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.

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.

Electrochemical Micro-Immunosensor of Cubic AuPt Dendritic Nanocrystals/Ti3C2-MXenes for Exosomes Detection.

Exosomes are extracellular vesicles that exist in body circulation as intercellular message transmitters. Although the potential of tumor-derived exosomes for non-invasive cancer diagnosis is promising, the rapid detection and effective capture of exosomes remains challenging. Herein, a portable electrochemical aptasensor of cubic AuPt dendritic nanocrystals (AuPt DNs)/Ti3C2 assisted in signal amplification, and aptamer CD63 modified graphene oxide (GO) was immobilized on a screen-printed carbon electrode (SPCE) as the substrate materials for the direct capture and detection of colorectal carcinoma exosomes. Cubic AuPt DNs/Ti3C2 was synthesized according to a simple hydrothermal procedure, and the AuPt DNs/Ti3C2-Apt hybrid demonstrated an efficient recognition of exosomes. Under optimal conditions, a detection limit of down to 20 exosomes µL-1 was achieved with the linear range from 100 exosomes µL-1 to 5.0 × 105 exosomes µL-1. The proposed immunosensor could be suitable for the analysis of exosomes and has clinical value in the early diagnosis of cancer.

New Ultrasensitive Sandwich-Type Immunoassay of Dendritic Tri-Fan Blade-like PdAuCu Nanoparticles/Amine-Functionalized Graphene Oxide for Label-Free Detection of Carcinoembryonic Antigen.

The early detection of tumor markers has an effective role in the treatment of cancer. Here, a new sandwich-type electrochemical immunosensor for early label-free detection of the cancer biomarker carcinoembryonic antigen (CEA) was developed. Dendritic tri-fan blade-like PdAuCu nanoparticles (PdAuCu NPs)/amine functionalized graphene oxide (NH2-GO) were the label of secondary antibodies (Ab2), and Au nanoparticle-decorated polydopamines (Au/PDA) were immobilized on a screen-printed carbon electrode (SPCE) as the substrate materials. Dendritic tri-fan blade-like PdAuCu NPs/NH2-GO was synthesized according to a simple hydrothermal procedure and used to immobilize antibodies (Ab2) with large surfaces areas, increased catalytic properties and good adsorption to amplify the current signals. Subsequently, Ab2/PdAuCu NPs/NH2-GO catalyzed the reduction of H2O2 in the sandwich-type immunoreactions. Under optimal conditions, the immunosensor exhibited a satisfactory response to CEA with a limit detection of 0.07 pg mL-1 and a linear detection range from 0.1 pg mL-1 to 200 ng mL-1. The proposed immunosensor could be suitable enough for a real sample analysis of CEA, and has clinical value in the early diagnosis of cancer.

Enhanced Antibacterial and Anti-Biofilm Activities of Antimicrobial Peptides Modified Silver Nanoparticles.

BACKGROUND: The biofilms could protect bacteria from antibiotics and promote the production of drug-resistant strains, making the bacteria more difficult to be eradicated. Thus, we developed an AMP@PDA@AgNPs nanocomposite, which is formed by modifying silver nanoparticles (AgNPs) with antimicrobial peptides (AMP) modified nanocomposite to destroy biofilm in this study. METHODS: The AMP@PDA@AgNPs nanocomposite was prepared with polymerization method and characterized by using ultraviolet-visible (UV-vis) spectroscopy, dynamic light scattering (DLS), Fourier transform-infrared spectroscopy (FT-IR), and transmission electron microscope (TEM). The antibacterial effects of the nanocomposite were investigated by using agar diffusion method and minimum inhibitory concentration (MIC) test. The quantitative analysis of the biofilm formation by the nanocomposite was conducted using crystal violet staining and confocal laser scanning microscope (CLSM). RESULTS: The DLS and TEM analysis showed it was a spherical nanocomposite with 200 nm size and well dispersed . The results of UV-vis and FT-IR confirmed the presence of AMP and AgNPs. The nanocomposite had an excellent biocompatibility at 100 µg/mL. And the AMP@PDA@AgNPs nanocomposite showed superior antimicrobial activity against both Gram-negative (E. coli, P. aeruginosa) and Gram-positive (S. aureus) bacteria than AgNPs or AMP. Importantly, the mRNA expression of biofilm-related genes were decreased under the action of the nanocomposites. CONCLUSION: An AMP@PDA@AgNPs nanocomposite with good biocompatibility was successfully prepared. The nanocomposite could destruct bacterial biofilms by inhibiting the expression of biofilm-related genes. The synergistic strategy of AMPs and AgNPs could provide a new perspective for the treatment of bacterial infection.

