Needle-Free Immunization with Chitosan-Based Systems.

Despite successful use, needle-based immunizations have several issues such as the risk of injuries and infections from the reuse of needles and syringes and the low patient compliance due to pain and fear of needles during immunization. In contrast, needle-free immunizations have several advantages including ease of administration, high level of patient compliance and the possibility of mass vaccination. Thus, there is an increasing interest on developing effective needle-free immunizations via cutaneous and mucosal approaches. Here, we discuss several methods of needle-free immunizations and provide insights into promising use of chitosan systems for successful immunization.

Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study. RESULTS: We firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 µg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers. CONCLUSIONS: In this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.

Combinatorial Approach of Antigen Delivery Using M Cell-Homing Peptide and Mucoadhesive Vehicle to Enhance the Efficacy of Oral Vaccine.

Orally ingested pathogens or antigens are taken up by microfold cells (M cells) in Peyer's patches of intestine to initiate protective immunity against infections. However, the uptake of orally delivered protein antigens through M cells is very low due to lack of specificity of proteins toward M cells and degradation of proteins in the harsh environment of gastrointestinal (GI) tract. To overcome these limitations, here we developed a pH-sensitive and mucoadhesive vehicle of thiolated eudragit (TE) microparticles to transport an M cell-targeting peptide-fused model protein antigen. Particularly, TE prolonged the particles transit time through the GI tract and predominantly released the proteins in ileum where M cells are abundant. Thus, oral delivery of TE microparticulate antigens exhibited high transcytosis of antigens through M cells resulting in strong protective sIgA as well as systemic IgG antibody responses. Importantly, the delivery system not only induced CD4(+) T cell immune responses but also generated strong CD8(+) T cell responses with enhanced production of IFN-γ in spleen. Given that M cells are considered a promising target for oral vaccination, this study could provide a new combinatorial method for the development of M-cell-targeted mucosal vaccines.

Release and Cytokine Production of BmpB from BmpB-Loaded pH-Sensitive and Mucoadhesive Thiolated Eudragit Microspheres.

Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 µ. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.

Chemical modification of chitosan with pH-sensitive molecules and specific ligands for efficient DNA transfection and siRNA silencing.

Successful gene therapy depends on the development of efficient and cell-specific gene delivery systems. Currently, animal viral vectors have been mostly used for in vivo and in clinical trials owing to their high transduction efficiency. However, they suffer from numerous limitations such as biosafety, immunogenicity, gene packaging capacity, complicated production and cell specificity. Therefore non-viral vectors are attractive alternatives to viral gene delivery systems due to their low toxicity, relatively easy production and greater diversity. Among non-viral vectors, chitosan and chitosan derivatives have been extensively utilized as gene carriers owing to their low immunogenicity, biocompatibility, biodegradability, low toxicity and ease of chemical modifications. However, low transfection efficiency of DNA (or low gene silencing of siRNA) and low cell specificity of chitosan should be overcome before clinical trials. The objective of this review is to summarize several parameters affecting the transfection efficiency of DNA (or gene silencing of siRNA) for the promising use of chitosan as gene carriers. Besides, chemical modifications of chitosan with pH-sensitive molecules and specific ligands so as to enhance the transfection efficiency of DNA (or gene silencing of siRNA) and cell specificity will be covered.

Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational way to prepare a suitable vector for targeted gene delivery to vimentin-expressing cells.

Enhanced osteoblast proliferation and corrosion resistance of commercially pure titanium through surface nanostructuring by ultrasonic shot peening and stress relieving.

