"Lighting up" fluoride: cellular imaging and zebrafish model interrogations using a simple ESIPT-based mycophenolic acid precursor-based probe.

The discovery and implementation of media that derive from bioinspired designs and bear optical readouts featuring large Stokes shifts are of continued interest to a wide variety of researchers and clinicians. Myco-F, a novel mycophenolic acid precursor-based probe features a cleavable tert-butyldimethylsiloxy group to allow for fluoride detection. Myco-F exhibits high selectivity and specificity towards F- (Stokes shift = 120 nm). All measurements were performed in complete aqueous media (LOD=0.38 µM). Myco-F enables detection of fluoride ions in living HEK293 cells and localizes in the eye region (among other regions) of the zebrafish. DFT calculations support the proposed ESIPT working photomechanism.

Plasmid DNA Nanoparticles for Nonviral Oral Gene Therapy.

Herein, a bile acid-inspired triple padlock oral gene delivery platform is developed, facilitating the protection of the therapeutic gene from gastrointestinal degradation, selective intestinal accumulation through a bile acid-specific transporter, and transportation of pDNA NPs through the enterohepatic recycling system. This nonviral oral gene delivery nanoparticle exhibits excellent gene expression kinetics in in vitro, in vivo, and ex vivo studies. A single oral dose leads to maintaining normoglycemia for up to 7 days in three different diabetes mouse models and 14 days in diabetic monkeys. Also, the optimized dosage form can reduce nonfast blood glucose levels and hemoglobin A1C within a normal range from the last stage diabetes conditions with a reduction of weight gain from changes of food uptake behavior after treatment once weekly for 20 weeks. Taken together, the current findings could improve the current painful treatment experience of diabetics and thus improve their quality of life.

Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8.

BACKGROUND: Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. RESULTS: We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. CONCLUSIONS: We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

Identification of novel immunogenic proteins against Streptococcus parauberis in a zebrafish model by reverse vaccinology.

Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available S. parauberis genomes. The membrane-anchored protein SEC10/PgrA and the metal ABC transporter substrate-binding lipoprotein mtsA were potent antigenic proteins based on western blotting with mouse-derived antiserum against whole bacteria of S. parauberis serotypes I and II. In particular, metal ABC transporter substrate-binding lipoprotein (mtsA) showed similar protective immunity to that of whole-cell bacterins against S. parauberis in a zebrafish model. These results suggest that mtsA may be considered as a novel candidate in the development of vaccines against S. parauberis.

High-level production of N-terminal pro-brain natriuretic peptide, as a calibrant of heart failure diagnosis, in Escherichia coli.

Heart failure (HF) is a coronary disease that affects people worldwide and has a high mortality rate. N-terminal pro-brain natriuretic peptide (NT-proBNP) has been proven to be a useful and accurate biomarker for diagnosing systolic HF. Here, we report a strategy for the high-level production of recombinant (r)NT-proBNP in Escherichia coli. An Fh8 tag with six histidines was fused to the N terminus of NT-proBNP along with the recognition site of tobacco etch virus (TEV) protease; the 6HFh8-NT-proBNP fusion peptide was expressed in flask cultures of E. coli in almost completely soluble form. The peptide was purified by HisTrap affinity chromatography, and the N-terminal tag was cleaved by TEV protease. After a second round of HisTrap affinity chromatography to remove the TEV protease and N-terminal tag, rNT-proBNP was isolated with high purity (≥ 98%) by carboxymethyl cation exchange chromatography. The final yield of purified rNT-proBNP (97.5 mg/l of bacterial culture; 3.25 mg/g of wet cell) was 55-fold higher than that reported in previous studies (0.5-1.75 mg/l of bacterial culture). Furthermore, the high cell density E. coli fed-batch culture enabled high-level production of rNT-proBNP in the order of grams per liter. The purified rNT-proBNP was detected by enzyme-linked immunosorbent assay and chemiluminescence enzyme immunoassay using commercial monoclonal antibodies recognizing different epitopes, showing a linear dose-response relationship in the range of tested concentrations (slope = 3.58 and r2 = 0.995). These results demonstrate the efficiency of our process for mass producing (gram-to-liter level) rNT-proBNP with acceptable analytical performance.

Enhanced Transdermal Drug Delivery by Sonophoresis and Simultaneous Application of Sonophoresis and Iontophoresis.

