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Infectious diseases induced by multidrug-resistant bacteria are a challenging problem in medicine because of global rise in the drug resistance to pathogenic bacteria. Despite great efforts on the development of antibiotics and antimicrobial agents, there is still a great need to develop a strategy to early detect bacterial infections and eradicate bacteria effectively and simultaneously. The innate immune systems of various organisms produce antimicrobial peptides, which kill a broad range of bacteria with minimal cytotoxicity to mammalian cells. Therefore, antimicrobial peptides have recently attracted increasing attention as an alternative to conventional antibiotics in antibacterial medications. Here, we report a new family of antibacterial agents, which is formulated from self-assembly of a chimeric antimicrobial lipopeptide (DSPE-HnMc) and amphiphilic biodegradable polymers. HnMc micelles could effectively bind the bacterial membrane to kill a wide spectrum of bacteria and bacterial biofilms. In the studies of mouse models of drug-resistant bacterial infections, HnMc micelles could target bacterial infections with high specificity and also kill drug-resistant bacteria effectively, demonstrating the great potential of HnMc micelles as imaging and targeted antibacterial agents. These findings also provide new insight into the design of antimicrobial peptide-based nanomedicine for detection and treatment of bacterial infections.

Recent advancements in gene delivery systems that specifically target a variety of cancer types have increased demand for tissue-specific gene therapy. The current study describes the synthesis of a copolymer (GPgWSC) composed of a polyethylenimine (PEI)-grafted water-soluble chitosan (WSC) and gambogic acid (GA). It was validated as a ligand capable of enabling targeted attachment to transferrin receptors in HCT116 cancer cell lines. GPgWSC demonstrated superior antitumor activity in vitro in HCT116 compared to LoVo or MCF-7 cell lines, facilitated by the apoptotic activity of psiRNA-hBCL2. Pre-incubation of transferrin significantly inhibited GFP expression in the GPgWSC polyplex, demonstrating that GA is an extremely effective transferrin receptor targeting molecule. Additionally, in the HCT116-bearing mouse model, the tumor mass of PBS-treated mice increased to 2270 mm2 after 22 days, but the injection of GPgWSC polyplex significantly reduced the mass-increasing rate as a mass size of 248 mm2.

Background It remains an open question whether plant phloem sap proteins are functionally involved in plant defense mechanisms. Methods The antifungal effects of two profilin proteins from Arabidopsis thaliana, AtPFN1 and AtPFN2, were tested against 11 molds and 4 yeast fungal strains. Fluorescence profiling, biophysical, and biochemical analyses were employed to investigate their antifungal mechanism. Results Recombinant AtPFN1 and AtPFN2 proteins, expressed in Escherichia coli, inhibited the cell growth of various pathogenic fungal strains at concentrations ranging from 10 to 160 μg/mL. The proteins showed significant intracellular accumulation and cell-binding affinity for fungal cells. Interestingly, the AtPFN proteins could penetrate the fungal cell wall and membrane and act as inhibitors of fungal growth via generation of cellular reactive oxygen species and mitochondrial superoxide. This triggered the AtPFN variant-induced cell apoptosis, resulting in morphological changes in the cells. Conclusion PFNs may play a critical role as antifungal proteins in the Arabidopsis defense system against fungal pathogen attacks. General significance The present study indicates that two profilin proteins, AtPFN1 and AtPFN2, can act as natural antimicrobial agents in the plant defense system.

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, ‘XWZX’ basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.

Sung In Jeong,1,* Seong-Cheol Park,2,* Sun-Jeong Park,2,* Eun-Ji Kim,2 Hun Heo,2 Jong-Seok Park,1 Hui-Jeong Gwon,1 Youn-Mook Lim,1 Mi-Kyeong Jang2 1Research Division for Industry and Environment, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, 2Department of Polymer Science and Engineering, College of Engineering, Sunchon National University, Suncheon, Korea *These authors contributed equally to this work Introduction: Although numerous studies have been conducted with the aim of developing drug-delivery systems, chemically synthesized gene carriers have shown limited applications in the biomedical fields due to several problems, such as low-grafting yields, undesirable reactions, difficulties in controlling the reactions, and high-cost production owing to multi-step manufacturing processes. Materials and methods: We developed a 1-step synthesis process to produce 2-aminoethyl methacrylate-grafted water-soluble chitosan (AEMA-g-WSC) as a gene carrier, using gamma irradiation for simultaneous synthesis and sterilization, but no catalysts or photoinitiators. We analyzed the AEMA graft site on WSC using 2-dimensional nuclear magnetic resonance spectroscopy (2D NMR; 1H and 13C NMR), and assayed gene transfection effects in vitro and in vivo. Results: We revealed selective grafting of AEMA onto C6-OH groups of WSC. AEMA-g-WSC effectively condensed plasmid DNA to form polyplexes in the size range of 170 to 282 nm. AEMA-g-WSC polyplexes in combination with psi-hBCL2 (a vector expressing short hairpin RNA against BCL2 mRNA) inhibited tumor cell proliferation and tumor growth in vitro and in vivo, respectively, by inducing apoptosis. Conclusion: The simple grafting process mediated via gamma irradiation is a promising method for synthesizing gene carriers. Keywords: water-soluble chitosan, 2-aminoethyl methacrylate, gamma irradiation, gene delivery

