Cloning and characterization of Aiia, an acylhomoserine lactonase from Bacillus cereus RC1 to control soft rot causing pathogen Lelliottia amnigena RCE.

Bacterial pathogenesis-associated characteristics such as biofilm formation, synthesis of hydrolyzing enzymes, and toxins are regulated by Acyl Homoserine Lactones (AHLs), small peptides and diffusing signal factors (DSF). Lelliottia amnigena is gram negative bacteria and its pathogenicity is regulated by the luxR and luxI class of quorum sensing. The signaling molecules and their concentrations are essential for the virulence of the pathogenic bacterium. To suppresses the pathogenicity; the concentration of signalling molecules must be controlled or degraded. The lactonase have the ability to hydrolyze lactones of different chain length. The present study deals with a newer approach to control the pathogenesis of Lelliottia amnigena through isolation and characterization of Aiia lactonase from Bacillus cereus RC1. Aiia lactonase specific primers were used to amplify the gene, and the sequence thus obtained was submitted to the Genbank database under accession # OK643884.1. The gene was cloned in pBE-S shuttle vector and transformed in the recombinant host. The expressed and purified protein had a molecular weight of 28.00 KDa and exhibited its optimum activity at 37℃ by inhibiting the violacein pigment of the monitor strain Chromobacterium violaceum MTCC 2656. The proteinaceous nature of the purified molecule was confirmed by incubating it in the presence of proteinase K for 1 h. The activity of the pathogenesis-related protein, polygalacturonase was drastically reduced in the presence of the purified Aiia protein. The purified protein also showed a zone of inhibition when plated together with Lelliottia amnigena RCE (MZ712952.1). Searches of the Conserved Domain Database suggested that this protein belonged to the Metallo-beta-lactamase superfamily and is closely related to Aiia from B. thuringiensis serovar kurstaki. Modeling of the protein structure was done using I-TASSER; a C-score of 0.55 suggested that the model was of good quality. To be used commercially, this recombinant protein needs to be purified at an industrial scale; it can then be used to repress the growth of soft rot causing bacteria in horticultural crops during their storage period.

Reduction Sensitive PEG Hydrogels for Codelivery of Antigen and Adjuvant To Induce Potent CTLs.

Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257-264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8+ effector T cells in mice (∼6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.

Rapamycin-encapsulated nanoparticle delivery in polycystic kidney disease mice.

Rapamycin slows cystogenesis in murine models of polycystic kidney disease (PKD) but failed in clinical trials, potentially due to insufficient drug dosing. To improve drug efficiency without increasing dose, kidney-specific drug delivery may be used. Mesoscale nanoparticles (MNP) selectively target the proximal tubules in rodents. We explored whether MNPs can target cystic kidney tubules and whether rapamycin-encapsulated-MNPs (RapaMNPs) can slow cyst growth in Pkd1 knockout (KO) mice. MNP was intravenously administered in adult Pkd1KO mice. Serum and organs were harvested after 8, 24, 48 or 72 h to measure MNP localization, mTOR levels, and rapamycin concentration. Pkd1KO mice were then injected bi-weekly for 6 weeks with RapaMNP, rapamycin, or vehicle to determine drug efficacy on kidney cyst growth. Single MNP injections lead to kidney-preferential accumulation over other organs, specifically in tubules and cysts. Likewise, one RapaMNP injection resulted in higher drug delivery to the kidney compared to the liver, and displayed sustained mTOR inhibition. Bi-weekly injections with RapaMNP, rapamycin or vehicle for 6 weeks resulted in inconsistent mTOR inhibition and little change in cyst index, however. MNPs serve as an effective short-term, kidney-specific delivery system, but long-term RapaMNP failed to slow cyst progression in Pkd1KO mice.

Genome-Wide Identification, Quantification, and Validation of Differentially Expressed miRNAs in Eggplant (Solanum melongena L.) Based on Their Response to Ralstonia solanacearum Infection.

