Antioxidant and antibacterial activities of eugenol and carvacrol‐grafted chitosan nanoparticles

Essential oils are known to possess antimicrobial and antioxidant activity while chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. In this work, nanoparticles with both antioxidant and antibacterial properties were prepared by grafting eugenol and carvacrol (two components of essential oils) on chitosan nanoparticles. Aldehyde groups were first introduced in eugenol and carvacrol, and the grafting of these oils to chitosan nanoparticles was carried out via the Schiff base reaction. The surface concentration of the grafted essential oil components was determined by X‐ray photoelectron spectroscopy (XPS). The antioxidant activities of the carvacrol‐grafted chitosan nanoparticles (CHCA NPs) and the eugenol‐grafted chitosan nanoparticles (CHEU NPs) were assayed with diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a representative gram‐negative bacterium, Escherichia coli (E. coli) and a gram‐positive bacterium, Staphylococcus aureus (S. aureus). The grafted eugenol and carvacrol conferred antioxidant activity to the chitosan nanoparticles, and the essential oil component‐grafted chitosan nanoparticles achieved an antibacterial activity equivalent to or better than that of the unmodified chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the cytotoxicity of CHEU NPs and CHCA NPs were significant lower than those of the pure essential oils. Biotechnol. Bioeng. 2009; 104: 30–39 © 2009 Wiley Periodicals, Inc.     

Carbon nanotube clusters as universal bacterial adsorbents and magnetic separation agents

The magnetic susceptibility and high bacterial affinity of carbon nanotube (CNT) clusters highlight their great potential as a magnetic bio‐separation agent. This article reports the CNT clusters' capability as “universal” bacterial adsorbents and magnetic separation agents by designing and testing a multiwalled carbon nanotube (MWNT) cluster‐based process for bacterial capturing and separation. The reaction system consisted of large clusters of MWNTs for bacterial capture and an external magnet for bio‐separation. The designed system was tested and optimized using Escherichia coli as a model bacterium, and further generalized by testing the process with other representative strains of both gram‐positive and gram‐negative bacteria. For all strains tested, bacterial adsorption to MWNT clusters occurred spontaneously, and the estimated MWNT clusters' adsorption capacities were nearly the same regardless of the types of strains. The bacteria‐bound MWNT clusters also responded almost instantaneously to the magnetic field by a rare‐earth magnet (0.68 Tesla), and completely separated from the bulk aqueous phase and retained in the system. The results clearly demonstrate their excellent potential as highly effective “universal” bacterial adsorbents for the spontaneous adsorption of any types of bacteria to the clusters and as paramagnetic complexes for the rapid and highly effective magnetic separations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chitosan-Modified PLGA Nanoparticles with Versatile Surface for Improved Drug Delivery

Shortage of functional groups on surface of poly(lactide-co-glycolide) (PLGA)-based drug delivery carriers always hampers its wide applications such as passive targeting and conjugation with targeting molecules. In this research, PLGA nanoparticles were modified with chitosan through physical adsorption and chemical binding methods. The surface charges were regulated by altering pH value in chitosan solutions. After the introduction of chitosan, zeta potential of the PLGA nanoparticle surface changed from negative charge to positive one, making the drug carriers more affinity to cancer cells. Functional groups were compared between PLGA nanoparticles and chitosan-modified PLGA nanoparticles. Amine groups were exhibited on PLGA nanoparticle surface after the chitosan modification as confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The modified nanoparticles showed an initial burst release followed by a moderate and sustained release profile. Higher percentage of drugs from cumulative release can be achieved in the same prolonged time range. Therefore, PLGA nanoparticles modified by chitosan showed versatility of surface and a possible improvement in the efficacy of current PLGA-based drug delivery system.     

Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance

Protein adsorption to nanoparticles is described as a chemical reaction in which proteins attach to binding sites on the nanoparticle surface. This process is defined by a dissociation coefficient, which tells how many proteins are adsorbed per nanoparticle in dependence of the protein concentration. Different techniques to experimentally determine dissociation coefficients of protein adsorption to nanoparticles are reviewed. Results of more than 130 experiments in which dissociation coefficients have been determined are compared. Data show that different methods, nanoparticle systems, and proteins can lead to significantly different dissociation coefficients. However, we observed a clear tendency of smaller dissociation coefficients upon less negative towards more positive zeta potentials of the nanoparticles. The zeta potential thus is a key parameter influencing protein adsorption to the surface of nanoparticles. Our analysis highlights the importance of the characterization of the parameters governing protein–nanoparticle interaction for quantitative evaluation and objective literature comparison.     

Antimicrobial activity of Fe‐loaded chitosan nanoparticles

To increase the antimicrobial activities of chitosan, chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were prepared by surfactant‐assisted chitosan chelating Fe²⁺, Fe³⁺ and ionic gelation chitosan. Their chelating rates were determined by spectrophotometry. The particle sizes and zeta potentials of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were measured by size and zeta potential analysis. The nanoparticles antimicrobial activities were evaluated by different concentration against Escherichia coli, Staphylococcus aureus, Candida albicans in vitro. Results showed that the mean diameter of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ were 206.4 and 195.2 nm, respectively. Their zeta potentials were +28.82 and +28.26 mV, respectively. The chelating rate of chitosan nanoparticles loaded with Fe²⁺ was greatly higher than with Fe³⁺. Their antimicrobial activity was showed greatly higher at lower concentrations compared to chitosan, and the antibacterial effect of chitosan nanoparticles loaded with Fe²⁺ or Fe³⁺ was preliminary observed.     