The highly conserved SGT1 (suppressor of the G2 alleles of skp1) proteins from Arabidopsis are known to contribute to plant resistance to pathogens. While SGT1 proteins respond to fungal pathogens, their antifungal activity is not reported and the mechanism for this inhibition is not well understood. Therefore, recombinant Arabidopsis SGT1 proteins were cloned, expressed, and purified to evaluate their antifungal activity, resulting in their potent inhibition of pathogen growth. Dye-labeled proteins are localized to the cytosol of Candida albicans cells without the disruption of the cell membrane. Moreover, we showed that entry of the proteins into C. albicans cells resulted in the accumulation of reactive oxygen species (ROS) and cell death via altered mitochondrial potential. Morphological changes of C. albicans cells in the presence of proteins were visualized by scanning electron microscopy. Our data suggest that AtSGT1 proteins play a critical role in plant resistance to pathogenic fungal infection and they can be classified to a new plant antifungal protein.

Targeting antitumor drugs to specific tissues or cells has attracted considerable interest in the recent tumor therapy because it can reduce the side effects in the body and the treated drug dosage through homing drug to the desired tissues. To utilize the high affinity of biotin to avidin as a drug carrier for doxorubicin (DOX), biotinylated targeting material (biotin-polyethylene glycol (PEG)-folic acid, BPF) and biotinylated drug with a bioreducible linker (biotin-SS-PEG-DOX, BSPD) were designed in this study. The in vitro cellular uptake of the avidin/biotinylated drug complexes showed that the complexes with BPF were internalized dose-dependently into HeLa cells, while their uptake was not detected in the absence of BPF. Although the anti-tumor activity of the drug complexes with BPF and BSPD were similar to that of free DOX in cervical cancer cells, the cytotoxic effect of DOX was significantly reduced in normal cells. Their targeting antitumor effect revealed the significant inhibition of the increasing tumor size in HeLa-xenograft mouse model. These results show that the strong complexing between avidin and biotin acted as a targeting moiety and the antitumor drug is an adaptable tool in the fields of tumor therapy.

An antifungal protein, AtUSP protein (At3g53990), was isolated from Arabidopsis thaliana leaves by ion and size chromatography and sequenced by N-terminal sequencing. The AtUSP gene amplified from an Arabidopsis leaf cDNA library was transformed to Escherichia coli to express the AtUSP protein. The recombinant protein inhibited the cell growth of various pathogenic fungal strains. The levels of the AtUSP transcripts were increased by various stresses, including pathogenic infection and salt stress. These results suggest that Arabidopsis AtUSP plays a critical role in the plant tolerance to diverse pathogenic infections. The potent antifungal action, which is a new function of AtUSP, was attributed to fungal reactive oxygen species (ROS) generation and mitochondrial potential alteration.

A plant protein with effective antifungal activity was isolated from Arabidopsis thaliana leaves. The results of N-terminal sequencing analysis revealed that the isolated protein matches with a partial fragment of A. thaliana oxidoreductase thioredoxin h type 5; AtTrx-h5 (GenBank accession number AT1G45145). The AtTrx-h5 gene isolated from an Arabidopsis leaf cDNA library was transformed into Escherichia coli to investigate the functional characterizations of AtTrx-h5 protein. Recombinant AtTrx-h5 protein inhibited the conidia germination in 7 filamentous fungal cells and the proliferation in 3 pathogenic yeast cells. In addition, the cellular distribution of recombinant AtTrx-h5 protein in Fusarium solani showed pH-dependent cytosolic accumulation. Interestingly, AtTrx-h5 acted as an inhibitor of fungal growth via cellular reactive oxygen species (ROS) generation. We propose the novel functions of AtTrx-h5 as a potential candidate for natural antifungal material in the study.