Specific Anti-biofilm Activity of Carbon Quantum Dots by Destroying P. gingivalis Biofilm Related Genes.

INTRODUCTION: Biofilms protect bacteria from antibiotics and this can produce drug-resistant strains, especially the main pathogen of periodontitis, Porphyromonas gingivalis. Carbon quantum dots with various biomedical properties are considered to have great application potential in antibacterial and anti-biofilm treatment. METHODS: Tinidazole carbon quantum dots (TCDs) and metronidazole carbon quantum dots (MCDs) were prepared by a hydrothermal method with the clinical antibacterial drugs tinidazole and metronidazole, respectively. Then, TCDs and MCDs were characterized by transmission electron microscopy, UV-visible spectroscopy, infrared spectroscopy and energy-dispersive spectrometry. The antibacterial effects were also investigated under different conditions. RESULTS: The TCDs and MCDs had uniform sizes. The results of UV-visible and energy-dispersive spectrometry confirmed their important carbon polymerization structures and the activity of the nitro group, which had an evident inhibitory effect on P. gingivalis, but almost no effect on other bacteria, including Escherichia coli, Staphylococcus aureus and Prevotella nigrescens. Importantly, the TCDs could penetrate the biofilms to further effectively inhibit the growth of P. gingivalis under the biofilms. Furthermore, it was found that the antibacterial effect of TCDs lies in its ability to impair toxicity by inhibiting the major virulence factors and related genes involved in the biofilm formation of P. gingivalis, thus affecting the self-assembly of biofilm-related proteins. CONCLUSION: The findings demonstrate a promising new method for improving the efficiency of periodontitis treatment by penetrating the P. gingivalis biofilm with preparations of nano-level antibacterial drugs.

An Electrochemical Immunosensor for Sensitive Detection of the Tumor Marker Carcinoembryonic Antigen (CEA) Based on Three-Dimensional Porous Nanoplatinum/Graphene.

Carcinoembryonic antigen (CEA) is an important broad-spectrum tumor marker. The quantitative detection of a low concentration of CEA has important medical significance. In this study, three-dimensional porous graphene-oxide-supported platinum metal nanoparticles (3DPt/HGO) composites were prepared by a wet chemical method and modified on an electrode with enhanced conductivity, a large surface area, and good adsorption of immobilizing antibodies (Ab1). Horseradish peroxidase (HRP)-functionalized Au nanoparticles were fabricated to label the secondary antibodies (Ab2). The proposed immunosensor showed a good linear relationship in the range of 0.001-150 ng/mL for CEA and a detection limit of 0.0006 ng/mL. The immunosensor had high sensitivity, good stability and reproducibility, and has great application prospects for the clinical diagnosis of cancer.

Preparation and Evaluation of Upconversion Nanoparticles Based miRNA Delivery Carrier in Colon Cancer Mice Model.

