Aerosolization Performance of Immunoglobulin G by Jet and Mesh Nebulizers.

Recently, many preclinical and clinical studies have been conducted on the delivery of therapeutic antibodies to the lungs using nebulizers, but standard treatment guidelines have not yet been established. Our objective was to compare nebulization performance according to the low temperature and concentration of immunoglobulin G (IgG) solutions in different types of nebulizers, and to evaluate the stability of IgG aerosols and the amount delivered to the lungs. The output rate of the mesh nebulizers decreased according to the low temperature and high concentration of IgG solution, whereas the jet nebulizer was unaffected by the temperature and concentration of IgG. An impedance change of the piezoelectric vibrating element in the mesh nebulizers was observed because of the lower temperature and higher viscosity of IgG solution. This affected the resonance frequency of the piezoelectric element and lowered the output rate of the mesh nebulizers. Aggregation assays using a fluorescent probe revealed aggregates in IgG aerosols from all nebulizers. The delivered dose of IgG to the lungs in mice was highest at 95 ng/mL in the jet nebulizer with the smallest droplet size. Evaluation of the performance of IgG solution delivered to the lungs by three types of nebulizers could provide valuable parameter information for determination on dose of therapeutic antibody by nebulizers.

Controllable Nitric Oxide Storage and Release in Cu-BTC: Crystallographic Insights and Bioactivity.

Crystalline metal-organic frameworks (MOFs) are extensively used in areas such as gas storage and small-molecule drug delivery. Although Cu-BTC (1, MOF-199, BTC: benzene-1,3,5-tricarboxylate) has versatile applications, its NO storage and release characteristics are not amenable to therapeutic usage. In this work, micro-sized Cu-BTC was prepared solvothermally and then processed by ball-milling to prepare nano-sized Cu-BTC (2). The NO storage and release properties of the micro- and nano-sized Cu-BTC MOFs were morphology dependent. Control of the hydration degree and morphology of the NO delivery vehicle improved the NO release characteristics significantly. In particular, the nano-sized NO-loaded Cu-BTC (NO⊂nano-Cu-BTC, 4) released NO at 1.81 µmol·mg-1 in 1.2 h in PBS, which meets the requirements for clinical usage. The solid-state structural formula of NO⊂Cu-BTC was successfully determined to be [CuC6H2O5]·(NO)0.167 through single-crystal X-ray diffraction, suggesting no structural changes in Cu-BTC upon the intercalation of 0.167 equivalents of NO within the pores of Cu-BTC after NO loading. The structure of Cu-BTC was also stably maintained after NO release. NO⊂Cu-BTC exhibited significant antibacterial activity against six bacterial strains, including Gram-negative and positive bacteria. NO⊂Cu-BTC could be utilized as a hybrid NO donor to explore the synergistic effects of the known antibacterial properties of Cu-BTC.

In vitro delivery efficiencies of nebulizers for different breathing patterns.

BACKGROUND: Nebulizers are medical devices that deliver aerosolized medication directly to lungs to treat a variety of respiratory diseases. However, breathing patterns, respiration rates, airway diameters, and amounts of drugs delivered by nebulizers may be respiratory disease dependent. METHOD: In this study, we developed a respiratory simulator consisting of an airway model, an artificial lung, a flow sensor, and an aerosol collecting filter. Various breathing patterns were generated using a linear actuator and an air cylinder. We tested six home nebulizers (jet (2), static (2), and vibrating mesh nebulizers (2)). Nebulizers were evaluated under two conditions, that is, for the duration of nebulization and at a constant output 1.3 mL using four breathing patterns, namely, the breathing pattern specified in ISO 27427:2013, normal adult, asthmatic, and COPD. RESULTS: One of the vibrating mesh nebulizers had the highest dose delivery efficiency. The drug delivery efficiencies of nebulizers were found to depend on breathing patterns. CONCLUSION: We suggest a quantitative drug delivery efficiency evaluation method and calculation parameters that include considerations of constant outputs and residual volumes. The study shows output rates and breathing patterns should be considered when the drug delivery efficiencies of nebulizers are evaluated.