Styrenated phenols are usually synthesized by the reaction of styrene and phenol under acid catalysts. Styrenated phenol involving high content of di-styrenated phenol (DSP) was synthesized, which can be used to prepare styrenated phenol alkoxylate. The solid catalyst used in this study was prepared by impregnation method. SO4 2- on SO4 2-/ZrO2 catalyst was introduced from an aqueous 1M-H2SO4 solution. The catalysts were characterized by SEM images, XRD patterns, and FT-IR spectra. The catalytic activity was examined by measuring the conversion of phenol and styrene in a liquid-phase batch reactor. Almost 100% conversion of both phenol and styrene over 15-SO4 2-/ZrO2 catalyst were obtained at reaction temperature of 100 °C and reaction time of 6 hr. Amount of catalyst to the reactants was 15 wt%. At same reaction conditions, selectivity of MSP, DSP, and TSP were 23.6%, 52.1%, and 5.4%, respectively. It was known that the selectivity to DSP was increased as IR absorption peak of 1236 cm-1 corresponding to O–S–O bonds was increased.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant Pseudomonas aeruginosa and Staphylococcus aureus. The effects of PS1-2, PS1-5, and PS1-6 were also tested in vivo using a mouse model. All peptides inhibited planktonic cell growth and biofilm formation in a dose-dependent manner. They also reduced preformed biofilm masses by removing the carbohydrates, extracellular DNA, and lipids that comprised extracellular polymeric substances (EPSs) but did not affect proteins. In vivo, PS1-2 showed the greatest efficacy against preformed biofilms with no cytotoxicity. Our findings indicate that the PS1-2 peptide has potential as a next-generation therapeutic drug to overcome multidrug resistance and to regulate inflammatory response in biofilm-associated infections.

Plants are constantly subjected to a variety of environmental stresses and have evolved regulatory responses to overcome unfavorable conditions that might reduce or adversely change a plant&rsquo, s growth or development. Among these, the regulated production of reactive oxygen species (ROS) as a signaling molecule occurs during plant development and pathogen defense. This study demonstrates the possible antifungal activity of Oryza sativa Tetratricopeptide Domain-containing thioredoxin (OsTDX) protein against various fungal pathogens. The transcription of OsTDX was induced by various environmental stresses known to elicit the generation of ROS in plant cells. OsTDX protein showed potent antifungal activity, with minimum inhibitory concentrations (MICs) against yeast and filamentous fungi ranging between 1.56 and 6.25 and 50 and 100 &micro, g/mL, respectively. The uptake of SYTOX-Green into fungal cells and efflux of calcein from artificial fungus-like liposomes suggest that its killing mechanism involves membrane permeabilization and damage. In addition, irregular blebs and holes apparent on the surfaces of OsTDX-treated fungal cells indicate the membranolytic action of this protein. Our results suggest that the OsTDX protein represents a potentially useful lead for the development of pathogen-resistant plants.

Recently gene delivery has been designed newly using bioactive biomaterial and applied in the various field by many researchers. In this study, we proposed a new gene delivery system which has the capability of targeting effect in the specific tissue and remarkably enhanced transfection efficiency. We investigated 1H-NMR spectroscopy, particle size analyzer and gel retardation to confirm the correct preparation of gene delivery. Also, we identified the hemo-compatibility of gene delivery by hemolysis assay, non-cytotoxicity by MTT test and transfection efficiency. The uptake mechanism of the gene carrier was confirmed using inhibitor agent such as sodium azide, indomethacin, quercetin, colchicine, and chloropromazine. As a results, it was identified that gene carrier prepared by in this study entered in the cell by the microtubule-dependent, energy-dependent and clathrin-mediated endocytosis pathway.

The hydrophobicity of NADPH-thioredoxin reductase C (AtNTRC) of Arabidopsis was determined at the carboxyl terminal based on a Kyte-Dolittle hydropathy plot. A carboxyl-terminal deletion mutant of AtNTRC was made in this study to determine whether such deletion could affect the structure and function of AtNTRC. The mutant protein with 14 amino acids deleted at the carboxyl terminus (designated as AtNTRCΔC14) was purified. It was found that AtNTRCΔC14 protein had higher hydrophobicity compared to AtNTRC. Such increase in hydrophobicity of AtNTRCΔC14 affected its native structure and functions. In addition, AtNTRCΔC14 had higher number of high oligomeric complexes compared to AtNTRC based on native-gel electrophoresis and size exclusion chromatography. Although the chaperone activity of AtNTRCΔC14 was enhanced, its NADPH-dependent reductase activity was lower compared to AtNTRC. Therefore, the hydrophobicity of the thioredoxin domain of AtNTRC is important for forming high molecular weight complexes and for maintaining functional balance between chaperone activity and thioredoxin reductase activity.

