Immunoregulation and antioxidant activities of a novel acidic polysaccharide from Radix Paeoniae Alba.

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, immunomodulatory, cancer, and other diseases. In this paper, a novel acidic polysaccharide RPAPS purified from Radix Paeoniae Alba was evaluated for its structural features and potential of immunomodulatory and antioxidant activities. RPAPS (molecular weight: 1.0× 105 Da) was mainly composed of α-(1 → 4)-Glcp, α-Arap, α-Galp, α-Rhap, ß-D-Glcp, α-(1 → 6)-linked Glcp and GalA. Immunological tests indicated that RPAPS could improve RAW264.7 phagocytic activity and LPS-induced splenocyte proliferation. For antioxidant activities, RPAPS showed reducing power and DPPH scavenging activity in dose dependent. Moreover, RPAPS could significantly protect the PC12 cells from H2O2 damage. These data implied polysaccharides RPAPS had the potential to be novel natural antioxidative and immunopotentiating agents for using in functional foods or medicine.

Hyaluronic acid conjugation facilitates clearance of intracellular bacterial infections by streptomycin with neglectable nephrotoxicity.

Antibiotics such as ß-lactams and aminoglycosides are often subtherapeutic to intracellular infections due to their high hydrophilicity, resulting in low effectiveness against intracellular pathogens and the emergence of antibiotic resistance. Here we reported that an endogenous aminoglycan, hyaluronic acid could be an effective carbohydrate carrier of the aminoglycoside antibiotic, streptomycin against intracellular pathogens. This conjugation could enhance phagocytic activity, and facilitated the entry of streptomycin into host cells via a CD44-mediated pathway. It appeared that this conjugate could clear intracellular bacteria in phagocytic or nonphagocytic cells in a short-term therapy (4 h) at a lower effective dose. In addition, this conjugate was more efficient in reducing bacteria burden in an in vivo acute infection model than streptomycin did. Interestingly, subcutaneous injection of this conjugate at an excess amount had undetectable side effects such as nephrotoxicity. These results suggested that hyaluronic acid might be an efficient Trojan horse for the delivery of hydrophilic antibiotics to deal with intracellular infections.

Synthesis, characterization and anticancer activity of tanshinone I grafted low molecular chitosan.

In this study, we synthesized a novel water-soluble low molecular chitosan (LMC) derivative through Vilsmeier reaction and reductive amination reaction. The derivative was characterized by UV-visible spectroscopy, 1H NMR, FTIR and SEM techniques. The results showed that the derivative effectively reduced the cell viability rate, inhibited cell metastasis, induced cell apoptosis and dissipated mitochondrial membrane potential (ΔΨm). Moreover, the antitumor activity was strengthened with the increase of the degree of substitution of tanshinone I (TanI). These findings provided important support for developing new water-soluble antitumor agent and expand the scope of application of LMC.

Lysozyme Associated Liposomal Gentamicin Inhibits Bacterial Biofilm.

Bacteria on living or inert surfaces usually form biofilms which make them highly resistant to antibiotics and immune clearance. Herein, we develop a simple approach to overcome the above conundrum through lysozyme-associated liposomal gentamicin (LLG). The association of lysozyme to the surface of liposomes can effectively reduce the fusion of liposomes and undesirable payload release in regular storage or physiological environments. The LLG was more effective at damaging established biofilms and inhibiting biofilm formation of pathogens including Gram-positive and Gram-negative bacteria than gentamicin alone. This strategy may provide a novel approach to treat infections due to bacterial biofilm.

Chitosan conjugation enables intracellular bacteria susceptible to aminoglycoside antibiotic.

