshmukh,1 Suhash Reddy Chavva,1 Nikhil Tyagi,1 Sanjeev Kumar Srivastava,1 Girijesh Kumar Patel,1 Ajay Pratap Singh,1,2 Seema Singh1,2 1Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA; 2Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA Background: Despite tremendous advancement, cancer still remains one of the leading causes of death worldwide. Inefficiency of current drug delivery regimens is one important factor that limits the therapeutic efficacy of existing drugs, thus contributing to cancer mortality. To address this limitation, synthetic nanotechnology-based delivery systems have been developed; however, they raise concern of inducing adverse immunogenic reactions. Exosomes (Exos) are nonimmunogenic nano-sized vesicles that have received significant attention as efficient drug delivery system. Methods: Drug loading in Exos were achieved by incubating different cell types viz pancreatic cancer cells (PCCs), pancreatic stellate cells (PSCs), and macrophages (MØs) with Doxorubicin (DOX). Differential ultracentrifugation was performed to isolate exosome and their size was determined by dynamic light scattering analysis. The efficacy of drug packaging into Exos was evaluated by HPLC. Flow cytometry was performed to examine the apoptosis. Cell viability was determined using the WST-1 assay.Results: PCCs shed the most Exos and were the most efficient in drug loading followed by MØs and PSCs as examined by HPLC quantification. However, when compared for antitumor efficacy, MØ-derived Exos loaded with DOX (MØ-Exo-DOX) showed highest activity followed by PSCs and PCCs. Conclusion: These varying antitumor activities likely resulted from nondrug contents of Exos since we did not observe any significant differences in their uptake by the cancer cells. Altogether, our data suggest that donor cell-specific differences exist in Exos, which could influence their utility as drug carrier for therapeutic purposes. Keywords: exosome, macrophage, pancreatic cancer, drug delivery, doxorubicin

Rishi P Singh, Sunil K Srivastava, Justis P Ehlers, Fabiana Q Silva, Rumneek Bedi, Andrew P Schachat, Peter K Kaiser Cole Eye Institute, Cleveland Clinic, Cleveland, OH, USA Summary statement: In subjects with active exudative age-related macular degeneration, treating with a fixed intravitreal aflibercept injection dosing regimen for 12 months demonstrated improved anatomic and vision endpoints from baseline.Purpose: Switching therapies in neovascular age-related macular degeneration (AMD) may offer an advantage for some patients. This study evaluates the efficacy of intravitreal aflibercept injection (IAI) in subjects previously treated with ranibizumab and/or bevacizumab.Methods: Subjects (n=26) were given monthly 2 mg of IAI for 3 months, followed by 2 mg once in every 2 months for up to 12 months. The mean absolute change from baseline in central subfield thickness (CST) measured by optical coherence tomography and the mean change from baseline in best-corrected visual acuity (BCVA) early treatment in diabetic retinopathy study (ETDRS) letter score were obtained. Additionally, the percentage of subjects who gained or lost ≥15 letters of vision and the percentage of subjects who are 20/40 or better or 20/200 or worse were evaluated.Results: There was a mean decrease in CST of -50.3 µm (P