Transdermal drug delivery has advantages of topical drug administration compared to the other conventional administration methods. However, the skin penetration of drugs is limited by the barrier properties of stratum corneum. The combinational strategy has been investigated to improve the skin permeability of the drug. For this study, we devised an improved device that can perform not only the single application of sonophoresis or iontophoresis but also the simultaneous application. The enhancement effect of sonophoresis was evaluated for various cosmeceutical drugs using a Franz diffusion cell. The enhancement ratio of niacinamide and retinol with sonophoresis was increased to 402% and 292%, respectively. The relationship was found between the enhancement effect of sonophoresis and the physicochemical properties of drugs. In particular, the simultaneous treatment of sonophoresis and iontophoresis enhanced skin penetration of glutamic acid to 240% using the fabricated device. The simultaneous application showed significantly higher enhancement ratio than application of sonophoresis or iontophoresis alone. Moreover, the improved device achieved skin penetration enhancement of various cosmeceutical drugs with lower intensity and a short application time. This combined strategy of transdermal physical enhancement methods is advantageous in terms of decline in energy density, thereby reducing the skin irritation. The miniaturized device with sonophoresis and iontophoresis is a promising approach due to enhanced transdermal drug delivery and feasibility of self-administration in cosmetic and therapeutic fields.

Drug-coated microneedles for rapid and painless local anesthesia.

This study showed that drug-coated PLLA (Poly (L-lactide)) microneedle arrays can induce rapid and painless local anesthesia. Microneedle arrays were fabricated using a micro-molding technique, and the needle tips were coated with 290.6 ± 45.9 µg of lidocaine, the most widely used local anesthetic worldwide. A dip-coating device was newly designed for the coating step using an optimized coating formulation. Lidocaine coated on the arrays was released rapidly into PBS within 2 min, and its stability in storage lasted 3 weeks at 4, 25, and 37°C. Furthermore, the microneedle arrays showed consistent in vitro skin penetration and delivered 200.8 ± 43.9, 224.2 ± 39.3, and 244.1 ± 19.6 µg of lidocaine into the skin 1, 2, and 5 min after application with a high delivery efficiency of 69, 77, and 84%. Compared to a commercially available topical anesthetic EMLA® cream, a 22.0, 13.6, and 14.0-fold higher amount of lidocaine was delivered into the skin. Note, in vitro skin permeation of Lidocaine was also notably enhanced by a 2-min-application of the lidocaine-coated microneedle arrays. Altogether, these results suggest that the biocompatible lidocaine-coated PLLA microneedle arrays could provide significantly rapid local anesthesia in a painless manner without any of the issues from topical applications or hypodermic injections of local anesthetics.

Oral Gavage Delivery of PR8 Antigen with ß-Glucan-Conjugated GRGDS Carrier to Enhance M-Cell Targeting Ability and Induce Immunity.

Oral gavage is known as one of most convenient routes for therapeutic administration in comparison with other available routes such as intravenous, intra muscular, suppository, etc. An oral vaccine delivery system has additional potential as it may provide a convenient way to prevent infectious diseases by introducing optimum immunization in mucus. Although oral vaccine delivery has attracted tremendous interest in vaccine delivery research, various limitations have prevented its rate of progress up to the level that was initially expected. However, the major problems of oral vaccine delivery are vaccine instability and lack of absorbability, resulting from degradation of the sophisticated antigens in the acidic medium in the stomach. In order to obtain adequate microfold-cell (M-cell) targeting and uptake, the therapeutic material is required to pass through the stomach and reach the small intestine without degradation. In this project, we have introduced a conjugate of ß-glucan and Glycine-Arginine-Glycine-Aspartic acid-Serine (GRGDS) that is effective for simultaneous protection of the antigen (PR8) and M-cell targeting. According to the experimental results, the cationic ß-glucan-GRGDS conjugate can encapsulate a certain amount of anionic PR8 through electrostatic interaction, which forms nanoparticles with a range of diameter of 200-250 nm. Also, the PR8 incorporated nanoparticles showed high cell viability and stability in diverse environments. Finally, excellent M-cell targeting ability was verified in an in vitro M-cell model. Most importantly, the in vivo test obviously demonstrated the superiority of this system, which significantly increases antibody concentration in serum, intestine, and mucus as measured 21 days after immunization.