This investigation was carried out to study the effect of a novel process of surface modification, surface nanostructuring by ultrasonic shot peening, on osteoblast proliferation and corrosion behavior of commercially pure titanium (c p-Ti) in simulated body fluid. A mechanically polished disc of c p-Ti was subjected to ultrasonic shot peening with stainless steel balls to create nanostructure at the surface. A nanostructure (<20 nm) with inhomogeneous distribution was revealed by atomic force and scanning electron microscopy. There was an increase of approximately 10% in cell proliferation, but there was drastic fall in corrosion resistance. Corrosion rate was increased by 327% in the shot peened condition. In order to examine the role of residual stresses associated with the shot peened surface on these aspects, a part of the shot peened specimen was annealed at 400°C for 1 hour. A marked influence of annealing treatment was observed on surface structure, cell proliferation, and corrosion resistance. Surface nanostructure was much more prominent, with increased number density and sharper grain boundaries; cell proliferation was enhanced to approximately 50% and corrosion rate was reduced by 86.2% and 41% as compared with that of the shot peened and the as received conditions, respectively. The highly significant improvement in cell proliferation, resulting from annealing of the shot peened specimen, was attributed to increased volume fraction of stabilized nanostructure, stress recovery, and crystallization of the oxide film. Increase in corrosion resistance from annealing of shot peened material was related to more effective passivation. Thus, the surface of c p-Ti, modified by this novel process, possessed a unique quality of enhancing cell proliferation as well as the corrosion resistance and could be highly effective in reducing treatment time of patients adopting dental and orthopedic implants of titanium and its alloys.

Exploration of two epimerase homologs in Streptomyces peucetius ATCC 27952.

Streptomyces peucetius ATCC 27952 is a potent producer of the therapeutically important antitumor drug, doxorubicin. S. peucetius contains two deoxythymidine diphospho (dTDP)-4-keto-6-deoxyglucose 3,5-epimerase-encoding genes, dnmU and rmbC, in its genome. While dnmU from the doxorubicin biosynthesis gene cluster is involved in the biosynthesis of dTDP-L-daunosamine, rmbC is involved in the biosynthesis of dTDP-L-rhamnose, a precursor of cell wall biosynthesis. The proteins encoded by dnmU and rmbC share 47 % identity and 64 % similarity with each other. Both enzymes converted the same substrate, dTDP-4-keto-6-deoxy-D-glucose, into dTDP-4-keto-L-rhamnose in vitro. However, when disruption of dnmU or rmbC was carried out, neither gene in S. peucetius compensated for each other's loss of function in vivo. These results demonstrated that although dnmU and rmbC encode for similar functional proteins, their native roles in their respective biosynthetic pathways in vivo are specific and independent of one other. Moreover, the disruption of rmbC resulted in fragmented mycelia that quickly converted into gray pigmented spores. Additionally, the production of doxorubicin, a major product of S. peucetius, appeared to be abolished after the disruption of rmbC, demonstrating its pleiotropic effect. This adverse effect might have switched on the genes encoding for spore formation, arresting the expression of many genes and, thereby, preventing the production of other metabolites.

Effect of microencapsulation of Lactobacillus plantarum 25 into alginate/chitosan/alginate microcapsules on viability and cytokine induction.

Lactobacillus plantarum 25 (LP25) encapsulated into alginate/chitosan/alginate (ACA) microcapsules (LP25-ACA MCs) prepared by an extrusion methods were characterized to assess their efficacy in oral delivery. The particle sizes of LP25-ACA MCs were 1.11 +/- 0.32 mm. The loading content of LP25 was 1.11 x 10(7) colony forming unit (cfu)/microcapsule and encapsulation efficiency was above 98%. The viability of LP25 in ACA MCs was more than 65% in simulated gastric fluid (SGF, pH 2.0) and 75% in simulated small intestinal fluid (SIF, pH 7.2) up to 2 h. Encapsulated LP25 were completely released from LP25-ACA MCs in SIF and simulated colon fluid (SCF, pH 6.0) within 12 h and 8 h respectively. The viability of LP25 in ACA MCs till 5 weeks was above 58%, whereas viability of free LP25 stored at room temperature up to 5 weeks was zero. Besides, LP25-ACA MCs induced the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) from macrophages and dendritic cells showing the immunomodulatory effect of LP25. These findings demonstrate that the encapsulation of LP25 by ACA is a suitable strategy for oral delivery of probiotics.