Therapeutic efficacy of solid tumor is often severely hampered by poor penetration of therapeutics into diseased tissues and lack of tumor targeting. In this study, the functionalized upconversion nanoparticles (UCNP)-based delivery vector targeting cancer cells was developed. Firstly, NaYF4:Yb/Tm (UCNP) was prepared with the solvothermal method for the uniform nanoparticle size and brilliant lattice structure. The SiO2 coated UCNP was demonstrated a high upconversion emission and good monodispersity, which was coupled with polyetherimide (PEI) and miR-145 vector. Then, it was further functionalized via hyaluronic acid (HA) (UCNP/PEI/HA Nanocomplex, UCNPs) coating for the targeted delivery and improved biocompatibility. The UCNPs/miR-145 displays an excellent biocompatibility, a high level of cellular uptake and miR-145 expression, which results in a significant cell cycle arrest in G1, and induces CCND1, CDK6 and CCNE2 proteins downregulation. In vivo, the HA-coated UCNPs were enriched at the tumor site by targeting and retention effects, which resulted in a significant inhibition of tumor growth. Histological experiments demonstrated that UCNPs did not show significant toxicity in mice colon cancer model. Taken together, a UCNPs-based delivery platform was successfully constructed and used for miRNA target delivery, which provided a new method and idea for bioengineering and nanotechnology-based tumor therapy.

Bioinspired Drug Delivery Carrier for Enhanced Tumor-Targeting in Melanoma Mice Model.

As a widely used first-line chemotherapy drug for tumor, Doxorubicin (DOX) can induce various side effects on normal tissues because of its non-specific distribution in the body. Emerging evidence has shown that platelets have the capability to recognize and interact with tumor cells. Inspired by this, the platelet-based drug delivery system was constructed by loading of DOX in platelet cytoplasm and modification of transferrin on the surface of platelet (Tf-P-DOX). The encapsulation efficiency of DOX in platelet was the highest at the DOX concentration of 0.05 mM, and reached to 64.9%. Fluorescence microscopy showed that the Tf-P-DOX facilitated cell uptakes and enhanced intracellular drug accumulation in B16F10 cells. Compared with free DOX, Tf-P-DOX exhibited an enhanced effect on cell apoptosis at the same concentration of DOX. In vivo imaging system showed that the near-infrared fluorescence of B16F10 tumor-bearing mice was mainly accumulated in the tumor site, which caused the inhibition of tumor growth in mice. The morphological changes of tumor tissue in Tf-P-DOX group was significant in comparison with those of the control group, including the small nucleus, the insufficiency of cancerous nest, and the infiltration of inflammatory cells, while Tf-P-DOX did not show significant adverse effects on normal tissues. Compared with the control group, the levels of caspase 9 and caspase 3 protein expressions were increased significantly in Tf-P-DOX group. Our studies suggest platelets can be repurposed as promising carriers for efficient targeting and treatment of solid tumors.

Three-Dimensional Holey-Graphene Architectures for Highly Sensitive Enzymatic Electrochemical Determination of Hydrogen Peroxide.

Three-dimensional (3D) graphene with high specific surface area, excellent conductivity and designed porosity is essential for many practical applications. Herein, holey graphene oxide with nano pores was facilely prepared via a convenient mild defect-etching reaction and then fabricated to 3D nanostructures via a reduction method. Based on the 3D architectures, a novel enzymatic hydrogen peroxide sensor was successfully fabricated. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) were used to characterize the 3D holey graphene oxide architectures (3DHGO). Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of 3DHGO at glassy carbon electrode (GCE). Excellent electrocatalytic activity to the reduction of H2O2 was observed, and a linear range of 5.0×10-8~5.0×10-5 M with a detection limit of 3.8×10-9 M was obtained. These results indicated that 3DHGO have potential as electrochemical biosensors.

Three-Dimensional Au/Holey-Graphene as Efficient Electrochemical Interface for Simultaneous Determination of Ascorbic Acid, Dopamine and Uric Acid.

The quantification of ascorbic acid (AA), dopamine (DA), and uric acid (UA) has been an important area of research, as these molecules' determination directly corresponds to the diagnosis and control of diseases of nerve and brain physiology. In our research, graphene oxide (GO) with nano pores deposited with gold nanoparticles were self-assembled to form three-dimensional (3D) Au/holey-graphene oxide (Au/HGO) composite structures. The as-prepared 3DAu/HGO composite structures were characterized for their structures by X-ray diffraction, Raman spectrum, scanning electron microscopy, and transmission electron microscopy coupled with cyclic voltammograms. Finally, the proposed 3DAu/HGO displayed high sensitivity, excellent electron transport properties, and selectivity for the simultaneous electrochemical determination of AA, DA and UA with linear response ranges of 1.0⁻500 µM, 0.01⁻50 µM and 0.05⁻50 µM respectively. This finding paves the way for graphene applications as a biosensor for detecting three analytes in human serum.