Norfloxacin is a broad-spectrum synthetic antibacterial agent that has been used to treat complicated urinary tract infections. However, the poor water-solubility of norfloxacin limits its bioavailability. The aim of the present study was to synthesize water-soluble norfloxacin derivate through adding isosorbide via enzymatic catalyst (Green chemistry) and to evaluate its physiological and biological properties. Isosorbide-dinorfloxacin (ISDNf) was synthesized by enzymatic esterification between the carboxyl group of norfloxacin and two hydroxyl groups of isosorbide. Chemical structure and water-solubility of ISDNf purified by HPLC was investigated by using 1H NMR and MALDITOF, and optical spectrometry, respectively. Antibacterial effects of ISDNf were evaluated against 7 drug-susceptible and 7 drug-resistant bacteria. ISDNf, which was successfully synthesized, was water-soluble at 10,000 mg/L, compared that free norfloxacin was water-insoluble at 100 mg/L. The antibacterial efficiency of ISDNf was maintained in drugsusceptible bacteria, while that was enhanced in drugresistant bacteria. Additionally, its cytotoxicity in mammalian cells was remarkably reduced and its solid diffusion capacity was increased. The increased water-solubility, antibacterial efficacy, and diffusion capacity and reduced cytotoxicity of ISDNf suggests that it could potentially be developed as a novel prodrug.

Increases in the numbers of immunocompromised patients and the emergence of drug-resistance fungal pathogens have led to the need for new, safe, efficacious antifungal agents. In this study, we designed a histidine-lysine-lysine (HKK) motif and synthesized six HKK peptides with repetitions of the motif. These peptides showed length-dependent antifungal activity against drug-susceptible and drug-resistant fungal pathogens via membranolytic or non-membranolytic action. None of the peptides were cytotoxic to rat erythrocytes or NIH3T3 mouse embryonic fibroblasts. Short-length peptides were directly translocated in fungal cytosol and reacted with mitochondria, resulting in apoptosis. Membrane-permeabilizing activity occurred in the presence of long peptides, and peptides were able to transfer to the cytosol and induce reactive oxygen species. Our results suggest that peptides composed only of cationic amino acids may be good candidates as antifungal agents.

To biochemically characterize synthetic peptides to control harmful algal blooms (HABs) that cause red tides in marine water ecosystems. We present an analysis of several short synthetic peptides and their efficacy as algicidal agents. By altering the amino acid composition of the peptides we addressed the mode of algicidal action and determine the optimal balance of cationic and hydrophobic content for killing. In a controlled setting, these synthetic peptides disrupted both plasma and chloroplast membranes of several species known to result in HABs. This disruption was a direct result of the hydrophobic and cationic content of the peptide. Furthermore, by using an anti-HAB bioassay in scallops, we determined that these peptides were algicidal without being cytotoxic to other marine organisms. These synthetic peptides may prove promising for general marine ecosystem remediation where HABs have become widespread and resulted in serious economic loss.

Reactive oxygen species (ROS) produced by host phagocytes exert antibacterial action against a variety of pathogens and ROS-induced oxidative stress is the governing mechanism for the antibacterial activity of major bactericidal antibiotics. In particular, hydroxyl radical is a strong and nonselective oxidant which can damage biomolecules such as DNA, proteins and lipids. Ferrous ion is known to convert mild oxidant hydrogen peroxide (H2O2) into highly reactive and toxic hydroxyl radicals, referred to as Fenton reaction. Herein, we report a new class of antibacterial agents based on Fenton reaction-performing nanostructures, composed of H2O2-generating polymer (PCAE) and iron-containing ferrocene. Amphiphilic PCAE was designed to incorporate H2O2-generating cinnamaldehyde through acid-cleavable linkages and self-assemble to form thermodynamically stable micelles which could encapsulate ferrocene in their hydrophobic core. All the experiments in vitro display that ferrocene-loaded PCAE micelles produce hydroxyl radicals to kill Escherichia coli and Pseudomonas aeruginosa through membrane damages. Intraperitoneally injected ferrocene-loaded PCAE micelles significantly reduced the lung damages and therefore increased the survival rate of mice infected with drug resistant P. aeruginosa. Given their potent antibacterial activity, ferrocene-loaded PCAE micelles hold great potential as a new class of ROS-manipulating antibacterial agents.