Size-Dependent Photodynamic Anticancer Activity of Biocompatible Multifunctional Magnetic Submicron Particles in Prostate Cancer Cells.

In this study, newly designed biocompatible multifunctional magnetic submicron particles (CoFe2O4-HPs-FAs) of well-defined sizes (60, 133, 245, and 335 nm) were fabricated for application as a photosensitizer delivery agent for photodynamic therapy in cancer cells. To provide selective targeting of cancer cells and destruction of cancer cell functionality, basic cobalt ferrite (CoFe2O4) particles were covalently bonded with a photosensitizer (PS), which comprises hematoporphyrin (HP), and folic acid (FA) molecules. The magnetic properties of the CoFe2O4 particles were finely adjusted by controlling the size of the primary CoFe2O4 nanograins, and secondary superstructured composite particles were formed by aggregation of the nanograins. The prepared CoFe2O4-HP-FA exhibited high water solubility, good MR-imaging capacity, and biocompatibility without any in vitro cytotoxicity. In particular, our CoFe2O4-HP-FA exhibited remarkable photodynamic anticancer efficiency via induction of apoptotic death in PC-3 prostate cancer cells in a particle size- and concentration-dependent manner. This size-dependent effect was determined by the specific surface area of the particles because the number of HP molecules increased with decreasing size and increasing surface area. These results indicate that our CoFe2O4-HP-FA may be applicable for photodynamic therapy (PDT) as a PS delivery material and a therapeutic agent for MR-imaging based PDT owing to their high saturation value for magnetization and superparamagnetism.

Photodynamic Anticancer Activity of CoFe2O4 Nanoparticles Conjugated with Hematoporphyrin.

This work reports the synthesis and the characterization of water-soluble and biocompatible photosensitizer (PS)-conjugated magnetic nanoparticles composed of a cobalt ferrite (CoFe2O4) magnetic core coated with a biocompatible hematoporphyrin (HP) shell. The photo-functional cobalt ferrite magnetic nanoparticles (CoFe2O4@HP) were uniform in size, stable against PS leaching, and highly efficient in the photo-generation of cytotoxic singlet oxygen under visible light. With the CoFe2O4@HP, we acquired in vitro MR images of cancer cells (PC-3) and confirmed good biocompatibility of the CoFe2O4@HP in both normal and cancer cells. In addition, we confirmed the potential of the CoFe2O4@HP as an agent for photodynamic therapy (PDT) applications. The photodynamic anticancer activities in 25, 50, and 100 µg/mL of CoFe2O4@HP were measured and found to exceed 99% (99.0, 99.4, and 99.5%) (p < 0.002). The photodynamic anticancer activity was 81.8% (p < 0.003). From these results, we suggest that our CoFe2O4@HP can be used safely as a type of photodynamic cancer therapy with potential as a therapeutic agent having good biocompatibility. Moreover, these photo-functional magnetic nanoparticles are highly promising for applications in versatile imaging diagnosis and as a therapy tool in biomedical engineering.

Facile synthesis, cytotoxicity and bioimaging of Fe(3+) selective fluorescent chemosensor.

The designing and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of alanine substituted rhodamine B derivative 3 having specific binding affinity toward Fe(3+) with micro molar concentration level. Through fluorescence titration at 599nm, we were confirmed that ligand 3 exhibited ratiometric fluorescence response with remarkable enhancement in emission intensity by complexation between 3 and Fe(3+) while it appeared no emission in case of the competitive ions (Sc(3+), Yb(3+), In(3+), Ce(3+), Sm(3+), Cr(3+), Sn(2+), Pb(2+), Ni(2+), Co(2+), Cu(2+), Ba(2+), Ca(2+), Mg(2+), Ag(+), Cs(+), Cu(+), K(+)) in aqueous/methanol (60:40, v/v) at neutral pH. However, the fluorescence as well as colorimetric response of ligand-iron complex solution was quenched by addition of KCN which snatches the Fe(3+) from complex and turn off the sensor confirming the recognition process was reversible. Furthermore, bioimaging studies against L-929 cells (mouse fibroblast cells) and BHK-21 (hamster kidney fibroblast), through confocal fluorescence microscopic experiment indicated that ligand showed good permeability and minimum toxicity against the tested cell lines.