MicroRNAs (miRNAs), a type of short noncoding RNA molecule (21-23 nucleotides), mediate repressive gene regulation through RNA silencing at the posttranscriptional level and play an important role in the defense response to abiotic and biotic stresses. miRNAs of the plant system have been studied in model crops for their diverse regulatory role while less is known about their significance in other plants whose genome and transcriptome data are scarce in the database, including eggplant (Solanum melongena L.). In the present study, a next-generation sequencing platform was used for the sequencing of miRNA, and real-time quantitative PCR for miRNAs was used to validate the gene expression patterns of miRNAs in Solanum melongena plantlets infected with the bacterial wilt-causing pathogen Ralstonia solanacearum (R. solanacearum). Sequence analyses showed the presence of 375 miRNAs belonging to 29 conserved families. The miR414 is highly conserved miRNA across the plant system while miR5658 and miR5021 were found exclusively in Arabidopsis thaliana surprisingly, these miRNAs were found in eggplants too. The most abundant families were miR5658 and miR414. Ppt-miR414, hvu-miR444b, stu-miR8020, and sly miR5303 were upregulated in Pusa purple long (PPL) (susceptible) at 48 h postinfection, followed by a decline after 96 h postinfection. A similar trend was obtained in ath-miR414, stu-mir5303h, alymiR847-5p, far-miR1134, ath-miR5021, ath-miR5658, osa-miR2873c, lja-miR7530, stu-miR7997c, and gra-miR8741 but at very low levels after infection in the susceptible variety, indicating their negative role in the suppression of host immunity. On the other hand, osa-miR2873c was found to be slightly increased after 96 hpi from 48 hpi. Most of the miRNAs under study showed relatively lower expression in the resistant variety Arka Nidhi after infection than in the susceptible variety. These results shed light on a deeper regulatory role of miRNAs and their targets in regulation of the plant response to bacterial infection. The present experiment and their results suggested that the higher expression of miRNA leads to a decline in host mRNA and thus shows susceptibility.

Nontargeted Metabolite Profiling of the Most Prominent Indian Mango (Mangifera indica L.) Cultivars Using Different Extraction Methods.

Aroma has a crucial role in assessing the quality of fresh fruit and its processed versions, which serve as reliable indications for advancing local cultivars in the mango industry. The aroma of mango is attributed to a complex of hundreds of volatile, polar, and nonpolar metabolites belonging to different chemical classes like monoterpenes, sesquiterpenes, nonterpene hydrocarbons (alkanes), alcohols, esters, fatty acids, aldehydes, lactones, amides, amines, ethers, and many more. This study looked at the volatile, nonpolar, and polar metabolites from 16 mango cultivars to determine their relative quantities and intervarietal changes using hexane, ethanol, and solid-phase microextraction (SPME), followed by gas chromatography-mass spectrometry (GC-MS) analysis. In total, 58 volatile compounds through SPME, 50 nonpolar metabolites from hexane extract, and 52 polar metabolites from ethanol extract were detected from all of the cultivars, belonging to various chemical classes. Through the SPME method, all 16 mango cultivars except Dashehari and Neelum exhibited abundant monoterpenes with maximum concentration in Kesar (91.00%) and minimum in Amrapali (60.66%). However, the abundance of fatty acids and sesquiterpenes was detected in Dashehari (37.91%) and Neelum (74.80%), respectively. In the hexane extract, 23 nonterpene hydrocarbons exhibited abundance in all 16 mango cultivars except Baneshan, with a higher concentration in Dashehari (95.45%) and lower in Ratna (77.63%). The ethanol extraction of 16 mango cultivars showed a higher concentration of esters, aldehydes, alcohols, and amides in Jamadar (52.16%), Dadamio (74.30%), Langra (64.38%), and Kesar (37.10%), respectively. There have been a lot of metabolite variations observed and analyzed using hierarchical cluster analysis (HCA) and principal component analysis (PCA) based on the similarity of various chemical compounds. Cluster analysis revealed the true similarity and pedigree of different mango cultivars, viz., Neeleswari, Dashehari, Neelum, Alphonso, Baneshan, Sonpari, and Neeleshan. They occupied the same cluster during analysis.

Pseudomonas aeruginosa inhibits quorum-sensing mechanisms of soft rot pathogen Lelliottia amnigena RCE to regulate its virulence factors and biofilm formation.