Most chronic infections are difficult to eradicate because bacteria capable of surviving in host-infected cells may be protected from the killing actions of antibiotics, leading to therapy failures and disease relapses. Here we demonstrated that covalent-coupling chitosan to streptomycin significantly improved intracellular bactericidal capacity towards multiple organisms within phagocytic or nonphagocytic cells. Structure-activity relationship investigations indicated that antibiotic contents, molecular size and positive charges of the conjugate were the key to retain this intracellular bactericidal activity. Mechanistic insight demonstrated the conjugate was capable to target and eliminate endocytic or endosomal escaped bacteria through facilitating the direct contact between the antibiotic and intracellular organism. In vivo acute infection models indicated that compared to equal dose of the antibiotic, chitosan-streptomycin (C-S) conjugate and especially the human serum album binding chitosan-streptomycin conjugate (HCS) complex formed by human serum album and C-S conjugate greatly decreased the bacteria burden in the spleen and liver in both wild type and immuno-suppressive mice. Furthermore, the HCS complex remarkably reduced mortality of infected TLR2 deficient mice, mimicking immune-compromised persons who were more susceptible to bacterial infections. These findings might open up a new avenue to combat intracellular bacterial infection by aminoglycosides antibiotics at a lower effective dose.

Structural characterization and biological activities of two α-glucans from radix paeoniae alba.

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, cancer, and other diseases. In this study, two polysaccharides RPAPW1 and RPAPW2 were isolated from Radix Paeoniae Alba by DEAE-52 cellulose chromatography and G-25 sephadex. According to physicochemical methods, NMR and methylation analysis, RPAPW1 and RPAPW2 were established to be α-glucans consisting of predominant 4-linked α- Glc residues branched at O-6 and contained trace amount of protein and uronic acid. Immunological tests indicated that RPAPW1, RPAPW2 and could promote splenocyte proliferation and RAW264.7 phagocytic activity. In vitro, RPAPW1 and RPAPW2 elicited a week reducing power, DPPH scavenging activity and could not protect the PC12 cells from H2O2 damage. These data implied polysaccharides RPAPW1 and RPAPW2 had the potential to be a natural immunopotentiating and antioxidant supplement for preparing functional foods and nutraceuticals.

Unveiling the Mode of Action of Two Antibacterial Tanshinone Derivatives.

In this study, 2-(N-pyrrolidine-alkyl) tanshinones bearing pyrrolidine groups were synthesized and the antibacterial mechanism was explored. These derivatives selectively elicited antibacterial activity against Gram-positive bacteria. Moreover, their antibacterial activities were time-, concentration-dependent and persistent. It appeared that Fenton-mediated hydroxyl radicals were involved, and the disruption of cell membranes was observed. This study indicates that 2-(N-pyrrolidine-alkyl) tanshinones might be potential candidates as antibacterial agents.

Alteration in immune responses toward N-deacetylation of hyaluronic acid.

Hyaluronic acid (HA) is an ubiquitous nonsulfated glycosaminoglycan of the extracellular matrix in all mammalian connective tissues. Along with the age growth, HA will lose its N-acetyl groups in vivo; however, the significance of this physiological process remains largely unknown. Herein, three highly N-deacetylated HAs (dHAs), dHA-5 kDa (Mw: 5 kDa, DD: 100%), dHA-16 kDa (Mw: 16 kDa, DD: 94%) and dHA-110 kDa (Mw: 110 kDa, DD: 72%), were generated after hydrazinolysis. Their capability in the activation of antigen-presenting cells (APCs) was compared with that of their respective HAs. Our results demonstrated that both HAs and dHAs could activate the nuclear factor-kappa B (NF-κB) transcription factor in APCs and induced cytokine production through the Toll-like receptor (TLR)/MyD88 pathway. Notably, the capacity of dHAs in cytokine induction was much lower than that of HAs. In addition, the TLR-2 pathway was much involved following the appearance of zwitterionic motifs in dHAs. Thus, our findings highlight that N-deacetylation renders HA divergences in immune response, which might be implicated in age-induced functional change in endogenous glycosaminoglycans due to the structural modification in vivo.

Chitosan improves anti-biofilm efficacy of gentamicin through facilitating antibiotic penetration.