Optimization of volatile fatty acids and hydrogen production from Saccharina japonica: acidogenesis and molecular analysis of the resulting microbial communities.

Response surface methodology (RSM) was used to optimize the production of volatile fatty acids (VFAs) and hydrogen from mixed anaerobic cultures of Saccharina japonica with respect to two independent variables: methanogenic inhibitor concentration and temperature. The effects of four methanogenic inhibitors on acidogenic processes were tested, and qualitative microbial analyses were carried out. Escherichia, Acinetobacter, and Clostridium were the most predominant genera in samples treated with chloroform (CHCl3), iodoform (CHI3), 2-bromoethanesulfonate (BES), or ß-cyclodextrin (ß-CD), respectively. RSM showed that the production of VFAs reached a peak of 12.5 g/L at 38.6 °C in the presence of 7.4 g/L ß-CD; these were the conditions under which hydrogen production was also nearly maximal. The quantitative polymerase chain reaction (qPCR) showed that shifts in the bacterial community population correlated with the concentrations of ß-CD indicating that this compound effectively inhibited methanogens.

A comprehensive study on volatile fatty acids production from rice straw coupled with microbial community analysis.

Rice straw is one of the most abundant renewable energy sources available. Through anaerobic acidogenesis, the substance of rice straw can be converted to volatile fatty acids (VFAs). VFAs itself is of value and is a precursor to biofuels. Hence, it can be converted to mixed alcohols by addition of hydrogen, and biodiesel can be produced as a carbon source for oleaginous microorganism. To maximize VFAs production during anaerobic digestion (AD), response surface analysis (RSM) was carried out with respect to temperature, substrate concentration, and pH variables. Optimization results showed maximal VFAs concentration of 12.37 g/L at 39.23 °C, 52.85 g/L of rice straw, and pH 10. In quantification of microbial community by quantitative polymerase chain reaction, the bacterial profile showed that the growth of methanogens was effectively inhibited by methanogenic inhibitors. Furthermore, 454 pyrosequencing showed that members of the Ruminococcaceae family, capable of hydrolyzing lignocellulosic biomass, were the most dominant species in many RSM trials. This study provided a useful insight on the biological improvement of AD performance through the combinational linkage between process parameters and microbial information.

Enhancement of volatile fatty acids production from rice straw via anaerobic digestion with chemical pretreatment.

Rice straw is one of the most abundant renewable biomass sources and was selected as the feedstock for the production of volatile fatty acids (VFAs) from which microbial biodiesel can be produced. Two kinds of chemical pretreatments involving nitric acid and sodium hydroxide were investigated at 150 °C with 20 min of reaction time. The nitric acid pretreatment generated the most hemicellulose hydrolyzate, while significant reduction of the lignin occurred with sodium hydroxide pretreatment. Anaerobic digestion of 20 g/L rice straw yielded 6.00 and 7.09 g VFAs/L with 0.5% HNO3 and 2% NaOH, respectively. The VFAs yield with 2% NaOH was 0.35 g/g.

Tumor phagocytosis-driven STING activation invigorates antitumor immunity and reprograms the tumor micro-environment.

Immunogenic cell death (ICD) holds the potential for in situ tumor vaccination while concurrently eradicating tumors and stimulating adaptive immunity. Most ICD inducers, however, elicit insufficient immune responses due to negative feedback against ICD biomarkers, limited infiltration of antitumoral immune cells, and the immunosuppressive tumor micro-environment (TME). Recent findings highlight the pivotal roles of stimulators of interferon gene (STING) activation, particularly in stimulating antigen-presenting cells (APCs) and TME reprogramming, addressing ICD limitations. Herein, we introduced 'tumor phagocytosis-driven STING activation', which involves the activation of STING in APCs during the recognition of ICD-induced cancer cells. We developed a polypeptide-based nanocarrier encapsulating both doxorubicin (DOX) and diABZI STING agonist 3 (dSA3) to facilitate this hypothesis in vitro and in vivo. After systemic administration, nanoparticles predominantly accumulated in tumor tissue and significantly enhanced anticancer efficacy by activating tumor phagocytosis-driven STING activation in MC38 and TC1 tumor models. Immunological activation of APCs occurred within 12 h, subsequently leading to the activation of T cells within 7 days, observed in both the TME and spleen. Furthermore, surface modification of nanoparticles with cyclic RGD (cRGD) moieties, which actively target integrin αvß3, enhances tumor accumulation and eradication, thereby verifying the establishment of systemic immune memory. Collectively, this study proposes the concept of tumor phagocytosis-driven STING activation and its effectiveness in generating short-term and long-term immune responses.