Imiquimod-gemcitabine nanoparticles harness immune cells to suppress breast cancer.

Monotherapy with a single chemotherapeutic regimen has met with significant hurdles in terms of clinical efficacy. The complexity of cancer accentuates the need for an alternative approach with a combination of two or more therapeutic regimens to win the battle. However, it is still a challenge to develop a successful combination of drugs with high efficiency and low toxicity to control cancer growth. While gemcitabine monotherapy remains a choice of standard treatment for advanced breast cancer, the approach has not prolonged the median survival time of metastatic breast cancer patients. Here, we report a hyaluronic acid (HA)-based drug combination of gemcitabine (GEM) with imiquimod (IMQ) to stimulate immune cells for anticancer activity. Treatment of the drug combination (IMQ-HA-GEM) showed enhanced anticancer activity against 4T1 breast tumor cells in vitro. Our study with a microfluidics-based 3D, compartmentalized cancer model showed that infiltration of THP-1 monocytes occurred particularly at the site of cancer cells treated with IMQ-HA-GEM. Moreover, IMQ-HA-GEM significantly suppressed the volume of 4T1 breast tumor of mice in vivo. Flow cytometry study displayed a significantly higher activation of CD11b+ immune cells in the blood of mice treated with IMQ-HA-GEM, whereas immunohistochemistry study revealed greater prevalence of CD68+ tumor-associated macrophages in the tumor. Histological examination of isolated tumors of mice treated with IMQ-HA-GEM further confirmed the efficacy of drug combination on cancer cells. This study supports the conclusion that imiquimod potentiates the effect of gemcitabine by activating immune cells to suppress tumors in the form of combination nanoparticles.

Saliva as a propitious diagnostic biofluid, biomarker, and bodies first line of defense against COVID-19: A review.

This review aims to recognize the role of saliva not just as a transmitting agent of COVID 19, but also comprehend its role in the diagnosis, and as a biomarker. A systematic literature search was performed in the PubMed database and eligible studies were included if they addressed the key issues i.e saliva as a diagnostic aid. As of January 10, 2021, a total of 309 articles across the PubMed database were identified of which 28 studies met the inclusion criteria. They were carefully examined for the type of study, sample size, parameters used, sample collection technique, and conclusions drawn. Diagnostic properties of saliva, the role of ACE 2 receptors, antibody formation ability, and antiviral characteristics were also explored. Comparisons among methods of sample collection like nasopharyngeal swabs and oropharyngeal swabs to saliva were also investigated. The observations and important deductions among the different studies were compared. Results indicated that saliva could be a reliable and financially viable option in both testing viral titers as well as marking for bio analytes due to its propitious specificity and sensitivity results reported in most of the studies. However, the inferences drawn from many of these studies should be interpreted with caution due to small sample sizes, inadequate detailing on the sample handling, laboratory processing, and rush in Corona-related publication. Scientific research with larger sample sizes, in diverse populations and age groups, at different phases of disease progression of COVID-19 are essential to reach any conclusion regarding its multi-facet use in the future.

A prospective case-control study to evaluate oral health status before and after intervention using different dental aids in children with visual impairment.