Antibiotic overuse is one of the major drivers in the generation of antibiotic resistant "super bugs" that can potentially cause serious effects on health. In this study, we reported that the polycationic polysaccharide, chitosan could improve the efficacy of a given antibiotic (gentamicin) to combat bacterial biofilms, the universal lifestyle of microbes in the world. Short- or long-term treatment with the mixture of chitosan and gentamicin resulted in the dispersal of Listeria monocytogenes (L. monocytogenes) biofilms. In this combination, chitosan with a moderate molecular mass (~13 kDa) and high N-deacetylation degree (~88% DD) elicited an optimal anti-biofilm and bactericidal activity. Mechanistic insights indicated that chitosan facilitated the entry of gentamicin into the architecture of L. monocytogenes biofilms. Finally, we showed that this combination was also effective in the eradication of biofilms built by two other Listeria species, Listeria welshimeri and Listeria innocua. Thus, our findings pointed out that chitosan supplementation might overcome the resistance of Listeria biofilms to gentamicin, which might be helpful in prevention of gentamicin overuse in case of combating Listeria biofilms when this specific antibiotic was recommended.

An "in control" hyaluronic acid nanogel with light-cleavable for rational use of antibiotics.

The consequences caused by bacterial resistance are becoming more and more serious. The rate of antibiotic development is far behind the rate of bacterial resistance, so it is urgent to develop a new drug system. In this study, photoresponsive nanogels based on hyaluronic acid were prepared and loaded with ciprofloxacin as a model molecule. The results showed that the nanogels had the advantages of high stability and good cytocompatibility. The inhibition effect of drug-loaded nanogels after light irradiation on the growth of Staphylococcus aureus and Salmonella typhimurium was significantly better than that before light irradiation, and ciprofloxacin could be released on demand and in control. This strategy is of great significance to reduce the unnecessary use of antibiotics and weaken bacterial resistance.

The application of pH-responsive hyaluronic acid-based essential oils hydrogels with enhanced anti-biofilm and wound healing.

pH could play vital role in the wound healing process due to the bacterial metabolites, which is one essential aspect of desirable wound dressings lies in being pH-responsive. This work has prepared a degradable hyaluronic acid hydrogel dressing with wound pH response-ability. The aldehyde-modified hyaluronic acid (AHA) was obtained, followed by complex mixture formation of eugenol and oregano antibacterial essential oil in the AHA-CMCS hydrogel through the Schiff base reaction with carboxymethyl chitosan (CMCS). This hydrogel composite presents pH-responsiveness, its disintegration mass in acidic environment (pH = 5.5) is 4 times that of neutral (pH = 7.2), in which the eugenol release rate increases from 37.6 % to 82.1 %. In vitro antibacterial and in vivo wound healing investigations verified that hydrogels loaded with essential oils have additional 5 times biofilm removal efficiency, and significantly accelerate wound healing. Given its excellent anti-biofilm and target-release properties, the broad application of this hydrogel in bacteria-associated wound management is anticipated.

Acetolactate Decarboxylase as an Important Regulator of Intracellular Acidification, Morphological Features, and Antagonism Properties in the Probiotic Lactobacillus reuteri.

SCORE: This study identifies the coding gene (aldB) of acetolactate decarboxylase (ALDC) as an important regulatory gene of the intracellular pH in Lactobacillus reuteri (L. reuteri), uncovering the important role of ALDC in regulating intracellular pH, morphological features, and antagonism properties in the probiotic organism L. reuteri. METHODS AND RESULTS: The aldB mutant (ΔaldB) of L. reuteri is established using the homologous recombination method. Compare to the wild-type (WT) strain, the ΔaldB strain shows a smaller body size, grows more slowly, and contains more acid in the cell cytoplasm. The survival rate of the ΔaldB strain is much lower in low pH and simulated gastric fluid (SGF) than that of the WT strain, but higher in simulated intestinal fluid (SIF). The antagonism test demonstrates the ΔaldB strain can inhibit Listeria monocytogenes (L. monocytogenes) and Salmonella more effectively than the WT strain. Additionally, there is a dramatic decrease in the adhesion rate of Salmonella to Caco-2 and HT-29 cells in the presence of the ΔaldB strain compared to the WT strain. Simultaneously analyze, the auto-aggregation, co-aggregation, cell surface hydrophobicity (CSH), hemolytic, temperature, NaCl, oxidative stress, and antibiotic susceptibility of the ΔaldB strain are consistent with the features of probiotics. CONCLUSION: This study highlights that the aldB gene plays a significant role in the growth and antibacterial properties of L. reuteri.