Translocation of cell penetrating peptides on Chlamydomonas reinhardtii.

Engineering of algal cells by delivering macromolecules through cell wall and plasma membrane presents many difficulties with the conventional methods. Recent research has shown that a new delivery method, namely cell penetrating peptide (CPP), has the ability to translocate into animal, plant, fungal, and bacterial cells. This study reports the apparent translocation of CPPs into algal cells of Chlamydomonas reinhardtii and the successful delivery of the conjugated fluorochrome. Although translocation efficiency was specific to each CPP studied, pVEC (peptide vascular endothelial cadherin) showed the highest translocation efficiency in comparison with penetratin (PEN), trans-activating transcriptional (TAT) peptide, and transportan (TRA). The maximum translocation of pVEC into the algal cell was reached in 15 min of incubation at 25°C. More importantly, translocation with pVEC demonstrated an absence of cytotoxicity. Thus, we suggested that pVEC is an attractive candidate for delivering macromolecules into algal cells for use in industrial applications.

DNA vaccination in the skin using microneedles improves protection against influenza.

In this study, we tested the hypothesis that DNA vaccination in the skin using microneedles improves protective immunity compared to conventional intramuscular (i.m.) injection of a plasmid DNA vaccine encoding the influenza hemagglutinin (HA). In vivo fluorescence imaging demonstrated the expression of a reporter gene delivered to the skin using a solid microneedle patch coated with plasmid DNA. Vaccination at a low dose (3 µg HA DNA) using microneedles generated significantly stronger humoral immune responses and better protective responses post-challenge compared to i.m. vaccination at either low or high (10 µg HA DNA) dose. Vaccination using microneedles at a high (10 µg) dose further generated improved post-challenge protection, as measured by survival, recall antibody-secreting cell responses in spleen and bone marrow, and interferon (IFN)-γ cytokine T-cell responses. This study demonstrates that DNA vaccination in the skin using microneedles induces higher humoral and cellular immune responses as well as improves protective immunity compared to conventional i.m. injection of HA DNA vaccine.

Apoptosis/Necroptosis Inducing Thiazole-Containing Artificial Polypeptide for Immunogenic Cell Death of Cancer.

Immunogenic cell death (ICD) has emerged as a promising approach to cancer immunotherapy. During ICD, cancer cell death and the release of damage-associated molecular pattern (DAMP) signals occur simultaneously. Increased production of reactive oxygen species (ROS) and severe endoplasmic reticulum stress are necessary for enhanced ICD. Furthermore, the levels of ROS and reduced glutathione (GSH) are involved in various cell death mechanisms. The thiazole ring structure has gained considerable interest as a functional moiety for anticancer agents. This study designed and synthesized a positively charged cell-penetrating polypeptide with a thiazole functional moiety (NS). The NS internalizes into the cancer cells through direct penetration and endo-lysosomal escape. The NS induces mitochondrial depolarization and ER stress in a concentration-dependent manner, leading to a significant ROS production and GSH depletion. Consequently, the ICD of cancer cells is activated, resulting in the release of DAMP signals. Furthermore, NS causes a shift in the cell death pathway from apoptosis to necroptosis as the concentration increases. In this study, we confirmed the possibility of NS as a promising ICD inducer that can be used while varying the concentration according to the cancer type.

Intracellular protein delivery and gene transfection by electroporation using a microneedle electrode array.

The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, intracellular delivery of molecules and transfection with plasmid DNA by electroporation is presented using a novel microneedle electrode array designed for the targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50% of cells and transfection of 12% of cells with a gene for green fluorescent protein is demonstrated at high cell viability. It is concluded that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA, and other biopharmaceuticals.

Carbon-based nanostructures for cancer therapy and drug delivery applications.