The quorum-sensing (QS) cascade is responsible for the colonization and phenotypic behavior of the pathogenic organism and the regulation of diverse signal molecules. The disruption of the quorum-sensing system is an effective strategy to overcome the possibility of antibiotic resistance development in the pathogen. The quorum quenching does not kill the microbes. Instead, it hinders the expression of pathogenic traits. In the present experiment, Pseudomonas aeruginosa RKC1 was used to extract the metabolites responsible for quorum-sensing inhibition in soft rot pathogen Lelliottia amnigena RCE. During the initial screening, P. aeruginosa RKC1 was found to be most promising and inhibits violacein of Chromobacterium violaceum MTCC2656 pyocyanin, swarming-swimming motility of P. aeruginosa MTCC2297. The characterization of metabolites produced by the microbes which are responsible for quorum-sensing inhibition through GC-MS is very scarce in scientific literature. The ethyl acetate extract of P. aeruginosa RKC1 inhibits biofilm formation of L. amnigena RCE while inhibiting growth at higher concentrations. The GC-MS analysis suggested that Cyclic dipeptides (CDPs) such as Cyclo (L-prolyl-L-valine), Cyclo (Pro-Leu), and Cyclo(D-phenylalanyl-L-prolyl) were predominantly found in the ethyl acetate extract of the P. aeruginosa RKC1 (93.72%). This diketopiperazine (DKPs) exhibited quorum-sensing inhibition against the pathogen in liquid media during the active growth phase and regulated diverse metabolites of the pathogen. Moreover, the metabolites data from the clear zone around wells showed a higher concentration of DKSs (9.66%) compared to other metabolites. So far, very few reports indicate the role of DKPs or CDPs in inhibiting the quorum-sensing system in plant pathogenic bacteria. This is one such report that exploits metabolites of P. aeruginosa RKC1. The present investigation provided evidence to use quorum-sensing inhibitor metabolites, to suppress microbes' pathogenesis and thus develop an innovative strategy to overcome antibiotic resistance.

Evaluation of Plant Growth-Promoting and Salinity Ameliorating Potential of Halophilic Bacteria Isolated From Saline Soil.

Among the biotic and abiotic stress affecting the physical, chemical, and biological properties of soil, salinity is a major threat that leads to the desertification of cultivable land throughout the world. The existence of diverse and versatile microbial populations inhabiting the nutrient-rich soil and varied soil conditions affects the soil dynamism. A normal soil constitutes 600 million bacteria belonging to about 20,000 species, which is reduced to 1 million with 5,000-8,000 species in stress conditions. Plant growth-promoting rhizobacteria (PGPR) are in symbiotic association with the plant system, which helps in combating the abiotic stress and increases the overall productivity and yield. These microorganisms are actively associated with varied cellular communication processes through quorum sensing and secondary metabolites such as the production of Indole-3-acetic acid (IAA), exopolysaccharide (EPS) siderophore, ammonia, ACC deaminase, and solubilization of phosphate. The present study focused on the isolation, identification, and characterization of the microorganisms isolated from the seacoast of Dandi, Navsari. Twelve isolates exhibited PGP traits at a high salt concentration of 15-20%. AD9 isolate identified as Bacillus halotolerans showed a higher ammonia production (88 ± 1.73 µg/mL) and phosphate solubilization (86 ± 3.06 µg/mL) at 15% salt concentration, while AD32* (Bacillus sp. clone ADCNO) gave 42.67 ±1.20 µg/mL IAA production at 20% salt concentration. AD2 (Streptomyces sp. clone ADCNB) and AD26 (Achromobacter sp. clone ADCNI) showed ACC deaminase activity of 0.61 ± 0.12 and 0.60 ± 0.04 nM α-ketobutyrate/mg protein/h, respectively. AD32 (Bacillus sp. clone ADCNL) gave a high siderophore activity of 65.40 ± 1.65%. These isolates produced salinity ameliorating traits, total antioxidant activities, and antioxidant enzymes viz. superoxide dismutase (SOD), Glutathione oxidase (GSH), and catalase (CAT). Inoculation of the multipotent isolate that produced PGP traits and salinity ameliorating metabolites promoted the plant growth and development in rice under salinity stress conditions. These results in 50% more root length, 25.00% more plant dry weight, and 41% more tillers compared to its control.

Quorum Sensing Inhibitory and Quenching Activity of Bacillus cereus RC1 Extracts on Soft Rot-Causing Bacteria Lelliottia amnigena.