pH-Responsive Co-Assembled Peptide Hydrogel to Inhibit Drug-Resistant Bacterial Infection and Promote Wound Healing.

Drug-resistant bacterial infection and biofilm formation are the key inhibitors of wound healing, and new strategies are urgently needed to address these issues. In this study, we designed a pH-responsive co-assembled peptide hydrogel to inhibit Methicillin-resistant Staphylococcus aureus (MRSA) infection and promote wound healing. We synthesized a cationic short peptide (Nap-FFKKK) and a co-assembled hydrogel with curcumin at pH ∼ 7.8. The loaded curcumin was continuously released in a weak acid environment (pH ∼ 5.5). The lysine-rich cationic peptide inhibited biofilm formation in MRSA via electrostatic interaction with the negatively charged bacterial cell surface and, thus, provided a reinforcing antibacterial effect with curcumin. In vitro antibacterial experiments showed that the co-assembled system considerably reduced the minimum inhibitory concentration of curcumin against MRSA by 10-fold and promoted wound healing in a mouse model of MRSA-infected wounds. This study provides a simple and promising strategy to treat drug-resistant bacterial infections in wounds.

Evaluation of living bacterial therapy assisted by pH/reactive oxygen species dual-responsive sodium alginate-based hydrogel for wound infections.

The enhancement of antimicrobial wound dressings is of utmost importance in light of the escalating risk of antibiotic resistance caused by excessive antibiotic usage. Conventional antimicrobial materials eradicate pathogenic bacteria while impeding the proliferation of beneficial bacteria during the management of wound infections, thereby disturbing the equilibrium of the skin micro-ecosystem and engendering recurrent cutaneous complications. Lactobacillus rhamnosus (L.rha) is a probiotic that can inhibit the growth of certain pathogenic bacteria by secreting a large number of metabolites. In this paper, we synthesized a cross-linker (SPBA) with a boric acid molecule from succinic acid and 4-(bromomethyl)phenylboronic acid, which formed a boric acid ester bond with a diol on the natural polysaccharide sodium alginate (SA), and obtained a pH/reactive oxygen species (ROS) dual-responsive hydrogel (SA-SPBA) for loading L.rha to treat wound infections. The SA-SPBA@L.rha hydrogel improves the survival of L.rha during storage and has good injectability as well as self-healing properties. The hydrogel showed good biocompatibility, the antimicrobial effect increases in a dose-dependent manner, and it has a certain antioxidant and anti-inflammatory capacity, accelerating wound repair. The use of SA-SPBA@L.rha hydrogel provides a safe and effective strategy for the repair of skin wound infections.

Microenvironment-triggered cascade metal-polyphenolic nanozyme for ROS/NO synergistic hyperglycemic wound healing.