Optical properties of some novel 2,5-disubstituted 1,3,4-oxadiazole derivatives and their application as an efficient cell staining azo dyes.

A series of six 2,5-disubstituted 1,3,4-oxadiazole derivatives with various length of conjugation have been synthesized and their optical properties including UV-visible absorption spectra, fluorescence emission spectra, molar absorption co-efficient, Stokes shift and the relative fluorescence quantum yield were measured in a variety of organic solvents. Correlation of the absorption spectra and fluorescence emission response of the 2,5-disubstituted 1,3,4-oxadiazole derivatives with the substituent effect revealed that the optical response can easily be tuned toward red shift by increasing conjugation length. The synthesized compounds were further employed in bioimaging assay in order to investigate their potential as an efficient cell staining agent using L-929 cells under confocal fluorescence microscope which showed scrupulous cell permeability and no toxicity as assessed by MTT assay.

The smart 2-(2-fluorobenzoyl)-N-(2-methoxyphenyl)hydrazinecarbothioamide functionalized as Ni(II) sensor in micromolar concentration level and its application in live cell imaging.

In recent years, fluorescent probes for the detection of environmentally and biologically important metal cations have received extensive attention for designing and development of fluorescent chemosensors. Herein, we report the photophysical results of 2-(2-fluorobenzoyl)-N-(2-methoxyphenyl) hydrazinecarbothioamide (4) functionalized as Ni (II) sensor in micromolar concentration level. Through fluorescence titration at 488 nm, we were confirmed that ligand 4 showed the remarkable emission by complexation between 4 and Ni (II) while it appeared no emission in case of the competitive ions (Cr(3+), Fe(2+), Co(2+), Ba(2+), Cu(2+), Ca(2+), Na(+), K(+), Cu(+), Cs(+)). Furthermore, ligand 4 exhibited no toxicity with precise cell permeability toward normal living cells using L929 cell lines in bio imaging experiment investigated through confocal fluorescence microscope. The non-toxic behavior of ligand 4 (assessed by MTT assay) and its ability to track the Ni(2+) in living cells suggest its possibility to use in biological system as nickel sensor.

Evaluation of antibody drug delivery efficiency via nebulizer in various airway models and breathing patterns.

BACKGROUND: Nebulizers are commonly used to treat respiratory diseases, which are a major cause of morbidity and mortality. While inhalation therapy with antibodies has been evaluated in preclinical studies and clinical trials for respiratory diseases, it has not yet been approved for treatment. Moreover, there is limited information regarding the delivery efficiency of therapeutic antibodies via nebulizer. METHODS: In this study, the nebulization characteristics and drug delivery efficiencies were compared when immunoglobulin G (IgG) was delivered by five nebulizers using two airway models and five breathing patterns. The study confirmed that the delivered dose and drug delivery efficiency were reduced in the child model compared to those in the adult model and in the asthma pattern compared to those in the normal breathing pattern. RESULTS: The NE-SM1 NEPLUS vibrating mesh nebulizer demonstrated the highest delivery efficiency when calculated as a percentage of the loading dose, whereas the PARI BOY SX + LC SPRINT (breath-enhanced) jet nebulizer had the highest delivery efficiency when calculated as a percentage of the emitted dose. CONCLUSION: The results suggest that the total inspiration volume, output rate, and particle size should be considered when IgG nebulization is used. We, therefore, propose a method for evaluating the efficiency of nebulizer for predicting antibody drug delivery.