Synthesis, design, characterization, and application of carbon-based nanostructures (CBNSs) as drug carriers have attracted a great deal of interest over the past half of the century because of their promising chemical, thermal, physical, optical, mechanical, and electrical properties and their structural diversity. CBNSs are well-known in drug delivery applications due to their unique features such as easy cellular uptake, high drug loading ability, and thermal ablation. CBNSs, including carbon nanotubes, fullerenes, nanodiamond, graphene, and carbon quantum dots have been quite broadly examined for drug delivery systems. This review not only summarizes the most recent studies on developing carbon-based nanostructures for drug delivery (e.g. delivery carrier, cancer therapy and bioimaging), but also tries to deal with the challenges and opportunities resulting from the expansion in use of these materials in the realm of drug delivery. This class of nanomaterials requires advanced techniques for synthesis and surface modifications, yet a lot of critical questions such as their toxicity, biodistribution, pharmacokinetics, and fate of CBNSs in biological systems must be answered.

Enhanced Immunogenic Cell Death by Apoptosis/Ferroptosis Hybrid Pathway Potentiates PD-L1 Blockade Cancer Immunotherapy.

Even though chemotherapy regimens for treating cancer by inducing apoptosis are extensively utilized, their therapeutic effect is hindered by multiple limitations. Thus, a combination of other types of anticancer modalities is urgently needed. Herein, a tannic acid (TA)-Fe3+-coated doxorubicin (DOX)-encapsulated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (ammonium salt) (DSPE-PEG) micelle (TFDD) for apoptosis/ferroptosis-mediated immunogenic cell death (ICD) is reported. By coating TA-Fe3+ on the surface of DOX-loaded micelles, an apoptotic agent and a ferroptotic agent are simultaneously delivered into the cancer cells and induce cell death. Furthermore, the intracellular oxidative environment generated by the apoptosis/ferroptosis hybrid pathway stimulates the endoplasmic reticulum (ER) and leads to ICD induction. The in vivo results show that the combination treatment of TFDD and anti-programmed death-ligand 1 antibodies (anti-PD-L1) considerably inhibits tumor growth and improves antitumor immunity by activating CD4+ and CD8+ T cells and decreasing the ratio of regulatory T cells (Treg) to CD4+ T cells. This study suggests that the apoptosis/ferroptosis-mediated ICD inducer may offer a potent strategy for enhanced cancer immunotherapy.

Engineered Nanoparticles inside a Microparticle Oral System for Enhanced Mucosal and Systemic Immunity.

Antigen delivery through an oral route requires overcoming multiple challenges, including gastrointestinal enzymes, mucus, and epithelial tight junctions. Although each barrier has a crucial role in determining the final efficiency of the oral vaccination, transcytosis of antigens through follicle-associated epithelium (FAE) represents a major challenge. Most of the research is focused on delivering an antigen to the M-cell for FAE transcytosis because M-cells can easily transport the antigen from the luminal site. However, the fact is that the M-cell population is less than 1% of the total gastrointestinal cells, and most of the oral vaccines have failed to show any effect in clinical trials. To challenge the current dogma of M-cell targeting, in this study, we designed a novel tandem peptide with a FAE-targeting peptide at the front position and a cell-penetrating peptide at the back position. The tandem peptide was attached to a smart delivery system, which overcomes the enzymatic barrier and the mucosal barrier. The result showed that the engineered system could target the FAE (enterocytes and M-cells) and successfully penetrate the enterocytes to reach the dendritic cells located at the subepithelium dome. There was successful maturation and activation of dendritic cells in vitro confirmed by a significant increase in maturation markers such as CD40, CD86, presentation marker MHC I, and proinflammatory cytokines (TNF-α, IL-6, and IL-10). The in vivo results showed a high production of CD4+ T-lymphocytes (helper T-cell) and a significantly higher production of CD8+ T-lymphocytes (killer T-cell). Finally, the production of mucosal immunity (IgA) in the trachea, intestine, and fecal extracts and systemic immunity (IgG, IgG1, and IgG2a) was successfully confirmed. To the best of our knowledge, this is the first study that designed a novel tandem peptide to target the FAE, which includes M-cells and enterocytes rather than M-cell targeting and showed that a significant induction of both the mucosal and systemic immune response was achieved compared to M-cell targeting.

Intradermal vaccination with influenza virus-like particles by using microneedles induces protection superior to that with intramuscular immunization.

Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (