Artificial Neutrophil-Mediated CEBPA-saRNA Delivery to Ameliorate ALI/ARDS.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) still faces great challenges due to uncontrollable inflammation disorders, complicated causes of occurrence, and high mortality. Small-activating RNA (saRNA) has emerged as a novel and powerful gene-activating tool that may be useful in the treatment of ALI/ARDS. However, effective saRNA therapy is still challenged by the lack of effective and safe gene delivery vehicles. In this study, we develop a type of artificial neutrophil that is used to deliver saRNAs for ALI/ARDS treatment. The saRNA targeting CCAAT-enhancer binding protein α (CEBPA-saRNA) is complexed with H1 histone and further camouflaged with neutrophil membranes (NHR). Interestingly, we are the first to find that the H1 histone possesses the most effective binding capability to saRNA, compared to other subtypes. The prepared NHR shows excellent physicochemical properties, effective cellular uptake by the inflammatory M1 macrophages, and efficient activation of CEBPA, leading to significant M2 polarization. NHR shows an extended circulation lifetime and high-level accumulation in the inflamed lungs. The in vivo experiments indicate that NHR ameliorates ALI in a mouse model. This type of artificial neutrophil shows powerful inflammatory inhibition both in vitro and in vivo, which opens a new avenue for the treatment of ALI/ARDS.

Self-crosslinking hyaluronic acid-based hydrogel with promoting vascularization and ROS scavenging for wound healing.

Skin wound dressings are commonly utilized for the treatment of skin injuries, as they effectively facilitate wound healing and possess anti-inflammatory and antibacterial properties. However, conventional dressings fail to inhibit ROS production and promote vascularization, leading to delayed wound healing. Here, we developed injectable self-crosslinking hydrogels through thiolated hyaluronic acid (HASH/rhCOLIII) with enhancing the ROS inhibitory capacity while preserving the cell adhesion ability of hyaluronic acid. Additionally, recombinant humanized collagen type III (rhCOLIII) is incorporated via electrostatic adsorption to further enhance mechanical strength and angiogenesis properties of the hydrogel. The HASH/rhCOLIII demonstrated excellent biocompatibility, remarkable ROS scavenging ability, as well as hemostatic and angiogenic properties. Cell experiment results show that HASH/rhCOLIII has excellent biocompatibility and can significantly promote angiogenesis. Animal experiments results showed that HASH/rhCOLIII exhibits anti-inflammatory effects, significantly accelerating wound healing in a full-thickness skin defect model. These findings highlight that HASH/rhCOLIII hydrogel holds great promise as an advanced dressing for effective wound healing.

Protamine-grafted carboxymethyl chitosan based hydrogel with adhesive and long-term antibacterial properties for hemostasis and skin wound healing.

In this study, we developed a tissue-adhesive and long-term antibacterial hydrogel consisting of protamine (PRTM) grafted carboxymethyl chitosan (CMC) (PCMC), catechol groups modified CMC (DCMC), and oxidized hyaluronic acid (OHA), named DCMC-OHA-PCMC. According to the antibacterial experiments, the PCMC-treated groups showed obvious and long-lasting inhibition zones against E. coli (and S. aureus), and the corresponding diameters varied from 10.1 mm (and 15.3 mm) on day 1 to 9.8 mm (and 15.3 mm) on day 7. The DCMC-OHA-PCMC hydrogel treated groups also exhibited durable antibacterial ability against E. coli (and S. aureus), and the antibacterial rates changed from 99.3 ± 0.21 % (and 99.6 ± 0.36 %) on day 1 to 76.2 ± 1.74 % (and 84.2 ± 1.11 %) on day 5. Apart from good mechanical and tissue adhesion properties, the hydrogel had excellent hemostatic ability mainly because of the grafted positive-charged PRTM. As the animal assay results showed, the hydrogel was conducive to promoting the deposition of new collagen (0.84 ± 0.03), the regeneration of epidermis (98.91 ± 6.99 µm) and wound closure in the process of wound repairing. In conclusion, the presented outcomes underline the prospective potential of the multifunctional CMC-based hydrogel for applications in wound dressings.

Quaternary ammonium carboxymethyl chitosan composite hydrogel with efficient antibacterial and antioxidant properties for promoting wound healing.

Multifunctional hydrogels have been developed to meet the various requirements of wound healing. Herein, an innovative hydrogel (QCMC-HA-PEG) was formed through the Schiff base reaction, composed of quaternary ammonium-modified carboxymethyl chitosan (QCMC), hyaluronic acid (HA), and 8-arms Polyethylene Glycol aldehyde (8-ARM-PEG-CHO). The resulting hydrogels exhibited good mechanical and adhesive properties with improved antibacterial efficacy against both Gram-positive and Gram-negative bacteria compared to CMC hydrogels. QCMC-HA-PEG hydrogels demonstrated remarkable adhesive ability in lap-shear test. Furthermore, the incorporation of MnO2 nanosheets into the hydrogel significantly enhanced its reactive oxygen species (ROS) scavenging and oxygen generation capabilities. Finally, experimental results from a full-thickness skin wound model revealed that the QCMC-HA-PEG@MnO2 hydrogel promoted skin epithelization, collagen deposition, and inflammatory regulation significantly accelerated the wound healing process. Therefore, QCMC-HA-PEG@MnO2 hydrogel could be a promising wound dressing to promote wound healing.

Functionalization of graphene oxide with amphiphilic block copolymer to enhance antibacterial activity.

Functionalization of GO with an amphiphilic block copolymer is designed with an aim to enhance its biocompatibility, however, long copolymer chains can screen the blade effect of GO to sacrifice its antimicrobial activities. To solve this problem, low molecular weight of poly(ethylene glycol) (PEG), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and their block copolymer were respectively introduced onto GO via an isophorone diisocyanate modified GO as a intermediate, followed by a solvent evaporation of an oil-in-water emulsion treatment (SE treatment) to induce block copolymer into polymer micelle via phase separation to refresh the sharp edges of GO. Block copolymer modified GO possessed similar dispersibility and stability to PEG modified GO, and even higher loading capacity of the hydrophobic drug than PHBV modified GO, illustrating its superior properties to homopolymer. PEG, PHBV and their block copolymer modified GO were nontoxic towards ATDC5 cells while cultured for 3 days and compatible with erythrocytes within 8 h. SE treatment enhanced greatly the loading capacity of the hydrophobic drug and the accumulative release reached 91.3% within 24 h. The inhibition zone of the block copolymer modified GO was 14.1 mm and 14.8 mm against E. coli and S. aureus, comparable to that of PEG modified GO. The bacterial reduction rate of the copolymer micelle modified GO was 87.1% and 82.7% towards E. coli and S. aureus, much greater than that of PEG, PHBV and their block copolymer modified GO at a concentration of 1 mg/mL. The antibiofilm capacity of the copolymer micelle modified GO were equal to that of PEG modified, demonstrating its great promise in tissue engineering application for repair of infected tissue defects.

PDGF-AA loaded photo-crosslinked chitosan-based hydrogel for promoting wound healing.

Chitosan-based hydrogels are considered to be ideal materials for promoting wound healing due to their nontoxic, biodegradable, and biocompatible properties. However, the weak mechanical strength, hemostatic properties, and adhesive properties of chitosan hydrogels limit their potential applications. In this study, we synthesized methacrylimide-chitosan (MAC)-4-arm polyethylene glycol (PEG)-dopamine (DMA) (MAC-PEG-DMA) hybrid hydrogels containing A-chain homodimers of platelet-derived growth factor (PDGF-AA) through one-pot photo-crosslinking. The resulting hydrogel exhibited improved mechanical strength and hemostatic properties as demonstrated by both in vitro blood clotting assay and rat liver hemorrhage assay. Furthermore, The PDGF-AA loaded hydrogel was also able to accelerate cell migration and proliferation. Data from skin wounds treated with this hybrid hydrogel showed faster wound closure and collagen maturation. Therefore, MAC-PEG-DMA (PDGF-AA) has great potential as a dressing to promote wound healing.

A PEG-CMC-THB-PRTM hydrogel with antibacterial and hemostatic properties for promoting wound healing.

The introduction of antibacterial and hemostatic hydrogels with good mechanical properties that display desirable impacts on wound healing process is still an unmet essential for clinical wound dressings. Herein, a multifunctional hydrogel PEG-CMC-THB-PRTM was fabricated via a one-pot method combining carboxymethyl chitosan (CMC), 2,3,4-trihydroxybenzaldehyde (THB), protamine (PRTM) and 4-arm polyethylene glycol aldehyde (PEG). The hydrogel was formed by the dynamic Schiff base reaction between amino groups of carboxymethyl chitosan and aldehyde groups of 4-arm polyethylene glycol aldehyde and exhibited excellent mechanical properties. The developed hydrogel also showed outstanding effects on anti-bacteria and hemostasis through the release of PRTM. Moreover, the hydrogel could promote extracellular matrix formation and wound closure in vivo, thereby accelerating the healing of skin wound. These results suggested that the multifunctional PEG-CMC-THB-PRTM hydrogel is a promising candidate for the clinical treatment of full-thickness wounds.