Therapeutic Potency of NO Loaded into Anticancer Copper Metal-Organic Framework through Nonclassical Hydrogen Bonding.

Metal-organic frameworks (MOFs) are potential exogenous scaffolds for therapeutic nitric oxide (NO) delivery because they can store drug or bioactive gas molecules within pores or on active metal sites. Herein, we employed a Cu-MOF coordinated with glutarate (glu) and 1,2-bis(4-pyridyl)ethane (bpa) to obtain NO-loaded Cu-MOF (NO⊂Cu-MOF). NO loading transformed the space group of Cu-MOF from monoclinic C2/c to triclinic P-1 through nonclassical hydrogen bonding with glu and bpa. Cu-MOF showed good stability in deionized water and phosphate-buffered saline. NO⊂Cu-MOF released up to 1.10 µmol mg-1 NO over 14.6 h at 37 °C, which is suitable for therapeutic applications. NO⊂Cu-MOF showed moderate biocompatibility with L-929 cells and significant anticancer activity against HeLa cells, suggesting an apoptosis-mediated cell death mechanism. These insights into NO bonding modes with Cu-MOF that enable controlled NO release can inspire the design of functional MOFs as hybrid NO donors for drug delivery.

Non-thermal plasma promotes hair growth by improving the inter-follicular macroenvironment.

Non-thermal plasma (NTP) is widely used in the disinfection and surface modification of biomaterials. NTP treatment can regenerate and improve skin function; however, its effectiveness on hair follicle (HF) growth and its underlying mechanisms need to be elucidated. Herein, we propose an air-based NTP treatment, which generates exogenous nitric oxide (eNO), as a therapeutic strategy for hair growth. The topical application of air-based NTP generates large amounts of eNO, which can be directly detected using a microelectrode NO sensor, in the dermis of mouse dorsal skin. Additionally, NTP-induced eNO has no cytotoxicity in normal human skin cells and promotes hair growth by increasing capillary tube formation, cellular proliferation, and hair/angiogenesis-related protein expression. Furthermore, NTP treatment promotes hair growth with adipogenesis and activation of CD34+CD44+ stem cells and improves the inter-follicular macroenvironment via increased perifollicular vascularity in the mouse hair regrowth model. Given the importance of the hair follicle (HF) cycle ratio (growth vs. regression vs. resting) in diagnosing alopecia, NTP treatment upregulates the stem cell activity of the HF to promote the anagen : catagen : telogen ratio, leading to improved hair growth. We confirmed the upregulation of increasing Wnt/ß-catenin signaling and activation of perifollicular adipose tissue and angiogenesis in HF regeneration. In conclusion, these results show that the eNO from NTP enhances the cellular activities of human skin cells and endothelial cells in vitro and stem cells in vivo, thereby increasing angiogenesis, adipogenesis, and hair growth in the skin dermis. Furthermore, the results of this study suggest that NTP treatment may be a highly efficient alternative in regenerative medicine for achieving enhanced hair growth.

Aerosol Delivery of Dornase Alfa Generated by Jet and Mesh Nebulizers.

Recent reports on mesh nebulizers suggest the possibility of stable nebulization of various therapeutic protein drugs. In this study, the in vitro performance and drug stability of jet and mesh nebulizers were examined for dornase alfa and compared with respect to their lung delivery efficiency in BALB/c mice. We compared four nebulizers: two jet nebulizers (PARI BOY SX with red and blue nozzles), a static mesh nebulizer (NE-U150), and a vibrating mesh nebulizer (NE-SM1). The enzymatic activity of dornase alfa was assessed using a kinetic fluorometric DNase activity assay. Both jet nebulizers had large residual volumes between 24% and 27%, while the volume of the NE-SM1 nebulizer was less than 2%. Evaluation of dornase alfa aerosols produced by the four nebulizers showed no overall loss of enzymatic activity or protein content and no increase in aggregation or degradation. The amount of dornase alfa delivered to the lungs was highest for the PARI BOY SX-red jet nebulizer. This result confirmed that aerosol droplet size is an important factor in determining the efficiency of dornase alfa delivery to the lungs. Further clinical studies and analysis are required before any conclusions can be drawn regarding the clinical safety and efficacy of these nebulizers.