The quorum sensing (QS) system of bacteria helps them to communicate with each other in a density-dependent manner and regulates pathogenicity. The concentrations of autoinducers, peptides, and signaling factors are required for determining the expression of virulence factors in many pathogens. The QS signals of the pathogen are regulated by the signal transduction pathway. The binding of signal molecules to its cognate receptor brings changes in the structure of the receptor, makes it more accessible to the DNA, and thus regulates diverse expression patterns, including virulence factors. Degrading the autoinducer molecules or disturbing the quorum sensing network could be exploited to control the virulence of the pathogen while avoiding multidrug-resistant phenotypes. The rhizosphere is a tremendous source of beneficial microbes that has not yet been explored properly for its anti-quorum sensing potential. Lelliottia amnigena causes soft rot diseases in onion, potato, and other species. The present investigation was carried out with the aim of isolating the anti-quorum sensing metabolites and elucidating their role in controlling the virulence factors of the pathogen by performing a maceration assay. The ethyl acetate extracts of various bacteria are promising for violacein inhibition assay using Chromobacterium violaceum MTCC2656 and pyocyanin inhibition of Pseudomonas aeruginosa MTCC2297. Therefore, the extract was used to deduce its role in attenuation of soft rot in potato, carrot, and cucumber. The maximum reduction of macerated tissue in carrot, potato, and cucumber was given by Bacillus cereus RC1 at 91.22, 97.59, and 88.78%, respectively. The concentration-dependent inhibition of virulence traits was observed during the entire experiment. The quorum quenching potential of the bacterial extract was used to understand the regulatory metabolites. The data of the diffusible zone and gas chromatography-mass spectrometry (GC-MS) analysis showed that diketopiperazines, viz. Cyclo(d-phenylalanyl-l-prolyl), Cyclo Phe-Val, Cyclo(Pro-Ala), Cyclo(l-prolyl-l-valine), Cyclo (Leu-Leu), and Cyclo(-Leu-Pro), are prominent metabolites that could modulate the pathogenicity in L. amnigena RCE. The interaction of bacterial extracts regulates various metabolites of the pathogens during their growth in liquid culture compared to their control counterparts. This study might help in exploiting the metabolites from bacteria to control the pathogens, with concurrent reduction in the pathogenicity of the pathogens without developing antibiotic resistance.

Production, statistical optimization, and functional characterization of alkali stable pectate lyase of Paenibacillus lactis PKC5 for use in juice clarification.

Pectate lyase is a hydrolytic enzyme used by diverse industries to clarify food. The enzyme occupies a 25% share of the total enzyme used in food industries, and their demand is increasing gradually. Most of the enzymes in the market belong to the fungal origin and take more time to produce with high viscosity in the fermentation medium, limiting its use. The bacteria belonging to the genus Bacillus have vast potential to produce diverse metabolites of industrial importance. The present experiment aimed to isolate pectate lyase-producing bacteria that can tolerate an alkaline environment at moderate temperatures. Bacillus subtilis PKC2, Bacillus licheniformis PKC4, Paenibacillus lactis PKC5, and Bacillus sonorensis ADCN produced pectate lyase. The Paenibacillus lactis PKC5 gave the highest protein at 48 h of incubation that was partially purified using 80% acetone and ammonium sulphate. Purification with 80% acetone resulted in a good enzyme yield with higher activity. SDS-PAGE revealed the presence of 44 kDa molecular weight of purified enzyme. The purified enzyme exhibits stability at diverse temperature and pH ranges, the maximum at 50 °C and 8.0 pH. The metal ions such as Mg2+, Zn2+, Fe2+, and Co2+ significantly positively affect enzyme activity, while increasing the metal ion concentration to 5 mM showed detrimental effects on the enzyme activity. The organic solvents such as methanol and chloroform at 25% final concentration improved the enzyme activity. On the other hand, detergent showed inhibitory effects at 0.05% and 1% concentration. Pectate lyase from Paenibacillus lactis PKC5 had Km and Vmax values as 8.90 mg/ml and 4.578 µmol/ml/min. The Plackett-Burman and CCD designs were used to identify the significant process parameters, and optimum concentrations were found to be pectin (5 gm%) and ammonium sulphate (0.3 gm%). During incubation with pectate lyase, the clarity percentage of the grape juice, apple juice, and orange juice was 60.37%, 59.36%, and 49.91%, respectively.

Nanoparticles combined with cefixime as an effective synergistic strategy against Salmonella enterica typhi.