Carboxymethyl Chitosan/Tannic Acid Hydrogel with Antibacterial, Hemostasis, and Antioxidant Properties Promoting Skin Wound Repair.

Local causes of slow wound healing include infection and wound hemorrhage. Using sodium bicarbonate as a neutralizer, a variety of carboxymethyl chitosan-tannic acid (CMC-TA) composite hydrogels solidify through hydrogen bonding in this study. The best-performing hydrogel was synthesized by altering the concentration of TA and exhibited remarkable mechanical properties and biocompatibility. Following in vitro characterization tests, the CMC-TA hydrogel exhibited remarkable antibacterial and antioxidant properties, as well as quick hemostasis capabilities. In the in vivo wound healing study, the results showed that the CMC-TA hydrogel could relieve inflammation and promote the recovery of skin incision, re-epithelialization, and collagen deposition. Overall, this multifunctional hydrogel could be an ideal wound dressing for the clinical therapy of full-thickness wounds.

An oral delivery vehicle based on konjac glucomannan acetate targeting the colon for inflammatory bowel disease therapy.

Orally administered colon-targeted delivery vehicles are of major importance in the treatment of inflammatory bowel disease (IBD). However, it remains a challenge to maintain the integrity of such delivery vehicles during treatment, particularly in the gastric environment, which may cause untimely drug release before reaching the targeted colon. Herein, an oral colon-targeted drug delivery system (OCDDS) based on acetylated konjac glucomannan (AceKGM) has been developed in this work, which accomplishes colonic localization release and targets local inflammatory macrophages. The AceKGM nanoparticle-loading curcumin (Cur) was successfully fabricated by emulsion solvent evaporation techniques. DLS, AFM, and SEM were used in order to evaluate the nanoparticles' diameter as well as their in vitro drug release profile, and reactive oxygen species (ROS) scavenging results showed that the OCDDS considerably retained the activity of Cur treated with simulated gastric fluid (SGF) and controllably released in simulated intestinal fluid (SIF). In addition, the adhesion experiment results indicated that the nanoparticle could accumulate on the colonic macrophages. Evaluations in colitis mice showed that the treatment significantly alleviated the symptoms of colitis by decreasing the local level of myeloperoxidase (MPO) and the disease activity index (DAI) score in mice. In summary, the results of our research demonstrate that Cur-AceKGM nanoparticles exhibit significantly improved therapeutic efficacy compared to orally administered free Cur and can be developed as an effective drug delivery vehicle for IBD treatment.

A zwitterionic polymer-inspired material mediated efficient CRISPR-Cas9 gene editing.

The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox. However, its applications are still limited by its inefficient transduction. Herein, we present a novel gene vector, the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery. Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells. The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout Plk1 gene, which was expected to inhibit the expression of PLK1. Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently. The transduction with ZEBRA was cell line dependent, which showed ∼10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones. Furthermore, ZEBRA induced high-level expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of Plk1 gene, and inhibited the tumor cell growth significantly. This zwitterionic polymer-inspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.

Prompting immunostimulatory activity of curdlan with grafting methoxypolyethylene glycol.

The immunostimulatory activity of polysaccharides can improve human immunity, but their activity is low and prompting the activity is a great challenge. Curdlan, is a linear beta-1,3-glucan and has the potential to induce immune responses. However, owing to its tight triple helix structure and insolubility in water, its immunostimulatory activity is weakened. The keyway to promote its immunostimulatory activity is to relax its tight triple helix structure. In this work, methoxypolyethylene glycol (mPEG) was grafted onto curdlan (curdlan-g-mPEG) to unwind its triple helix structure. With its grafting mPEG, the water solubility of curdlan was enhanced. Moreover, with curdlan-g-mPEG treatment, macrophages secreted more tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited favorable phagocytosis of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa). These results reveal that curdlan-g-mPEG as an immunostimulant has potential applications in immunology and antibiotics.

Aminated ß-Glucan with immunostimulating activities and collagen composite sponge for wound repair.