Photo-Functionalized Magnetic Nanoparticles as a Nanocarrier of Photodynamic Anticancer Agent for Biomedical Theragnostics.

Various theragnostic agents have been devised and developed as cancer treatments; however, existing agents are often limited by their specific functions and complexities. Here, we report multifunctional magnetite (Fe3O4) nanoparticles functionalized with chlorin e6 (Ce6) and folic acid (FA) using a simple fabrication process to be used as theragnostic agents in photodynamic therapy (PDT). The effectiveness of cellular uptake of Fe3O4-Ce6-FA nanoparticles (FCF NPs) and its visualization as well as the photodynamic anticancer activities were evaluated. The mechanism of cancer cell death by the FCF NPs was also verified with qualitative and quantitative methods. Results indicate that FCF NPs have good penetration efficacy, resulting in excellent in vitro fluorescence and magnetic resonance imaging in cancer cells. FCF NPs exhibited promising anticancer activity in an irradiation time- and FCF NPs-dose-dependent manner in various cancer cell lines, leading to apoptotic cell death via morphological changes in cell membrane, nuclear, and DNA damage, and via overexpression of apoptosis-related genes, such as ZFP36L1, CYR61, GADD45G, caspases-2, -3, -9, 10, and -14. This study suggests that FCF NPs may be safely used in cancer therapy via PDT and could be a versatile therapeutic tool and biocompatible theragnostic agent, which may be used in diagnostic imaging.

Effect of Substituents on the Photophysical Properties and Bioimaging Application of BODIPY Derivatives with Triphenylamine Substituents.

We investigated the intramolecular charge transfer characteristics in the S1 state of boron-dipyrromethene (BODIPY) derivatives with triphenylamine (TPA) substituents, depending on the substituted position and the number of substituents. Based on the spectroscopic and theoretical results, the ß-substitution of TPA on BODIPY hybridizes locally excited and intramolecular charge transfer characteristics in the S1 state because of strong coupling between the highest occupied molecular orbitals of BODIPY and TPA moieties, and consequently, the BODIPY derivatives with ß-substituted TPAs exhibit strong red-color fluorescence around 640 nm in nonpolar and moderately polar solvents. The TPA substituent with propeller-like nonplanar geometry could prevent H-type aggregation between neighboring BODIPY derivative units and induce aggregation-induced emission enhancement (AIEE) characteristics of the BODIPY derivatives with TPA substituents, which are helpful to maintain their emission efficiencies under highly concentrated and condensed conditions. Since the red-color emission and AIEE property of the BODIPY derivatives with ß-substituted TPAs are promising characteristics for a bioimaging application, we applied these derivatives to L-929 fibroblast cells for cellular imaging. The BODIPY derivative with a single ß-substituted TPA (compound 2) was effectively loaded into porous silica nanoparticles (SNPs). Consequently, we achieved good cellular uptake of 2-SNPs and good cellular imaging, which further confirmed the bioimaging ability of 2-SNPs.

Comparison of Salbutamol Delivery Efficiency for Jet versus Mesh Nebulizer Using Mice.