Wound infection of hyperglycemic patient often has extended healing period and increased probability due to the high glucose level. However, achieving precise and safe therapy of the hyperglycemic wound with specific wound microenvironment (WME) remains a major challenge. Herein, a WME-activated smart L-Arg/GOx@TA-Fe (LGTF) nanozymatic system composed of generally recognized as safe (GRAS) compound is engineered. The nanozymatic system combining metal-polyphenol nanozyme (tannic acid-Fe3+, TA-Fe) and natural enzyme (glucose oxidase, GOx) can consume the high-concentration glucose, generating reactive oxygen species (ROS) and nitric oxide (NO) in situ to synergistically disinfect hyperglycemia wound. In addition, glucose consumption and gluconic acid generation can lower glucose level to promote wound healing and reduce the pH of WME to enhance the catalytic activities of the LGTF nanozymatic system. Thereby, low-dose LGTF can perform remarkable synergistic disinfection and healing effect towards hyperglycemic wound. The superior biosafety, high catalytic antibacterial and beneficial WME regulating capacity demonstrate this benign GRAS nanozymatic system is a promising therapeutic agent for hyperglycemic wound.

Inorganic catalase-powered nanomotors with hyaluronic acid coating for pneumonia therapy.

Clinical therapy for widespread infections caused by Streptococcus pneumoniae (S. pneumoniae), such as community-acquired pneumonia, is highly challenging. As an important bacterial toxin, hydrogen peroxide (H2O2) secreted by S. pneumoniae can suppress the host's immune system and cause more severe disease. To address this problem, a hyaluronic acid (HA)-coated inorganic catalase-driven Janus nanomotor was developed, which can cleverly utilize and decompose H2O2 to reduce the burden of bacterial infection, and have excellent drug loading capacity. HA coating prevents rapid leakage of loaded antibiotics and improves the biocompatibility of the nanomaterials. The Janus nanomotor converted H2O2 into oxygen (O2), gave itself the capacity to move actively, and encouraged widespread dispersion in the lesion site. Encouragingly, animal experiments demonstrated that the capability of the nanomotors to degrade H2O2 contributes to diminishing the proliferation of S. pneumoniae and lung tissue damage. This self-propelled drug delivery platform provides a new therapeutic strategy for infections with toxin-secreting bacteria.

A decrease in moisture absorption-retention capacity of N-deacetylation of hyaluronic acid.

The linear non-sulfated glycosaminoglycan, hyaluronic acid (HA), is widely distributed throughout connective, epithelial and neural tissues etc., and is of great importance in tissue hydration, lubrication and cellular function. Along with the age growth, HA will lose its acetyl groups under action of HA N-deacetylase in vivo. However, the biological consequence of this physiological process remains largely unknown. Herein two highly N-deacetylated HAs, dHA-6 and dHA-10 were generated via the NH2NH2-HIO3 procedure. Their molecular weights were estimated to be 24 and 16 kDa by high performance gel-permeation chromatography (HPGPC), and the N-deacetylation degrees were 79.4 % and 93 % respectively, as determined by (1)H nuclear magnetic resonance (NMR). The study on moisture-absorption (Ra) and -retention (Rh) abilities demonstrated that the Ra values of dHAs under conditions of 81 % or 43 % relative humidity, as well as the Rh values of dHAs under dry condition or 43 % relative humidity, were significantly smaller than that of their respective re-N-acetylated products. The decline of moisture-absorption and -retention capacity after HA N-deacetylation were consistent with the appearance of unsolvated amides remained in the N-deacetylated products, as indicated by circular dichroism (CD) spectroscopy. Our findings implied that HA N-deacetylation, in addition to the decrease of HA contents in the elderly persons, might account for manifestations of naturally aged skin, such as laxity, sagging, and wrinkling.

Clearance of intracellular bacterial infections by hyaluronic acid-based ROS responsive drug delivery micelles.