A novel polymeric prodrug (PXPEG) was prepared to enhance the solubility of an anti-cancer drug, paclitaxel, in aqueous solutions and decrease the cytotoxicity by PEGylation, which means PEG attached to another molecule. In addition, the targeting ligand, transferrin (TF), was modified to PXPEG to enhance the therapeutic efficacy. The targeting ligand-modified PXPEG (TFPXPEG) was examined by 1 H-NMR to confirm the successful synthesis. The synthesized TFPXPEG had better solubility than the free drug against aqueous solution. The particle size of TFPXPEG was approximately 197.2 nm and it had a spherical shape. The MTT assay showed that the anti-tumor efficiency of TFPXPEG was better than that of TF-unmodified PXPEG. In the KB tumor-bearing mouse model, the tumor volume of TFPXPEG treated groups was decreased dramatically by more than 2 fold or 3 fold compared to the PBS or PXPEG treated groups. The in vitro and in vivo evaluation showed that TFPXPEG had better efficacy than that of PXPEG due to the targeting effect of targeting ligands, such as TF.

We developed a polyamidoamine (PAMAM) conjugated disulfide-linked chitosan (dsCS-PAM) for nonviral gene delivery. At weight ratio of 128, dsCS and dsCS-PAM showed positive zeta potential of around 9.5 mV and 14.5 mV, respectively, demonstrating their ability to form complexes with plasmid DNA (pDNA) for cell transfection. CS-PAM and dsCS-PAM formed complexes with pDNA in a reducing environment containing 5 mM DLdithiothreitol (DTT) at weight ratio 32 and 16, respectively. PAMAM conjugated chitosan derivatives, dsCS-PAM and CS-PAM, showed higher in vitro transfection efficiency than unconjugated derivatives dsCS and CS. Approximately 80% of A549 and 293 cells were viable in the presence of up to 100 μg/mL of dsCS-PAM. The results of this study demonstrate that dsCS-PAM has the potential to be a safe gene delivery carrier. Open image in new window

Atopic dermatitis (AD) is a chronic inflammatory skin disease that was influenced by complex interactions via genetic, environmental, immunologic, and biochemical factors, though the cause of AD is still unknown. It characterized by elevated serum immunoglobulin E (IgE) levels, immunological abnormalities, and eosinophilia in the tissues and peripheral blood. In the present study, we applied an antimicrobial moisturizing cream containing low-molecular weight water-soluble chitosan and herbal honey (AMCH) to remedy AD-like lesions. The inhibiting effect of AMCH on NC/Nga mice, that AD-like lesion was induced by 1-chloro-2,4-dinitrobenzene (DNCB), was evaluated by examining sensory evaluation scores, scratching behavior, immune cells in blood, serum IgE level, infiltration of mast cells, and skin histology. The total sensory evaluation scores, scratching behavior, the level of serum IgE, interlukin-4 (IL-4), and IL-12 in AD mouse model were significantly reduced by AMCH. Moreover, its suppressing effect resulted in decreased mast cell infiltration. Our results suggest that AMCH might be beneficial as a potent agent for treatment of AD-like lesion.

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening Oryza sativa leaves to isolate novel AFPs. We identified a thioredoxin protein with antioxidant properties. Being ubiquitous, thioredoxins (Trxs) function in the redox balance of all living organisms. Sequencing by Edman degradation method revealed the AFP to be O. sativa Thioredoxin m-type isoform (OsTrxm). We purified the recombinant OsTrxm and its cysteine mutant proteins (OsTrxm C/S) in Escherichia coli. The recombinant OsTrxm proteins inhibited the growth of various pathogenic fungal cells. Interestingly, OsTrxm C/S mutant showed higher antifungal activity than OsTrxm. A growth inhibitory assay against various fungal pathogens and yeasts confirmed the pertinent role of cysteine residues. The OsTrxm protein variants penetrated the fungal cell wall and membrane, accumulated in the cells and generated reactive oxygen species. Although the role of OsTrxm in chloroplast development is known, its biochemical and molecular functions have not been elucidated. These findings suggest that in addition to redox regulation, OsTrxm also functions as an antimicrobial agent.

To develop water-soluble chitosan as an effient gene delivery carrier, chitosan oligosaccharides (COSs) with various molecular weights (MW) were studied for gene transfection agents. MWs of COSs fractionated by ultrafiltration techniques were identified as narrow MW distributions with the average MW ranging from 1 to 10 kDa through gel permeation chromatography (GPC) measurement depending on the applied ultrafiltration membranes. Their structural characterizations were analyzed by FTIR spectrophotometer and NMR. The degree of deacetylation was determined by UV spectroscopy showing the degree of deacetylation above 90%. The relative cell viabilities were maintained over 100% (10 mg/mL), independent of the MW of the fractionated COSs. The fractionated COSs of 10 mg/mL concentration with narrow MW distributions showed non-cytotoxicity in Caco-2 cells.