Recent reports using a breathing simulator system have suggested that mesh nebulizers provide more effective medication delivery than jet nebulizers. In this study, the performances of jet and mesh nebulizers were evaluated by comparing their aerosol drug delivery efficiencies in mice. We compared four home nebulizers: two jet nebulizers (PARI BOY SX with red and blue nozzles), a static mesh nebulizer (NE-U22), and a vibrating mesh nebulizer (NE-SM1). After mice were exposed to salbutamol aerosol, the levels of salbutamol in serum and lung were estimated by ELISA. The residual volume of salbutamol was the largest at 34.6% in PARI BOY SX, while the values for NE-U22 and NE-SM1 mesh nebulizers were each less than 1%. The salbutamol delivery efficiencies of NE-U22 and NE-SM1 were higher than that of PARI BOY SX, as the total delivered amounts of lung and serum were 39.9% and 141.7% as compared to PARI BOY SX, respectively. The delivery efficiency of the mesh nebulizer was better than that of the jet nebulizer. Although the jet nebulizer can generate smaller aerosol particles than the mesh nebulizer used in this study, the output rate of the jet nebulizer is low, resulting in lower salbutamol delivery efficiency. Therefore, clinical validation of the drug delivery efficiency according to nebulizer type is necessary to avoid overdose and reduced drug wastage.

Immunotoxicity of nivalenol after subchronic dietary exposure to rats.

Immunobiological effects of nivalenol (NIV), a trichothecene mycotoxin produced by Fusarium nivale, were examined in male F344 rats after 90-day dietary exposure at doses of 0, 0.4, 1.5, and 6.9 mg/kg body weight/day (0, 6.25, 25 and 100 ppm, respectively) in a subchronic toxicity study. With regards to the serum immunoglobulin levels, a slight increase of IgM was observed only at 6.9 mg/kg (26% increase), while levels of IgG and IgA did not fluctuate at any dose. Flow cytometric analysis of splenic cells revealed a dose-dependent decrease of T lymphocyte/B lymphocyte (CD3(+)/B220(+)) ratio from 1.5mg/kg and an elevated CD4(+)helper/CD8(+)cytotoxic T lymphocyte ratio at 6.9 mg/kg. Furthermore, increases of natural killer (NK) activity of splenic lymphocytes against YAC-1 target cells were observed at all doses, while the magnitude of changes was similar between 1.5 and 6.9 mg/kg. At 6.9 mg/kg, the reduction of the ratio of NKR-P1A(+) splenic cells, which is an indicator of NK cells in the spleen, was apparent. As with other previous studies of NIV, decreased body weight was observed from 1.5 mg/kg during the experiment in the present study. In summary, NIV affected immune function in rats after 90-day dietary exposure, the effects being apparent from 0.4 mg/kg judging from the increase of NK activity, although nutritional suppression might have influenced the immunological changes appeared from 1.5mg/kg.

Enhanced Photodynamic Anticancer Activities of Multifunctional Magnetic Nanoparticles (Fe3O4) Conjugated with Chlorin e6 and Folic Acid in Prostate and Breast Cancer Cells.

Photodynamic therapy (PDT) is a promising alternative to conventional cancer treatment methods. Nonetheless, improvement of in vivo light penetration and cancer cell-targeting efficiency remain major challenges in clinical photodynamic therapy. This study aimed to develop multifunctional magnetic nanoparticles conjugated with a photosensitizer (PS) and cancer-targeting molecules via a simple surface modification process for PDT. To selectively target cancer cells and PDT functionality, core magnetic (Fe3O4) nanoparticles were covalently bound with chlorin e6 (Ce6) as a PS and folic acid (FA). When irradiated with a 660-nm long-wavelength light source, the Fe3O4-Ce6-FA nanoparticles with good biocompatibility exerted marked anticancer effects via apoptosis, as confirmed by analyzing the translocation of the plasma membrane, nuclear fragmentation, activities of caspase-3/7 in prostate (PC-3) and breast (MCF-7) cancer cells. Ce6, used herein as a PS, is thus more useful for PDT because of its ability to produce a high singlet oxygen quantum yield, which is owed to deep penetration by virtue of its long-wavelength absorption band; however, further in vivo studies are required to verify its biological effects for clinical applications.

Aggregation-Induced Emission of Tetraphenylethene-Conjugated Phenanthrene Derivatives and Their Bio-Imaging Applications.