Hyaluronate-Functionalized Graphene for Label-Free Electrochemical Cytosensing.

Electrochemical sensors for early tumor cell detection are currently an important area of research, as this special region directly improves the efficiency of cancer treatment. Functional graphene is a promising alternative for selective recognition and capture of target cancer cells. In our work, an effective cytosensor of hyaluronate-functionalized graphene (HG) was prepared through chemical reduction of graphene oxide. The as-prepared HG nanostructures were characterized with Fourier transform infrared spectroscopy and transmission electron microscopy coupled with cyclic voltammograms and electrochemical impedance spectroscopy, respectively. The self-assembly of HG with ethylene diamine, followed by sodium hyaluronate, enabled the fabrication of a label-free electrochemical impedance spectroscopy cytosensor with high stability and biocompatibility. Finally, the proposed cytosensor exhibited satisfying electrochemical behavior and cell-capture capacity for human colorectal cancer cells HCT-116, and also displayed a wide linear range, from 5.0 × 10² cells∙mL-1 to 5.0 × 106 cells∙mL-1, and a low detection limit of 100 cells∙mL-1 (S/N = 3) for quantification. This work paves the way for graphene applications in electrochemical cytosensing and other bioassays.

Incorporation of microfibrillated cellulose into collagen-hydroxyapatite scaffold for bone tissue engineering.

In this study, the composite of Collagen-Hydroxyapitite (COL-HA) with microfibrillated cellulose (MFC) was developed as a new bone substitute material. COL-HA was prepared by in-situ method and modified by dehydrothermal treatment. Microfibrillated cellulose (MFC), a nature polysaccharide with plenty of hydroxyl groups, was incorporated into COL-HA composites to improve the properties. The novel COL-HA-MFC scaffold with different ratios of COL-HA and MFC were fabricated by cold isostatic pressing technique and freeze-drying technology. During the forming process, a three-dimensional bone-like structure was shaped in hybrid scaffolds. The microstructural transitions of COL-HA-MFC composites were examined by Fourier transform infrared spectroscope (FTIR), Ultraviolet-visible spectrophotometer (UV), and X-ray diffraction (XRD), which indicated that HA deposited on collagen molecules and MFC bonded with COL-HA. Hydrophilicity, swelling property, mechanical property, and degradability of COL-HA-MFC composites were investigated. Biological properties, such as cytotoxicity and hemolysis, were also studied. The results showed a good swelling capacity for the scaffolds, keeping their original shapes after swelling. The compression strength and degradability of the scaffold materials could be regulated by the MFC content. The compression strength of COL-HA-MFC composite scaffords increased to 20-40 MPa, closing to that of the nature bone (1-200 MPa). The obtained scaffolds are good in biocompatibility with high level of cell growth rate (>70%) and suitable hemolysis rate (≦5%). The work might provide an efficient and alternative approach for collagen-based biomaterials with necessary properties. The COL-HA-MFC composite scaffold showed a potential application in bone tissue engineering.

Engineered exosome-mediated delivery of functionally active miR-26a and its enhanced suppression effect in HepG2 cells.