Enteric fever caused by Salmonella typhi has been the most crucial health issue in rural people, especially in Southeast Asia and Africa. Another disease, Salmonellosis, caused by a large group of bacteria of the genus Salmonella, cause substantial economic loss resulting from mortality and morbidity. Higher concentration and repeated use of antibiotics to treat these diseases will likely develop antibiotic resistance among the microbes. The nanoparticle has good penetration power and can kill microbes. Combining two strategies by using nanoparticles with antibiotics kills microbes and reduces the chances of the development of antibiotics resistance. Silver, Nickel, Copper, and Zinc oxide Nanoparticles were chemically synthesized and characterized in this study. Silver nanoparticles at a concentration of 10 µg/ml inhibit all the strains under study. In comparison, silver nanoparticles (16.90 µg/ml), Nickel nanoparticles (83 µg ml-1), Copper nanoparticles (249 µg ml-1), and Zinc oxide (1614 µg ml-1) along with 50 µg/ml cefixime gave maximum zone of inhibition of 35 mm, 19 mm, 31 mm and 23 mm respectively. The antimicrobial assay showed that silver nanoparticles presented good antibacterial performance against all multi-drug-resistant pathogenic Salmonella sp alone as well as in combinations. The present study proved that silver nanoparticles at the lowest concentration along with cefixime could be a possible alternative to control the multi-drug-resistant pathogens.

Layer-by-layer assembled PLGA nanoparticles carrying miR-34a cargo inhibit the proliferation and cell cycle progression of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) accounts for 15-25% of diagnosed breast cancers, and its lack of a clinically defined therapeutic target has caused patients to suffer from earlier relapse and higher mortality rates than patients with other breast cancer subtypes. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of multiple genes through RNA interference to maintain normal tissue function. The tumor suppressor miR-34a is downregulated in TNBC, and its loss-of-expression correlates with worse disease outcomes. Therefore, delivering miR-34a mimics into TNBC cells is a promising strategy to combat disease progression. To achieve this goal, we synthesized layer-by-layer assembled nanoparticles (LbL NPs) comprised of spherical poly(lactic-co-glycolic acid) cores surrounded by alternating layers of poly-L-lysine (PLL) and miR-34a. TNBC cells internalized these LbL NPs to a greater extent than polyplexes comprised of PLL and miRNA, and confocal microscopy showed that LbL NPs delivered a substantial fraction of miR-34a cargo into the cytosol. This yielded robust suppression of the miR-34a target genes CCND-1, Notch-1, Bcl-2, Survivin, and MDR-1, which reduced TNBC cell proliferation and induced cell cycle arrest. These data validate that miR-34a delivery can impair TNBC cell function and support continued investigation of this platform for treatment of TNBC.

Polymer nanocarriers for MicroRNA delivery.

Abnormal expression of microRNAs (miRNAs), which are highlyconserved noncoding RNAs that regulate the expression of various genes post transcriptionally to control cellular functions, has been associated with the development of many diseases. In some cases, disease-promoting miRNAs are upregulated, while in other instances disease-suppressive miRNAs are downregulated. To alleviate this imbalanced miRNA expression, either antagomiRs or miRNA mimics can be delivered to cells to inhibit or promote miRNA expression, respectively. Unfortunately, the clinical translation of bare antagomiRs and miRNA mimics has been challenging because nucleic acids are susceptible to nuclease degradation, display unfavorable pharmacokinetics, and cannot passively enter cells. This review emphasizes the challenges associated with miRNA mimic delivery and then discusses the design and implementation of polymer nanocarriers to overcome these challenges. Preclinical efforts are summarized, and a forward-looking perspective on the future clinical translation of polymer nanomaterials as miRNA delivery vehicles is provided.

Best Practices for Preclinical In Vivo Testing of Cancer Nanomedicines.

Significant advances have been made in the development of nanoparticles for cancer treatment in recent years. Despite promising results in preclinical animal models, cancer nanomedicines often fail in clinical trials. This failure rate could be reduced by defining stringent criteria for testing and quality control during the design and development stages, and by performing carefully planned preclinical studies in relevant animal models. This article discusses best practices for the evaluation of nanomedicines in murine tumor models. First, a recommended set of experiments to perform is introduced, including discussion of the types of data to collect during these studies. This is followed by an outline of various tumor models and their clinical relevance. Next, different routes of nanoparticle administration are overviewed, followed by a summary of important controls to include in in vivo studies of nanomedicine. Finally, animal welfare considerations are discussed, and an overview of the steps involved in achieving US Food and Drug Administration approval after animal studies are completed is provided. Researchers should use this report as a guideline for effective preclinical evaluation of cancer nanomedicine. As the community adopts best practices for in vivo testing, the rate of clinical translation of cancer nanomedicines is likely to improve.