In this study, a series of rationally designed emissive phenanthrene derivatives were synthesized and their aggregation-induced emission (AIE) properties in tetrahydrofuran (THF)/water mixtures were investigated. Two tetraphenylethene (TPE) segments were conjugated to both ends of the phenanthrene core at the para-positions and meta-positions, resulting in pTPEP and mTPEP derivatives, respectively. While the TPE-conjugated phenanthrene derivatives did not show any emission when dissolved in pure THF, they showed strong sky-blue emissions in water-THF mixtures, which is attributed to the restriction of intramolecular motions of TPE segments by aggregation. Furthermore, silica nanoparticles loaded with these AIE-active compounds were prepared and proved to be promising intracellular imaging agents.

Functionalized ZnO Nanoparticles with Gallic Acid for Antioxidant and Antibacterial Activity against Methicillin-Resistant S. aureus.

In this study, we report a new multifunctional nanoparticle with antioxidative and antibacterial activities in vitro. ZnO@GA nanoparticles were fabricated by coordinated covalent bonding of the antioxidant gallic acid (GA) on the surface of ZnO nanoparticles. This addition imparts both antioxidant activity and high affinity for the bacterial cell membrane. Antioxidative activities at various concentrations were evaluated using a 2,2'-azino-bis(ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging method. Antibacterial activities were evaluated against Gram-positive bacteria (Staphylococcus aureus: S. aureus), including several strains of methicillin-resistant S. aureus (MRSA), and Gram-negative bacteria (Escherichia coli). The functionalized ZnO@GA nanoparticles showed good antioxidative activity (69.71%), and the bactericidal activity of these nanoparticles was also increased compared to that of non-functionalized ZnO nanoparticles, with particularly effective inhibition and high selectivity for MRSA strains. The results indicate that multifunctional ZnO nanoparticles conjugated to GA molecules via a simple surface modification process displaying both antioxidant and antibacterial activity, suggesting a possibility to use it as an antibacterial agent for removing MRSA.

In Situ Forming and H2O2-Releasing Hydrogels for Treatment of Drug-Resistant Bacterial Infections.

Various types of commercialized wound dressings (e.g., films, foams, gels, and nanofiber meshes) have been clinically used as a physical barrier against bacterial invasion and as wound-healing materials. Although these dressings can protect the wounded tissue from the external environment, they cannot treat the wounds that are already infected with bacteria. Herein, we report in situ H2O2-releasing hydrogels as an active wound dressing with antibacterial properties for treatment of drug-resistant bacterial infection. In this study, H2O2 was used for two major purposes: (1) in situ gel formation via a horseradish peroxidase (HRP)/H2O2-triggered cross-linking reaction, and (2) antibacterial activity of the hydrogel via its oxidative effects. We found that there were residual H2O2 in the matrix after in situ HRP-catalyzed gelling, and varying the feed amount of H2O2 (1-10 mM; used to make hydrogels) enabled control of H2O2 release kinetics within a range of 2-509 µM. In addition, although the gelatin-hydroxyphenyl propionic acid (GH) gel called "GH 10" (showing the greatest H2O2 release, 509 µM) slightly decreased cell viability (to 82-84%) of keratinocyte (HaCaT) and fibroblast (L-929) cells in in vitro assays, none of the hydrogels showed significant cytotoxicity toward tissues in in vivo skin irritation tests. When the H2O2-releasing hydrogels that promote in vivo wound healing, were applied to various bacterial strains in vitro and ex vivo, they showed strong killing efficiency toward Gram-positive bacteria including Staphylococcus aureus, S. epidermidis, and clinical isolate of methicillin-resistant S. aureus (MRSA, drug-resistant bacteria), where the antimicrobial effect was dependent on the concentration of the H2O2 released. The present study suggests that our hydrogels have great potential as an injectable/sprayable antimicrobial dressing with biocompatibility and antibacterial activity against drug-resistant bacteria including MRSA for wound and infection treatment.