Sumit Arora,1 Suresh K Swaminathan,2 Ameya Kirtane,2 Sanjeev K Srivastava,1 Arun Bhardwaj,1 Seema Singh,1 Jayanth Panyam,2 Ajay P Singh1,3 1Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, USA; 2Department of Pharmaceutics, The University of Minnesota, Minneapolis, USA; 3Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile, Alabama, USA Abstract: MicroRNAs are small (18–22 nucleotide long) noncoding RNAs that play important roles in biological processes through posttranscriptional regulation of gene expression. Their aberrant expression and functional significance are reported in several human malignancies, including pancreatic cancer. Recently, we identified miR-150 as a novel tumor suppressor microRNA in pancreatic cancer. Furthermore, expression of miR-150 was downregulated in the majority of tumor cases, suggesting that its restoration could serve as an effective approach for pancreatic cancer therapy. In the present study, we developed a nanoparticle-based miR-150 delivery system and tested its therapeutic efficacy in vitro. Using double emulsion solvent evaporation method, we developed a poly (D,L-lactide-co-glycolide) (PLGA)-based nanoformulation of miR-150 (miR-150-NF). Polyethyleneimine (a cationic polymer) was incorporated in PLGA matrix to increase the encapsulation of miR-150. Physical characterization of miR-150-NF demonstrated that these nanoparticles had high encapsulation efficiency (~78%) and exhibited sustained release profile. Treatment of pancreatic cancer cells with miR-150-NF led to efficient intracellular delivery of miR-150 mimics and caused significant downregulation of its target gene (MUC4) expression. Inhibition of MUC4 correlated with a concomitant decrease in the expression of its interacting partner, HER2, and repression of its downstream signaling. Furthermore, treatment of pancreatic cancer cells with miR-150-NF suppressed their growth, clonogenicity, motility, and invasion. Together, these findings suggest that PLGA-based nanoformulation could potentially serve as a safe and effective nanovector platform for miR-150 delivery to pancreatic tumor cells. Keywords: PLGA nanoparticles, miR-150, MUC4, invasion, migration

Chandravilas Keshvan Prashant,1 Madhusudan Bhat,1 Sandeep Kumar Srivastava,1 Ankit Saxena,2 Manoj Kumar,3 Amar Singh,2 Mohammed Samim,4 Farhan Jalees Ahmad,5 Amit Kumar Dinda1 1Department of Pathology, 2Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, 3Centre for Biomedical Engineering, Indian Institute of Technology, 4Department of Chemistry, Faculty of Sciences, 5Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India Purpose: The aim of the study was to load a model antigen, tetanus toxoid (TT), in poly-ε-caprolactone nanoparticles (PCL NPs) of two size ranges, ie, mean 61.2 nm (small) and 467.6 nm (large), and study its effect on macrophage polarization as well as antigen presentation in human monocyte-derived macrophages in vitro, along with humoral and cell-mediated immune (CMI) response generated in Swiss albino mice following immunization with the TT-loaded NPs. Materials and methods: PCL NPs were synthesized by solvent evaporation. The antigen-loaded PCL NPs were characterized for size, zeta potential, and protein-release kinetics. Swiss albino mice were immunized with the antigen-loaded PCL NPs. Flow cytometry was used to quantify interferon-γ- and interleukin-4-secreting cluster of differentiation (CD)4+ and CD8+ T cells in the spleen, and enzyme-linked immunosorbent assay was used to quantify anti-TT antibody levels in the serum of immunized mice. Results: Small PCL NPs generated an M1/M2 type polarization of human blood monocyte-derived macrophages and T helper (Th)1/Th2 polarization of autologous CD4+ T cells. Efficient CD8+ T-cell responses were also elicited. Large PCL NPs failed to cause any type of macrophage polarization. They did not elicit efficient CD8+ T-cell responses. Conclusion: TT-loaded small PCL NPs were able to generate persistent and strong CMI and humoral responses against TT 2 months after single injection in mice without booster dose. This biodegradable nanoadjuvant system may help to develop single-shot immunization for prolonged immunity without booster doses. The capability of enhanced CMI response may have high translational potential for immunization against intracellular infection. Keywords: tetanus toxoid, antigen cross-presentation, poly-ε-caprolactone nanoparticles, vaccine, adjuvant