BACKGROUND: A major dental concern in children with special health-care needs is poor oral hygiene, which results in increased incidences of dental caries, gingivitis, and periodontal disease. AIMS: The study intended to determine if there was a difference in the oral health status of children with visual impairment and normal children and to evaluate the efficacy of the frequently used dental aids. SETTINGS AND DESIGN: The study population included 90 children, 45 children with visual impairment (study group) with age- and sex-matched 45 normal children (control group). Both the groups were further divided into three intervention subgroups. Subgroup A: manual toothbrushes, Subgroup B: manual toothbrush with medicated mouthwashes, and Subgroup C: powered toothbrushes. MATERIALS AND METHODS: For each subject, oral hygiene index simplified (OHIS), Turesky-Gilmore-Glickman modification of the Quigley-Hein Plaque Index (TQPHI), and decayed missing filled teeth (DMFT) indices were recorded at baseline, i.e., before any intervention. This was followed by oral prophylaxis by ultrasonic scaling. The three indices were recorded in 0 (baseline), 30 days (1 month), 90 days (3 months), and 180 days (6 months), respectively. STATISTICAL ANALYSIS USED: ANOVA test, Chi-square test, and student paired test were used for statistical analysis. RESULTS: The mean TQHPI and OHIS values of mouthwashes at the end of 6 months were 1.01 and 1.60, respectively, which were lower than manual and power brushes. No statistically significant reduction in the DMFT scores with the use of any of the adjuncts was noted. CONCLUSIONS: Among the dental aids used in the study, mouthwash showed a significant reduction in plaque and oral hygiene scores as compared to powered toothbrushes and manual brushes alone.

Evaluation of Ozone Therapy in Endodontic Treatment of Teeth with Necrotic Pulp and Apical Periodontitis: A Randomized Clinical Trial.

INTRODUCTION: The aim of this study was to compare the effect of different application techniques of ozone on the prevalence of postendodontic pain in patients undergoing single-visit root canal treatment. METHODS: hundred eight patients with necrotic pulp in single-rooted teeth and apical periodontitis participated in the trial. A standard single-visit endodontics protocol was followed with 5.25% sodium hypochlorite and rotary nickel-titanium files. After shaping and cleaning, patients were randomly allocated into the following groups: group 1 (n = 21), ozone treatment with no activation (NA); group 2 (n = 22), ozone treatment with manual dynamic activation (MDA); group 3, (n = 21), ozone treatment with passive ultrasonic activation (PUA); group 4 (n = 23), ozone treatment with sonic activation (SA); and group 5 (n = 21), no ozone treatment (the control group). Patient levels of discomfort were recorded at 6 different time intervals using the visual analog scale (VAS). Comparison of the mean difference between the groups and time intervals was performed using 2-way analysis of variance followed by a post hoc Bonferroni test. The level of significance was set at 5%. RESULTS: VAS scores were highest for the control > NA > MDA > SA > PUA groups. A statistically significant reduction in VAS scores was observed in the PUA and SA groups in comparison with the NA, control, and MDA groups. Timewise comparison showed a highly significant decline in VAS scores at all time intervals (P < .001). CONCLUSIONS: Ultrasonic and sonic activation of ozone resulted in less pain in patients undergoing single-visit endodontics compared with no ozone treatment.

Sustained, local delivery of the PARP inhibitor talazoparib prevents the development of mammary gland hyperplasia in Brca1-deficient mice.

Mutations in BRCA genes are the leading cause of hereditary breast cancer. Current options to prevent cancer in these high-risk patients, such as anti-estrogen drugs and radical mastectomy, are limited by lack of efficacy, undesirable toxicities, or physical and emotional challenges. We have previously shown that PARP inhibitors can significantly delay tumor development in BRCA1-deficient mice. Here, we fabricated the PARP inhibitor talazoparib (TLZ) into spacer implants (InCeT-TLZ) for localized and sustained delivery. We hypothesized that this novel formulation will provide an effective chemopreventive strategy with minimal toxicity. TLZ was released gradually over 30 days as implants degraded. InCeT-TLZ significantly decreased proliferation and increased DNA damage in the mammary glands of BRCA1-deficient mice. Notably, the number of mice that developed hyperplasia in the mammary glands was significantly lower with InCeT-TLZ treatment compared to the control group. Meanwhile, InCeT-TLZ was also better tolerated than oral TLZ, without loss of body weight or anemia. This study provides proof of concept for a novel and safe chemopreventive strategy using localized delivery of a PARP inhibitor for high-risk individuals. Future studies will directly evaluate the effects of InCeT-TLZ for preventing tumor development.

