A multifunctional platform with single-NIR-laser-triggered photothermal and NO release for synergistic therapy against multidrug-resistant Gram-negative bacteria and their biofilms.

BACKGROUND: Infectious diseases caused by multidrug-resistant (MDR) bacteria, especially MDR Gram-negative strains, have become a global public health challenge. Multifunctional nanomaterials for controlling MDR bacterial infections via eradication of planktonic bacteria and their biofilms are of great interest. RESULTS: In this study, we developed a multifunctional platform (TG-NO-B) with single NIR laser-triggered PTT and NO release for synergistic therapy against MDR Gram-negative bacteria and their biofilms. When located at the infected sites, TG-NO-B was able to selectively bind to the surfaces of Gram-negative bacterial cells and their biofilm matrix through covalent coupling between the BA groups of TG-NO-B and the bacterial LPS units, which could greatly improve the antibacterial efficiency, and reduce side damages to ambient normal tissues. Upon single NIR laser irradiation, TG-NO-B could generate hyperthermia and simultaneously release NO, which would synergistically disrupt bacterial cell membrane, further cause leakage and damage of intracellular components, and finally induce bacteria death. On one hand, the combination of NO and PTT could largely improve the antibacterial efficiency. On the other hand, the bacterial cell membrane damage could improve the permeability and sensitivity to heat, decrease the photothermal temperature and avoid damages caused by high temperature. Moreover, TG-NO-B could be effectively utilized for synergistic therapy against the in vivo infections of MDR Gram-negative bacteria and their biofilms and accelerate wound healing as well as exhibit excellent biocompatibility both in vitro and in vivo. CONCLUSIONS: Our study demonstrates that TG-NO-B can be considered as a promising alternative for treating infections caused by MDR Gram-negative bacteria and their biofilms.

Betaine Improves Polymer-Grade D-Lactic Acid Production by Sporolactobacillus inulinus Using Ammonia as Green Neutralizer.

The traditional CaCO3-based fermentation process generates huge amount of insoluble CaSO4 waste. To solve this problem, we have developed an efficient and green D-lactic acid fermentation process by using ammonia as neutralizer. The 106.7 g/L of D-lactic acid production and 0.89 g per g of consumed sugar were obtained by Sporolactobacillus inulinus CASD with a high optical purity of 99.7% by adding 100 mg/L betaine in the simple batch fermentation process. The addition of betaine was experimentally proven to protect cell at high concentration of ammonium ion, increase the D-lactate dehydrogenase specific activity and thus promote the production of D-lactic acid.

Effect of site-specific PEGylation on the fibrinolytic activity, immunogenicity, and pharmacokinetics of staphylokinase.

The bacterial plasminogen-activator staphylokinase (Sak) is a promising thrombolytic agent for treating acute myocardial infarction. To effectively reduce the immunogenicity of Sak while maintaining its fibrinolytic activity, site-specific PEGylation was performed in the present study. The chemoselective cysteine PEGylation site was selected within an immunodominant region (amino acid residues 71-87) using an in silico approach. The PEGylated Sak variants prepared in this study showed a purity of >97.0%. PEGylation at Position 80 resulted in a Sak variant Sak(E80C-PEG) which has similar fibrinolytic activity and thermostability compared with the native recombinant staphylokinase (r-Sak). The immunogenicity of Sak(E80C-PEG) in guinea pigs was greatly reduced compared with the native r-Sak. Furthermore, preliminary pharmacokinetic results suggested that the plasma clearance of Sak(E80C-PEG) from the blood stream of rabbit was significantly decreased compared with that of r-Sak, resulting in a 2.8-fold increase of initial half-life and a 3.8-fold increase of systemic availability. In summary, these results demonstrated that site-specific PEGylation yielded a novel Sak variant Sak(E80C-PEG) with remarkable advantages over the unmodified Sak.

Co-delivery of polyinosinic:polycytidylic acid and flagellin by poly(lactic-co-glycolic acid) MPs synergistically enhances immune response elicited by intranasally delivered hepatitis B surface antigen.

The aim of the present work was to investigate the synergistic effect between toll-like receptor (TLR) 3 ligand polyinosinic:polycytidylic acid (pI:C) and TLR5 ligand flagellin (FLN) on immune responses induced by nasally delivered hepatitis B virus surface antigen (HBsAg). Mannan and chitosan oligosaccharide-modified, pH-responsive poly(lactic-co-glycolic acid) (MC-PLGA) microparticles (MPs) containing HBsAg, FLN, pI:C or both ligands were prepared with a double-emulsion method. In vitro uptake experiments show that cellular uptake of MC-PLGA MPs by macrophages was through energy-dependent, receptor-mediated endocytosis mechanism. After uptake of MPs by macrophages, MC-PLGA MPs existed both in the endo-some and in the cytoplasm. FLN and pI:C in solution or MP formulation could synergize to activate macrophages and induce higher pro-inflammatory cytokines interleukin (IL)-6, IL-12, interferon-γ and anti-inflammatory cytokines IL-10 compared to single TLR ligand (P<0.05). In vivo immunogenicity studies indicated that co-delivery of FLN and pI:C within MC-PLGA MPs synergistically induced higher serum anti-HBsAg IgG levels and Th1 cytokine levels compared with MC-PLGA MPs encapsulated single TLR ligand plus MPs encapsulated HBsAg (P<0.05). These results suggest that synergic TLR3 and TLR5 stimulation might be a promising novel tool for nasally delivered HBsAg.

Protective efficacy of PLGA microspheres loaded with divalent DNA vaccine encoding the ompA gene of Aeromonas veronii and the hly gene of Aeromonas hydrophila in mice.

In the present study, poly (lactic-co-glycolic) acid (PLGA) was used as a carrier for a divalent fusion DNA vaccine encoding the Aeromonas veronii outer membrane protein A (ompA) and Aeromonas hydrophila hemolysins (hly) protein. The recombinant pET-28a-ompA-hly was constructed by inserting the ompA gene and hly gene into a pET-28a expression vector. Loading of ompA-hly antigen module on PLGA microspheres were accomplished by water-in-oil-in-water (W/O/W) encapsulation. The molecular weight and specificity of pET-28a-ompA-hly were detected by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The microspheres showed an average particle size of 100-150 µm and a loading efficiency (LE) of 68.8%. Mice received ompA-hly antigen-loaded PLGA microspheres by intraperitoneal or intragastric administration mounted strong and sustained IgG response, which was significantly higher (p<0.05) than those achieved by pET-28a-ompA-hly antigen alone. OmpA-hly antigen-loaded PLGA microsphere vaccine uniquely conferred a long lasting (30 days) sterile immunity against challenge infection. Results indicated that ompA-hly antigen-loaded PLGA microsphere vaccine is a qualified candidate vector system for sterile protective immunity against A. hydrophila and A. veronii infections.