Pulmonary Delivery of Nanoparticle-Bound Toll-like Receptor 9 Agonist for the Treatment of Metastatic Lung Cancer.

CpG oligodeoxynucleotides are potent toll-like receptor (TLR) 9 agonists and have shown promise as anticancer agents in preclinical studies and clinical trials. Binding of CpG to TLR9 initiates a cascade of innate and adaptive immune responses, beginning with activation of dendritic cells and resulting in a range of secondary effects that include the secretion of pro-inflammatory cytokines, activation of natural killer cells, and expansion of T cell populations. Recent literature suggests that local delivery of CpG in tumors results in superior antitumor effects as compared to systemic delivery. In this study, we utilized PRINT (particle replication in nonwetting templates) nanoparticles as a vehicle to deliver CpG into murine lungs through orotracheal instillations. In two murine orthotopic metastasis models of non-small-cell lung cancer-344SQ (lung adenocarcinoma) and KAL-LN2E1 (lung squamous carcinoma), local delivery of PRINT-CpG into the lungs effectively promoted substantial tumor regression and also limited systemic toxicities associated with soluble CpG. Furthermore, cured mice were completely resistant to tumor rechallenge. Additionally, nanodelivery showed extended retention of CpG within the lungs as well as prolonged elevation of antitumor cytokines in the lungs, but no elevated levels of proinflammatory cytokines in the serum. These results demonstrate that PRINT-CpG is a potent nanoplatform for local treatment of lung cancer that has collateral therapeutic effects on systemic disease and an encouraging toxicity profile and may have the potential to treat lung metastasis of other cancer types.

Role of Linker Length and Antigen Density in Nanoparticle Peptide Vaccine.

Multiple studies have been published emphasizing the significant role of nanoparticle (NP) carriers in antigenic peptide-based subunit vaccines for the induction of potent humoral and cellular responses. Various design parameters of nanoparticle subunit vaccines such as linker chemistry, the proximity of antigenic peptide to NPs, and the density of antigenic peptides on the surface of NPs play an important role in antigen presentation to dendritic cells (DCs) and in subsequent induction of CD8+ T cell response. In this current study, we evaluated the role of peptide antigen proximity and density on DC uptake, antigen cross-presentation, in vitro T cell proliferation, and in vivo induction of CD8+ T cells. To evaluate the role of antigen proximity, CSIINFEKL peptides were systematically conjugated to poly(ethylene glycol) (PEG) hydrogels through N-hydroxysuccinimide-PEG-maleimide linkers of varying molecular weights: 2k, 5k, and 10k. We observed that the peptides conjugated to NPs via the 2k and 5k PEG linkers resulted in higher uptake in bone marrow-derived DCs (BMDCs) and increased p-MHC-I formation on the surface of bone marrow-derived DCs (BMDCs) as compared to the 10k PEG linker formulation. However, no significant differences in vitro T cell proliferation and induction of in vivo CD8+ T cells were found among linker lengths. To study the effect of antigen density, CSIINFEKL peptides were conjugated to PEG hydrogels via 5k PEG linkers at various densities. We found that high antigen density NPs presented the highest p-MHC-I on the surface of BMDCs and induced higher proliferation of T cells, whereas NPs with low peptide density resulted in higher DC cell uptake and elevated frequency of IFN-γ producing CD8+ T cells in mice as compared to the medium- and high-density formulations. Altogether, findings for these experiments highlighted the importance of linker length and peptide antigen density on DC cell uptake, antigen presentation, and induction of in vivo CD8+ T cell response.

Spherical Nucleic Acid Nanoparticles: Therapeutic Potential.

Spherical nucleic acids (SNAs) are highly oriented, well organized, polyvalent structures of nucleic acids conjugated to hollow or solid core nanoparticles. Because they can transfect many tissue and cell types without toxicity, induce minimum immune response, and penetrate various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier), they have become versatile tools for the delivery of nucleic acids, drugs, and proteins for various therapeutic purposes. This article describes the unique structures and properties of SNAs and discusses how these properties enable their application in gene regulation, immunomodulation, and drug and protein delivery. It also summarizes current efforts towards clinical translation of SNAs and provides an expert opinion on remaining challenges to be addressed in the path forward to the clinic.

Extending antigen release from particulate vaccines results in enhanced antitumor immune response.

Tumor-specific CD8+ cytotoxic T lymphocytes (CTLs) play a critical role in an anti-tumor immune response. However, vaccination intended to elicit a potent CD8+ T cell responses employing tumor-associated peptide antigens, are typically ineffective due to poor immunogenicity. Previously, we engineered a polyethylene glycol (PEG) hydrogel-based subunit vaccine for the delivery of an antigenic peptide and CpG (adjuvant) to elicit potent CTLs. In this study, we further examined the effect of antigen release kinetics on their induced immune responses. A CD8+ T cell epitope peptide from OVA (CSIINFEKL) and CpG were co-conjugated to nanoparticles utilizing either a disulfide or a thioether linkage. Subsequent studies comparing peptide release rates as a function of linker, determined that the thioether linkage provided sustained release of peptide over 72h. Ability to control the release of peptide resulted in both higher and prolonged antigen presentation when compared to disulfide-linked peptide. Both NP vaccine formulations resulted in activation and maturation of bone marrow derived dendritic cells (BMDCs) and induced potent CD8+ T cell responses when compared to soluble antigen and soluble CpG. Immunization with either disulfide or thioether linked vaccine constructs effectively inhibited EG7-OVA tumor growth in mice, however only treatment with the thioether linked vaccine construct resulted in enhanced survival.

Layer-by-layer assembled gold nanoshells for the intracellular delivery of miR-34a.

INTRODUCTION: MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation. METHODS: LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-L-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles' ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively. RESULTS: Each successive coating reversed the nanoparticles' charge and increased their hydrodynamic diameter, resulting in a final diameter of 208±4 nm and a zeta potential of 53±5 mV. The LbL-NS released ~30% of their miR-34a cargo over 5 days in 1X PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46±3% and 35±3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects. CONCLUSION: LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.

Nanoparticulate immunotherapy for cancer.

Although surgery, radiation therapy, and chemotherapy have significantly improved as treatments for cancer, they can rarely control metastatic disease and cures remain scarce. Promising recent developments suggest that cancer immunotherapy may become a powerful new therapy that clinicians can offer cancer patients. The opportunity to orchestrate the body's own immune system to target, fight, and eradicate cancer cells without destroying healthy cells makes this an extremely attractive treatment modality. Our increased knowledge in anti-tumor immunity and the immunosuppressive tumor microenvironment (TME) has provided many therapeutic strategies to battle cancer. That combined with advancements in the field of particulate delivery systems provide a mechanism to deliver these immunotherapeutics to their specific targeted cells and the TME. In this review we will focus on the current status of immunotherapy and the potential advantages of utilizing nanocarriers within the field.

Nanoparticle delivery of chemosensitizers improve chemotherapy efficacy without incurring additional toxicity.

Chemosensitizers can improve the therapeutic index of chemotherapy and overcome treatment resistance. Successful translation of chemosensitizers depends on the development of strategies that can preferentially deliver chemosensitizers to tumors while avoiding normal tissue. We hypothesized that nanoparticle (NP) formulation of chemosensitizers can improve their delivery to tumors which can in turn improve their therapeutic index. To demonstrate the proof of principle of this approach, we engineered NP formulations of two chemosensitizers, the PI3-kindase inhibitor wortmanin (Wtmn) and the PARP inhibitor olaparib. NP Wtmn and NP olaparib were evaluated as chemosensitizers using lung cancer cells and breast cancer cells respectively. We found Wtmn to be an efficient chemosensitizer in all tested lung-cancer cell lines reducing tumor cell growth between 20 and 60% compared to drug alone. NP formulation did not decrease its efficacy in vitro. Olaparib showed less consistent chemosensitization as a free drug or in NP formulation. NP Wtmn was further evaluated as a chemosensitizer using mouse models of lung cancer. We found that NP Wtmn is an effective chemosensitizer and more effective than free Wtmn showing a 32% reduction in tumor growth compared to free Wtmn when given with etoposide. Importantly, NP Wtmn was able to sensitize the multi-drug resistant H69AR cells to etoposide. Additionally, the combination of NP Wtmn and etoposide chemotherapy did not significantly increase toxicity. The present study demonstrates the proof of principle of using NP formulation of chemosensitizing drugs to improve the therapeutic index of chemotherapy.