INTRODUCTION: Exosomes are closed-membrane nanovesicles that are secreted by a variety of cells and exist in most body fluids. Recent studies have demonstrated the potential of exosomes as natural vehicles that target delivery of functional small RNA and chemotherapeutics to diseased cells. METHODS: In this study, we introduce a new approach for the targeted delivery of exosomes loaded with functional miR-26a to scavenger receptor class B type 1-expressing liver cancer cells. The tumor cell-targeting function of these engineered exosomes was introduced by expressing in 293T cell hosts, the gene fusion between the transmembrane protein of CD63 and a sequence from Apo-A1. The exosomes harvested from these 293T cells were loaded with miR-26a via electroporation. RESULTS: The engineered exosomes were shown to bind selectively to HepG2 cells via the scavenger receptor class B type 1-Apo-A1 complex and then internalized by receptor-mediated endocytosis. The release of miR-26a in exosome-treated HepG2 cells upregulated miR-26a expression and decreased the rates of cell migration and proliferation. We also presented evidence that suggest cell growth was inhibited by miR-26a-mediated decreases in the amounts of key proteins that regulate the cell cycle. CONCLUSION: Our gene delivery strategy can be adapted to treat a broad spectrum of cancers by expressing proteins on the surface of miRNA-loaded exosomes that recognize specific biomarkers on the tumor cell.

Polyethyleneimine-coated quantum dots for miRNA delivery and its enhanced suppression in HepG2 cells.

Quantum dots (QDs) have been intensively investigated for bioimaging, drug delivery, and labeling probes because of their unique optical properties. In this study, CdSe/ZnS QDs-based nonviral vectors with the dual functions of delivering miR-26a plasmid and bioimaging were formulated by capping the surface of CdSe/ZnS QDs with polyethyleneimine (PEI). The PEI-coated QDs were capable of condensing miR-26a expression vector into nanocomplexes that can emit strong red luminescence when loaded with CdSe/ZnS QDs. Further results showed that PEI-modified nanoparticles (NPs) could transfect miR-26a plasmid into HepG2 cells in vitro. Meanwhile, imaging of living cells could be achieved based on the CdSe/ZnS QDs. Further study suggested that miR-26a transfection up-regulated miR-26a expression, induced cycle arrest, and triggered proliferation inhibition in HepG2 cells. The results indicated that PEI-coated QD NPs possess the capability of bioimaging and gene delivery and could be a promising vehicle with the engineering of QD NPs for gene therapy in the future.

Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish.

Although a white spot syndrome virus (WSSV) subunit vaccine could significantly enhance the immune response and benefit the shrimp host, its practical application is currently not feasible because of drawbacks in existing expression systems. We generated a transgenic Dunaliella salina (D. salina) strain by introducing the WSSV VP28 gene to produce a novel oral WSSV subunit vaccine. Following transformation of D. salina, VP28 gene expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) assays, enzyme-linked immunosorbent assays (ELISAs), and western blot analysis. The RT-PCR results indicated that the VP28 gene was successfully expressed in D. salina cells. The presence of recombinant VP28 proteins with natural bioactivity was confirmed by western blot analysis and ELISA. Animal vaccination experiments indicated that transgenic D. salina can induce protection against WSSV by oral delivery in crayfish. Our findings indicate that the VP28 gene can be successfully expressed in transgenic D. salina and can be applied as an oral vaccine to protect crayfish against WSSV. We have demonstrated that it is feasible to produce an oral vaccine using D. salina, and thereby provide a new method for controlling other viral diseases in crustaceans.

Binding of white spot syndrome virus to Artemia sp. cell membranes.

Using differential velocity centrifugation, cell membranes of Artemia sp. were prepared, and their binding to white spot syndrome virus (WSSV) was analyzed in vitro. The results indicated that WSSV can specifically bind to Artemia cell membranes, and that WSSV receptor very likely existed in this membrane, which suggested that Artemia sp. may be a reservoir of WSSV. This study investigated the specific WSSV binding site by performing competitive inhibition experiments using shrimp gill cell membranes to bind WSSV to Artemia cell membranes. The results showed that shrimp gill cell membranes had a distinct inhibition effect on the specific binding of Artemia cell membranes to WSSV. Thus, potentially similar WSSV receptors or binding sites existed on Artemia sp. cell membranes and shrimp gill cell membranes. Taken together, these findings may provide experimental basis for the development of an effective approach to controlling WSSV, and theoretical basis for the study of WSSV receptors.