To develop a paclitaxel carrier based on chitosan, chitosan oligosaccharide (COS) was chemically modified with bile acid (deoxycholic acid and lithocholic acid) as a hydrophobic group. Paclitaxel was loaded in bile acid conjugated chitosan oligosaccharide (CBs) nanoparticles by a dialysis method. We confirmed that the paclitaxel-loaded COS (CBs-Tx) nanoparticles could be successfully prepared with a yield of 80%–90% and paclitaxel encapsulation of 54%–70%. The size of CB nanoparticles was in the range of 200–300 nm, and it increased to 300–400 nm after paclitaxel loading with the narrow size distribution maintained. Paclitaxel-loaded CBs (CBs-Tx) nanoparticles showed remarkably high anticancer activity compared with paclitaxel in cremophore EL (CrEL)-ethanol against B16F10 cells. The antitumor efficacy in vivo was shown with significant inhibition of the tumor growth in both paclitaxel-treated groups. The effect on tumor size by the paclitaxel in the CrEL-ethanol formulation appeared to be slightly larger than that in CBs-Tx. The decrease in cytotoxicity and the increase in antitumor activity may lead to the improvement in the therapeutic index in clinical use compared to commercial paclitaxel. The efficacy of CBs-Tx nanoparticles suggests that bile acid as a hydrophobic group may have a potential application of effectively loading hydrophobic drugs such as paclitaxel.

Centrifugal partition chromatography (CPC) can be used to isolate various bioactive compounds from natural materials by one-step. We confirmed antioxidative compounds existed in chloroform (CHCl3) fraction of Sargassum siliquastrum using online-HPLC. Fractions (A, B, C, D and E) were separated from the CHCl3 fraction by preparative CPC (n-hexane:ethyl acetate:methanol:water, 5:5:7:3, v/v). In this study, we proved that the isolated compounds exhibit anti-inflammatory activities using lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. The fraction A which exhibited the strongest inhibitory effect on nitric oxide (NO) production level, was confirmed as sargachromanol E by LC-MS-ESI, (1)H NMR and (13)C NMR data. The sargachromanol E significantly reduced the inflammatory response in LPS induced macrophages, decreasing LPS-induced transcription factor of pro-inflammatory cyclooxygenase-2, NO synthase, phosphate P38, phosphate ERK1/2, LPS-stimulated tumor-necrosis factor alpha, interleukin-1 beta and prostaglandin E2 release. In conclusion, it was suggested that sargachromanol E inhibited inflammation in LPS induced RAW 264.7 cells via MAPK pathway.

In the present study, the protective effects of phlorotannins isolated from Ecklonia cava against ethanol-induced cell damage and apoptosis were investigated both in vitro and in vivo. Three phlorotannin compounds, namely phloroglucinol, eckol and dieckol, were successively isolated and identified from the extract. Dieckol showed the strongest protective effect against ethanol-induced cell apoptosis in Chang liver cells, with the lowest cytotoxicity. It was observed that dieckol reduced cell apoptosis through activation of Bcl-xL and PARP, and down-regulation of Bax and caspase-3 in Western blot analyses. In the in vivo study, the protective effect of ethanol induced by dieckol was investigated in a zebrafish model. The dieckol treated group scavenged intracellural reactive oxygen species and prevented lipid peroxidation and ethanol induced cell death in the zebrafish embryo. In conclusion, dieckol isolated from E. cava might possess a potential protective effect against ethanol-induced liver diseases.

The safe and effective delivery of genetic material into cells is a necessary factor for gene therapy. Although a wide range of materials, methods, and combinations have been reported, successful gene therapy has been limited. In the present study, a targeted gene carrier for α

Amphotericin B (AmB) has been widely used against fungal infections throughout almost the entire body, including the skin, nails, oral cavity, respiratory tract, and urinary tract. However, the development of AmB-loaded nanoparticles demands a novel technique that reduces its toxicity and other associated problems. Here, we developed a pH-responsive and redox-sensitive polymer-based AmB-delivery carrier system. In particular, this system was functionalized by conjugation with the antifungal peptide histatin 5, which acts both as a targeting ligand and a synergistic antifungal molecule against Candida albicans, a major systemic fungal pathogen of humans. Our results in vitro and in vivo suggest that this drug-delivery system may serve as a novel tool to facilitate the use of antimicrobial peptides as targeting ligands to pathogenic microbes, which would open new avenues of investigation in the field of drug delivery.