Indro Neil Ghosh,1,* Supriya Deepak Patil,1,* Tarun Kumar Sharma,1,2 Santosh Kumar Srivastava,1 Ranjana Pathania,1 Naveen Kumar Navani11Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, Uttarakhand, 2Center for Biodesign and Diagnostics, Translational Health Science and Technology Institute, Gurgaon Haryana, India*These authors contributed equally to this workAbstract: Silver has long been advocated as an effective antimicrobial. However, toxicity issues with silver have led to limited use of silver in nanoform, especially for food preservation. With the aim of exploring combinatorial options that could increase the antibacterial potency of silver nanoparticles and reduce the effective dosage of silver, we evaluated the extent of synergy that a combination of silver nanoparticles and an essential oil representative (cinnamaldehyde) could offer. A battery of gram-positive and gram-negative bacterial strains was utilized for antibacterial assays, and extents of synergism were calculated from fractional inhibitory concentration indices. The activity of nanoparticles was greatly enhanced when utilized in the presence of cinnamaldehyde. We observed combinatorial effects that were strongly additive against all the bacterial strains tested, and genuine synergy was found against spore forming Bacillus cereus and Clostridium perfringens – bacterial strains associated with release of cytotoxins in contaminated food and known for their persistence. Bacterial kill curve analysis revealed a very fast bactericidal action when a combination of two agents was used. The electron and atomic force microscopy also revealed extensive damage to the bacterial cell envelop in the presence of both agents. We also performed hemolysis assays to investigate and approximate the extent of toxicity exhibited by the two agents, and observed no adverse effect at the concentrations required for synergy. This study shows that safe levels of silver in nanoform in combination with essential oil component cinnamaldehyde can be effectively used for controlling the spore-forming bacterial species.Keywords: antibacterial activity, bacterial food spoilage, erythrocyte toxicity, essential oil, in vitro, synergy, toxin producing spore-forming bacteria

Singh RP, Srivastava SK, Ehlers JP, et al. Clin Ophthalmol. 2015;9:1759–1766.On page 1763, Figure 4 has been amended. Read the original article

AbstractEthylene propylene diene terpolymer (EPDM)/MgAl layered double hydroxide (LDH) nanocomposites have been synthesized by solution intercalation using organically modified LDH (DS-LDH). The molecular level dispersion of LDH nanolayers has been verified by the disappearance of basal XRD peak of DS-LDH in the composites. The internal structures, of the nanocomposite with the dispersion nature of LDH particles in EPDM matrix have been studied by TEM and AFM. Thermogravimetric analysis (TGA) shows thermal stability of nanocomposites improved by ≈40 °C when 10% weight loss was selected as point of comparison. The degradation for pure EPDM is faster above 380 °C while in case of its nanocomposites, it is much slower.

Prashant CK, Bhat M, Srivastava SK, et al. Int J Nanomedicine. 2014;9:937–950.On page 937, the author affiliations have been listed incorrectly. The correct author list is as follows:Chandravilas Keshvan Prashant1Madhusudan Bhat2Sandeep Kumar Srivastava2Ankit Saxena3Manoj Kumar4Amar Singh3Mohammed Samim5Farhan Jalees Ahmad6Amit Kumar Dinda21Faculty of Engineering and Interdisciplinary Sciences, Jamia Hamdard, New Delhi, India; 2Department of Pathology, 3Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India; 4Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi, India; 5Department of Chemistry, Faculty of Science, Jamia Hamdard, New Delhi, India; 6Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, New Delhi, IndiaRead the original article

Contents Antisperm antibodies have been found in repeat-breeding( RB) cows, and those causing agglutination and/or immobilization of sperm are considered to be closely related to unexplained infertility. However, a standard protocol for identifying antisperm antibodies ( ASA) in cattle is not validated. Therefore, an investigation was undertaken to evaluate sperm immobilization ( SIT), sperm agglutination ( SAT) and immunoperoxidase ( IPT)assays for detection of ASA in serum and their respective threshold levels for confirmation. Animals (heifers, normally breeding, repeat-breeding and pregnant animals) that were free from IBR, brucellosis and uterine infections (screened by clinical examination) were included in the study. Sperm agglutinating, sperm immobilizing and antisperm antibodies evaluated by respective assay were significantly higher ( p < .05) in RB cows compared to other groups. The SIT assay was able to identify 61% of RB caused by ASA, more than those employing SAT and IPT. Furthermore, a dilution rate of 1:5 and 1:80 (confirms 59.0 and 57.0% RB+ve)were sufficient to diagnose ASA by SAT and IPT, respectively. Results indicate the presence of __12.6% clumped spermatozoa and __ 2.6%(cut-off value) peroxidase-positive spermatozoa at 1:5 and 1:80 dilutions diagnosed with SAT and IPT, respectively, may be considered as repeaters arising out of ASA. Furthermore, study also showed the presence of lower incidence of ASA positivity in other groups of animals (heifer