Precursor for biosynthesis of sugar moiety of doxorubicin depends on rhamnose biosynthetic pathway in Streptomyces peucetius ATCC 27952.

The doxorubicin biosynthetic gene cluster in Streptomyces peucetius ATCC 27952 contains a TDP-D-glucose 4,6-dehydratase gene, dnmM, that is putatively involved in the biosynthesis of daunosamine, but the gene contains a frameshift in the DNA sequence that would cause premature termination of translation. In pursuit of another TDP-D-glucose 4,6-dehydratase in S. peucetius, a homologue gene, rmbB, was found, whose deduced product exhibits high sequence similarity to a number of TDP-D-glucose 4,6-dehydratases. The gene was located within a putative rhamnose biosynthetic gene cluster at another locus in the genome. RmbB was verified to be a functional TDP-D-glucose 4,6-dehydratase by enzyme assay as it catalyzed the conversion of TDP-D-glucose into TDP-4-keto-6-deoxy-D-glucose. Inactivation of rmbB in the S. peucetius genome abolished the production of doxorubicin while complementation of the same gene in an rmbB knockout mutant restored the doxorubicin production. Hence, rmbB provides TDP-4-keto-6-deoxy-D-glucose as a nucleotide sugar precursor for the biosynthesis of doxorubicin.

Harnessing cells to deliver nanoparticle drugs to treat cancer.

Clinical translation of nanoparticle drug (nanodrug) delivery systems for cancer therapy is primarily hindered by short half-life of nanodrugs in blood circulation and their poor ability of tumor targeting and penetration in vivo. Circulatory cells have garnered much attention in cancer therapy as drug delivery vehicles due to their biocompatibility, high mobility, biodegradability, tissue targeting capability, high drug loading capacity, ability to cross biological barriers and inherent ability to remain in blood circulation long enough to accumulate within the tumors. Here, we review the progress and potential of circulatory cells as nanodrug delivery vehicles, especially for cancer therapy.

Nanoparticle Formulations of Poly (ADP-ribose) Polymerase Inhibitors for Cancer Therapy.

A number of poly(ADP-ribose) polymerase (PARP) inhibitors have been recently approved for clinical use in BRCA mutated and other cancers. However, off-target toxicity of PARP inhibitors and the emergence of drug resistance following prolonged administration of these inhibitors indicate the need for improved methods of drug delivery to the tumors. Nanomedicines based upon nanoparticle formulations of conventional small molecule drugs and inhibitors offer many advantages, such as increased solubility and bioavailability of drugs, reduced toxicity and drug resistance, and improved tissue selectivity and therapeutic efficacy. This review highlights the current trends in formulations of PARP inhibitors developed by nanotechnology approaches and provides an insight into the applications and limitations of these PARP inhibitor nanomedicines for cancer therapies.

Author Correction: Selective Priming of Tumor Blood Vessels by Radiation Therapy Enhances Nanodrug Delivery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Roadmap for metal nanoparticles in radiation therapy: current status, translational challenges, and future directions.

This roadmap outlines the potential roles of metallic nanoparticles (MNPs) in the field of radiation therapy. MNPs made up of a wide range of materials (from Titanium, Z = 22, to Bismuth, Z = 83) and a similarly wide spectrum of potential clinical applications, including diagnostic, therapeutic (radiation dose enhancers, hyperthermia inducers, drug delivery vehicles, vaccine adjuvants, photosensitizers, enhancers of immunotherapy) and theranostic (combining both diagnostic and therapeutic), are being fabricated and evaluated. This roadmap covers contributions from experts in these topics summarizing their view of the current status and challenges, as well as expected advancements in technology to address these challenges.

Exploration of geosmin synthase from Streptomyces peucetius ATCC 27952 by deletion of doxorubicin biosynthetic gene cluster.

Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.