Immunostimulating activities of yeast ß(1 â†’ 3)-D-Glucan (ß-Glucan) mainly depended on its structures. However, due to the tight triple helix structure of ß-Glucan, its immunostimulating activity is greatly weakened. Therefore, in order to partially unwind the tight triple helix structure of ß-glucan and improve its solubility in the medium, we modified it by amination in this study (A-Glu). The results showed that A-Glu could stimulate Raw264.7 macrophages and significantly enhance its TNF-α, IL-6, and IL-10 cytokine expression levels in vitro. A-Glu could also induce a shift of M0 Raw264.7 toward M1, and M2 toward M1. To expand the application of A-Glu in wound repair, the composite sponge consisting of A-Glu and type I collagen via the formation of a stable polyion complex (PIC) was developed. Moreover, the composite sponge could accelerate wound repair significantly. These results reveal that soluble A-Glu as an immunostimulating agent has potential applications in biomedicine.

The esterified lentinan bilayer nanofibrous membrane for promoting wound healing.

Host reactions following implantation of biomaterials, especially the macrophages' responses could significantly affect the wound healing process. Therefore, it is meaningful to develop immunostimulatory active wound dressing to accelerate wound healing. In this study, lentinan (LNT) was esterified with different carboxylic acids, and the bilayer nanofibrous membrane (BilayerM) based on the esterified LNT was designed. BilayerM exhibited good water absorption, red blood cells (RBC), platelets, and fibronectin adhesion abilities. The ELISA results indicated that BilayerM stimulated macrophages to secrete pro-inflammatory and pro-regenerative cytokines. Moreover, cell migration results showed that BilayerM promoted the migration and proliferation of NIH3T3 and HUVECs. The wound healing efficacy studies demonstrated that BilayerM significantly accelerated the wound healing rate in a full-thickness skin defect model. Therefore, these results indicate that this bioactive dressing is a hopeful candidate for clinical treatment of cutaneous wounds.

Bio-orthogonally crosslinked catechol-chitosan hydrogel for effective hemostasis and wound healing.

Chitosan-based hydrogels have been widely used in biomedical applications owing to their versatile properties. A novel chitosan-poly (ethylene glycol)-hydrocaffeic acid (CS-PEG-HA) hybrid hydrogel was fabricated, with a four-fold improvement in the mechanical performance as compared to the pure chitosan hydrogel, while enhancing the mucoadhesiveness and hemostasis properties. The in vivo studies on the CS-PEG-HA hydrogel demonstrated that liver bleeding could be rapidly repaired because of the outstanding adhesion and hemostasis characteristics of the developed hydrogel. Additionally, the in vivo wound healing efficacy studies indicated that the CS-PEG-HA hydrogel significantly accelerated the healing rate in a full-thickness skin defect model, thereby helping to successfully reconstruct an intact and thickened epidermis in 14 days. Overall, the developed multifunctional hybrid hydrogel provides a novel method for in vivo visceral hemostasis and accelerated healing of cutaneous skin wounds.

Alpha-terpineol grafted acetylated lentinan as an anti-bacterial adhesion agent.

Biomedical implants-associated bacterial infections have become a major threat to human health. Therefore, it is meaningful to develop new antibacterial strategies to solve this problem. In this study, we conjugated acetylated lentinan (AceLNT) with α-terpineol (AceLNT-g-α-ter), a highly effective natural antibacterial compound, to constitute a novel AceLNT-g-α-ter membrane (AceLNT-g-α-terM). Compared with AceLNT membrane (AceLNTM), the adhesion amount of E. coli and P. aeruginosa in AceLNT-g-α-terM decreased by 80% and 85% after 7 d incubation in fluid bacterial medium. Moreover, the number of E. coli and P. aeruginosa biofilm on AceLNT-g-α-terM surface decreased by 70% and 71%. At the meanwhile, α-terpineol grafting modification of AceLNT had limited effect on its stimulating activity on macrophages and had no more cytotoxicity. In summary, our study firstly confirmed that AceLNT-g-α-terM could effectively inhibit gram-negative bacteria adhesion and biofilm formation, and provided a novel strategy for preventing infection of biomedical implants.

A γ-PGA/KGM-based injectable hydrogel as immunoactive and antibacterial wound dressing for skin wound repair.