Alpha-synuclein (α-Syn), a small (14 kDa) protein associated with Parkinson's disease, is abundant in human neural tissues. α-Syn plays an important role in maintaining a supply of synaptic vesicles in presynaptic terminals; however, the mechanism by which it performs this function are not well understood. In addition, there is a correlation between α-Syn over-expression and upregulation of an innate immune response. Given the growing body of literature surrounding antimicrobial peptides (AMPs) in the brain, and the similarities between α-Syn and a previously characterized AMP, Amyloid-β, we set out to investigate if α-Syn shares AMP-like properties. Here we demonstrate that α-Syn exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, we demonstrate a role for α-Syn in inhibiting various pathogenic fungal strains such as Aspergillus flavus, Aspergillus fumigatus and Rhizoctonia solani. We also analyzed localizations of recombinant α-Syn protein in E. coli and Candida albicans. These results suggest that in addition to α-Syn's role in neurotransmitter release, it appears to be a natural AMP.

In this study, polypeptide/polysaccharide graft copolymers pullulan-g-poly(L-lysine) (PULPLL) was prepared through the ring opening polymerization of ɛ-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of pullulan. The PULPLL copolymer nanoparticles, which are prepared by the diafiltration method, were investigated by using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectra (1H NMR), dynamic light scattering (DLS), transmission electron microscope (TEM), and fluorescence spectroscopy. The PULPLL copolymers formed self-aggregated nanoparticles in the aqueous milieu. The physicochemical characterization revealed that the nanoparticle size ranged between 60 and 500 nanometers, and the critical aggregation concentration (CAC) is 0.001∼0.1 g/L, depending on the degree of substitution. With negligible cytotoxicity and higher transfection efficiency, PULPLL nanoparticles may be safe gene carriers.

：The silica-silica interaction parameter (α F ) of the silane treated silica filled natural rubber (NR) compoundwas investigated. As silane (TESPT) concentration increased from 2 to 12% (2, 4, 6, 8, 10, 12%), the α F value increasedat the same silica concentration (10, 20, 30, and 40 phr). It seemed the sulfur atoms in TESPT acted as a crosslinking agent in the compound. As silica concentration increased from 10 to 40 phr, the α F value increased at the same silaneconcentration due to increased silica-silica interaction. Keywords ：α F (measure of the in-rubber structure of the filler), silica / silane, silica structure, Wolff's theory, naturalrubber(NR). † 대표저자 E-mail: kkim@dongahtire.co.kr Ⅰ. 서 론 충전제 입자간 상호관계를 관찰하기 위해서는 충전제 입자간 상호작용 계수 (α F ), 1,2 Payne effect 3,4 를 측정하는 방법 등 여러 가지가 있다. Wolff는 충전제 로딩량과 토크 상승값 (T max-min )의 함수관계에서 나타나는 기울기를 α F 로 정의하였고 입자가 충전된 복합소재와 비충전된 복합소재의 토크 상승값을 이용하여 측정하였으며 카본블랙과 실리카의 α F 를 비교하였다. 1,2 한편 Payne은 입자간 관계를 strain sweep으로 표현하였는데 입자간 상호작용은 입자들의 percolation point 이상에서 관찰된다. Wolff의 방법은 컴파운드의 토크 변화를 관찰하여 측정하는 것이기 때문에 입자간 상호작용 뿐만 아니라 컴파운드 내의 3차원적 사슬구조 (3-dimensional network)의 증가를 관찰할 수 있는 이점이 있다.

：The silica-silica interaction parameter (α F ) of the silane treated silica filled natural rubber (NR) compoundwas investigated. The measured α F values using mass fraction method following Wolff's theory were compared with volumefraction method. As silica concentration increased, the α F value increased for both methods. The value of α F expressedas volume fraction was higher than that of mass fraction, which resulted in large gaps between α F values. The effect ofaccelerator (MBT) concentration on α F values was compared. Keywords ：α F (measure of the in-rubber structure of the filler), silica / silane, silica structure, Wolff's theory, naturalrubber(NR). † 대표저자 E-mail: kkim@dongahtire.co.kr Ⅰ. 서 론 표면이 hydroxyl 그룹으로 이루어져 있는 실리카는 실란과 함께 고무복합소재에 첨가되었을 시 고무사슬과의 3차원적 사슬구조를 형성한다. 따라서, 카본블랙을 첨가했을 시 보다 회전저항력(rolling resistance) 및 노면접지력(traction) 등에서 우수한 물성들을 보인다. 따라서 실리카는 연비 향상과 눈이나 비가 왔을 시 노면접착력이 우수한 성능이 요구되는 타이어의 tread compound 제조에 사용되고 있다. 1 그 동안 실리카의 보강효과, 2 실리카-실란의 반응

To develop hyaluronic acid (HA)-based anticancer agent carrier, hyaluronic acid was chemically modified with the hydrophobic group of deoxycholic acid(DA). The physicochemical properties of the deoxycholic acid-conjugated HA (HADA) were investigated by using 1 H NMR, FTIR, spectrophotometer and TEM. Paclitaxel (Tx)-loaded HADA nanoparticles were prepared by a dialysis method. The loading efficiency of drug and drug contents of Tx-loaded HADA nanoparticles (HADA-Tx) were measured by HPLC. The anticancer activity of HADA-Tx was investigated by its cytotoxicity against KB cell in vitro. The HADA-Tx was shown to have the superior potential for the anticancer drug delivery.