Pathogenic bacteria residing inside cells could cause disruption of cellular metabolic balance. Therefore, basing on high oxidative stress response of the intracellular bacteria infected micro-environment, a novel amphipathic micelle (HATAD-TCS) was developed consisting of hyaluronic acid-derivative and reactive oxygen species (ROS) - responsive group and antibacterial agent triclosan (TCS). ROS-generating cinnamaldehyde (CA) was incorporated into ROS-cleavable linkages which are future linked to the 1-decylamine to form hydrophobicity. The cinnamaldehyde released did not just killed bacteria however, also maintained intracellular ROS levels. In this study, the HATAD-TCS micelles have been characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The HATAD-TCS micelles could release drug gradually upon exposure to endogenous ROS being caused by infected intracellular bacteria. Furthermore, the more promising therapeutic effect of the HATAD-TCS micelles was observed in a mouse pneumonia model. These results might highlight a ROS-responsive hyaluronic acid-based nanoparticle, which could effectively treat intracellular bacterial infections.

A 0D-2D Heterojunction Bismuth Molybdate-Anchored Multifunctional Hydrogel for Highly Efficient Eradication of Drug-Resistant Bacteria.

Due to the increasing antibiotic resistance and the lack of broad-spectrum antibiotics, there is an urgent requirement to develop fresh strategies to combat multidrug-resistant pathogens. Herein, defect-rich bismuth molybdate heterojunctions [zero-dimensional (0D) Bi4MoO9/two-dimensional (2D) Bi2MoO6, MBO] were designed for rapid capture of bacteria and synergistic photocatalytic sterilization. The as-prepared MBO was experimentally and theoretically demonstrated to possess defects, heterojunctions, and irradiation triple-enhanced photocatalytic activity for efficient generation of reactive oxygen species (ROS) due to the exposure of more active sites and separation of effective electron-hole pairs. Meanwhile, dopamine-modified MBO (pMBO) achieved a positively charged and rough surface, which conferred strong bacterial adhesion and physical penetration to the nanosheets, effectively trapping bacteria within the damage range and enhancing ROS damage. Based on this potent antibacterial ability of pMBO, a multifunctional hydrogel consisting of poly(vinyl alcohol) cross-linked tannic acid-coated cellulose nanocrystals (CPTB) and pMBO, namely CPTB@pMBO, is developed and convincingly effective against methicillin-resistant Staphylococcus aureus in a mouse skin infection model. In addition, the strategy of combining a failed beta-lactam antibiotic with CPTB@pMBO to photoinactivation with no resistance observed was developed, which presented an idea to address the issue of antibiotic resistance in bacteria and to explore facile anti-infection methods. In addition, CPTB@pMBO can reduce excessive proteolysis of tissue and inflammatory response by regulating the expression of genes and pro-inflammatory factors in vivo, holding great potential for the effective treatment of wound infections caused by drug-resistant bacteria.

Structural characterization of a homogalacturonan from Capparis spinosa L. fruits and anti-complement activity of its sulfated derivative.

A water-soluble polysaccharide CSPS-2B-2 with a molecular mass of 8.8 kDa, was obtained from the fruits of Capparis spinosa L. Chemical and NMR spectral analysis verified CSPS-2B-2 was a linear poly-(1-4)-α-D-galactopyranosyluronic acid in which 12.9±0.4% of carboxyl groups existed as methyl ester and 2.6±0.1% of D-GalpA residues were acetylated. A sulfated derivative Sul-2B-2 with a sulfation degree of 0.88±0.02 was prepared via the substitution of C-2 and/or C-3 of GalpA residues in CSPS-2B-2. Bioassay on the complement and coagulation system demonstrated that Sul-2B-2 (CH(50): 3.5±0.2 µg/mL) had a stronger inhibitory effect on the activation of complement system through the classic pathway than that of heparin (CH(50): 8.9±0.3 µg/mL). Interestingly, Sul-2B-2 at low dose even middle dose (for example 52 µg/mL) had no effect on coagulation system, which was totally different from heparin. Thus, our observation indicated that Sul-2B-2 was more efficient than heparin in inhibiting the activation of the complement system through classical pathway and exhibiting a relatively less anti-coagulant activity. These results suggested that the sulfated derivative Sul-2B-2 prepared from the homogalacturonan in the fruits of Capparis spinosa L, might be a promising drug candidate in case of necessary therapeutic complement inhibition.