Injectable hydrogels, of which the cover area and volume can be flexibly adjusted according to the shape and depth of the wound, are considered to be an ideal material for wound dressing. Konjac glucomannan (KGM) is a natural polysaccharide with immunomodulatory capability, while γ-polyglutamic acid (γ-PGA) is a single chain polyamino acid with moisturizing, water-retention and antibacterial properties. This work intended to combine the advantages of the two materials to prepare an injectable hydrogel (P-OK) by mixing the adipic acid dihydrazide (ADH) modified γ-PGA with oxidized KGM. The chemical structures, the physical and chemical properties, and the biological properties of the P-OK hydrogel were evaluated. The optimal conditions to form the P-OK hydrogel were fixed, and the cytotoxicity, qPCR, antibacterial and animal experiments were performed. Results showed that the P-OK hydrogel had a fast gelation time, good water-retention rate, little cytotoxicity, good immunomodulating and antibacterial capabilities, and could shorten the healing period in the rat full-thickness defect model, which makes it a potential candidate for wound repair dressing.

Intracellular Delivery of Proteins into Living Cells by Low-Molecular-Weight Polyethyleneimine.

INTRODUCTION: Intracellular protein delivery is emerging as a potential strategy to revolutionize therapeutics in the field of biomedicine, aiming at treating a wide range of diseases including cancer, inflammatory diseases and other oxidative stress-related disorders with high specificity. However, the current challenges and limitations are addressed to either synthetically or biologically through multipotency of engineering, such as protein modification, insufficient delivery of large-size proteins, deficiency or mutation of proteins, and high cytotoxicity. METHODS: We prepared the nanocomposites by mixing protein with PEI1200 at a certain molar ratio and demonstrated that it can deliver proteins into living cells in high efficiency and safety through the following experiments, such as dynamic light scattering, fluorescent detection, agarose gel electrophoresis, ß-Galactosidase activity detection, immunofluorescence staining, digital fluorescent detection, cell viability assay and flow cytometry. RESULTS: The self-assembly of PEI1200/protein nanocomposites with appropriate molar ratio (4:1 and 8:1) could provide efficiently delivery of active proteins to a variety of cell types in the presence of serum. The nanocomposites could continuously release protein up to 96 h in their desired intracellular locations. In addition, these nanocomposites were able to preserve protein activity while maintain low cytotoxicity (when final concentration <1 µg/mL). CONCLUSION: Collectively, PEI1200-based delivery system provided an alternative strategy to direct protein delivery in high efficiency and safety, offering increased potential applications in clinical biomedicine.

κ/ß-Carrageenan oligosaccharides promoting polarization of LPS-activated macrophage and their potential in diabetes wound healing.

Here we prepared a low-degree-sulfated κ/ß-carrageenan oligosaccharide (L-DS-KOS) by DMSO-methanol desulfation method, and fabricated a sponge dressing for the wound healing of diabetic rats. The immunomodulatory effects of L-DS-KOS on M1-like macrophages were evaluated. Results showed that L-DS-KOS could effectively promote the secretion of anti-inflammatory factors and accelerate polarization of LPS-activated macrophages from M1 to M2 type. The gross examination result showed that the sponge dressing with the mass ratio of L-DS-KOS: collagen = 3: 7 could effectively accelerate the repair process of the full-thickness excisional wound in diabetic rats; and H&E and Masson staining results disclosed that the L-DS-KOS/collagen dressing could better shorten the inflammation period of the wound site, and improve the process of collagen deposition and epithelial formation, thereby promote the repair of skin wounds in diabetic rats. These results demonstrate that L-DS-KOS has potential to be used as a new type of immunomodulatory biomaterial for diabetic wound dressings.

A carrageenan/agarose composite sponge and its immunomodulatory activities toward RAW264.7.

A kind of commercial hybrid carrageenan (HC)/agarose composite sponge containing κ-, µ-, ι-, and ν-carrageenan, which could turn into hydrogel and release carrageenan at human body temperature was fabricated for immune stimulation and modulation. Release behavior demonstrated that the hybrid carrageenan contained sponge was mechanically stable and could release carrageenan constantly. RT-PCR and ELISA experiments showed that the leaching liquor of the sponge could stimulate RAW264.7 from M0 state to a polarized state by secreting more anti-inflammatory factor IL-10 than pro-inflammatory ones, such as, IL-6 and TNF-α. Transwell experiments also indicated that the leaching liquor could promote the proliferation of NIH-3T3 by stimulating RAW264.7 of M0 state after 7 days. Results of particle size and intracellular concentration analyses suggested that the released carrageenan might enter into the cellular interior of RAW264.7 in the form of microgels or protein complexes. The sponge would be a promising candidate for skin wound dressing.