Doxorubicin (DOX)-conjugated poly(DL-lactide-co-glycolide)(PLGA) was synthesized and nanoparticles were prepared without the use of a surfactant. The DOX content in the nanoparticles ranged from 1.2 to 4.75% (w/w). The particle size of the DOX-conjugated PLGA (DOX-LG) nanoparticles increased when a higher molecular weight (M.W.) of polymer was used. TEM revealed the nanoparticles to have spherical shapes with a size of approximately 100∼200 nm. The drug was released continuously from the nanoparticles over a one month period. In particular, the overall drug release from the nanoparticles prepared with a higher M.W. was significantly faster than that from nanoparticles with a lower M.W. The cytotoxicity test in vitro using CT26 colon carcinoma cells showed that the free DOX had higher cytotoxicity than DOX-LG. These results suggest that DOX was released slowly from nanoparticles and was exposed to the tumor cells. Moreover, DOX-LG might show low cytotoxicity due to these properties of nanoparticles.

Insulin-incorporated nanoparticles were prepared by polyelectrolyte complex formation using low-molecular weight water soluble chitosan (LMWSC). Insulin-incorporated nanoparticles showed spherical shapes with a particle size of approximately 200 nm. The average particle size, drug content and loading efficiency increased with increasing insulin feed. The zeta potential decreased with increasing insulin feed, indicating that the electrostatic interaction increased with increasing insulin feed. At 1H nuclear magnetic resonance (NMR) spectra, insulin and LMWSC had intrinsic peaks while the peaks of the nanoparticles were similar to LMWSC itself at D2O. However, the specific peaks for insulin appeared as a result of the breakdown of nanoparticles using DCl. When polyelectrolyte complex had dissociated, fluorescein isothiocyanate (FITC)-labeled insulin in the nanoparticles showed similar UV absorption peaks to FITC-labeled insulin only. At the release study, insulin was released continuously from the nanoparticles over 120 h at all formulations. Higher insulin contents induced a decrease in the rate of insulin released from nanoparticles. Consequently, LMWSC and insulin successively formed polyelectrolyte complexes as a nanocarriers and LMWSC can be considered a good candidate for insulin delivery. Open image in new window

In this study, methotrexate (MTX)-encapsulated polymeric micelles using methoxy poly(ethylene glycol) (MPEG)-grafted chitosan (ChitoPEG) copolymer were prepared. The MTX-incorporated polymeric micelles of ChitoPEG copolymer has a particle size of around 50-100 nm. In 1H nuclear magnetic resonance (NMR) study, the specific peaks of MTX disappeared in heavy water (D2O) and only the specific peak of MPEG was observed, while all of the peaks were confirmed in dimethyl sulfoxide (DMSO). These results indicated that MTX was complexed with chitosan and then formed an ion complex inner-core of the polymeric micelle in an aqueous environment. The drug contents of the polymeric micelle were around 4∼12% and the loading efficiency of MTX in the polymeric micelles was higher than 60% (w/w) for all of the formulations. The cytotoxicity of MTX and MTX-incorporated polymeric micelle against CT26 tumor cells was not significantly changed.

Noble thermosensitive nanoparticles, based on a PNIPAAm-co-AA core and a chitosan shell structure, were designed and synthesized for the controlled release of the loaded drug. PNIPAAm nanoparticles containing a carboxylic group on their surface were synthesized using emulsion polymerization. The carboxylic groups were conjugated with the amino group of a low molecular weight, water soluble chitosan. The particle size of the synthesized nanoparticles was decreased from 380 to 25 nm as the temperature of the dispersed medium was increased. Chitosan-conjugated nanoparticles with 2∼5 wt% MBA, a crosslinking monomer, induced a stable aqueous dispersion at a concentration of 1 mg/1 mL. The chitosan-conjugated nanoparticles showed thermosensitive behaviors such as LCST and size shrinkage that were affected by the PNIPAAm core and induced some particle aggregation around LCST, which was not shown in the NIPAAm-co-AA nanoparticles. These chitosan-conjugated nanoparticles are also expected to be more biocompatible than the PNIPAAm core itself through the chitosan shell structures.