Carboxymethyl chitosan as a matrix material for platinum, gold, and silver nanoparticles

Carboxymethyl chitosan (CMC) was evaluated for its use in the synthesis and stabilization of catalytic nanoparticles for the first time. Many studies have reported on the ability of chitosan to bind with metal ions and support metal nanoparticles. CMC has a higher reported chelation capacity than chitosan, which has potential implications for improved catalyst formation and immobilization. Platinum, gold, and silver nanoparticles were synthesized in both chitosan and CMC. Particle size, morphology, and aggregation were examined using transmission electron microscopy (TEM). Complexation of nanoparticles was studied through Fourier transform infrared spectroscopy (FTIR). Similar nanoparticle size distributions were observed in the two polymers; however, CMC was observed to have higher rates of aggregation. This indicates that the carboxymethyl groups did not change nanoparticle formation; however, poor cross-linking and a limited anchoring ability of CMC led to the inability to immobilize the catalyst materials effectively.     

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: γ‐glutamylcysteine synthetase (γ‐GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over‐express either γ‐GCS or GS and found that γ‐GCS over‐expression resulted in inclusion body formation and impaired cell physiology, whereas GS over‐expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over‐expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild‐type cells in size (2–5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ≈2.5 times that attained in the wild‐type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli‐based biosynthesis offers a more energy‐efficient and eco‐friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

Preparation, characterization and transfection efficacy of chitosan nanoparticles containing the intestinal trefoil factor gene

Intestinal trefoil factor (ITF) is a novel polypeptide with potential pharmacological value for the prevention and healing of tissue injury; however, poor production capacity limits its clinical application. Chitosan, as a non-viral vehicle, has been successfully used in gene delivery for its intrinsic characteristics. In this context, we prepared chitosan nanoparticles enwrapping ITF cDNA and investigated its size, zeta potential, stability, release profiles, loading efficiency and loading capacity. Gene transfer capability was assessed in HEK293 cells. The data revealed that the chitosan/DNA nanoparticles were successfully prepared with sizes less than 500 nm and positive zeta potentials. The nanoparticles could protect DNA from nuclease degradation, and release profiles of DNA were dependent on N/P ratios. In addition, transfection efficiency of chitosan/DNA nanoparticles was equivalent to Lipofectamine (TM). Collectively, the results suggest that chitosan/DNA nanoparticles could be a promising method for ITF gene therapy.     

Chitosan-dextran Sulfate Nanoparticles for Delivery of an Anti-angiogenesis Peptide

Summary A novel nanoparticle delivery system has been developed by employing the oppositely charged polymers chitosan (CS) and dextran sulfate (DS), and a simple coacervation process. Under the conditions investigated, the weight ratio of the two polymers is identified as a determining factor controlling particle size, surface charge, entrapment efficiency and release characteristics of the nanoparticles produced. Particles of 223 nm mean diameter were produced under optimal conditions with a zeta potential of approximately −32.6 mV. A maximum of 75% anti-angiogenesis peptide entrapment efficiency was achieved with a CS:DS weight ratio of 0.59∶1. The same nanoparticle formulation also showed slow and sustained peptide release over a period of 6 days. In contrast, the formulation containing a lower ratio of CS:DS (0.5∶1) was found to have reduced entrapment efficiency and more rapid peptide release characteristics. The results of this study suggest that physicochemical and release characteristics of the CS-DS nanoparticles can be modulated by changing ratios of two ionic polymers. The novel CS-DS nanoparticles prepared by the coacervation process have potential as a carrier for small peptides.     

Adsorption of bovin serum albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a microemulsion system

The adsorption characteristics of BSA onto the magnetic chitosan nanoparticles have been investigated in this paper. The magnetic chitosan nanoparticles were prepared by adding the basic precipitant of NaOH solution into a W/O microemulsion system. The morphology of magnetic chitosan nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of magnetic chitosan nanoparticles was from 10nm to 20 nm, and the nanoparticles suspending in the aqueous solution could easily aggregate by a magnet, which suggested that the nanoparticles had good magnetic characteristics. The BSA adsorption experiment indicated that when pH of BSA solution was equal to 4, the maximum adsorption loading reached 110 mg/g. Through measuring the zeta potential of BSA solution and the magnetic nanoparticles, it was found that under this situation the surface of BSA took the negative charge, but the magnetic nanoparticles took the positive charge. Due to the small diameter, the adsorption equilibrium of BSA onto the nanoparticles reached very quickly within 10 min. The adsorption equilibrium of BSA onto the magnetic chitosan nanoparticles fitted well with the Freundlich model. The experimental results showed that the magnetic chitosan nanoparticles have potential to be used for the quick pretreatment in the protein analysis process.     

Xylan-mediated aggregation of Lactobacillus brevis and its relationship with the surface properties and mucin-mediated aggregation of the bacteria

Some Lactobacillus brevis strains were found to aggregate upon the addition of xylan after screening for lactic acid bacteria that interact with plant materials. The S-layer proteins of cell surface varied among the strains. The strains that displayed xylan-mediated aggregation retained its ability even after the removal of S-layer proteins. L. brevis had negative zeta potentials. A correlation between the strength of aggregation and zeta potential was not observed. However, partial removal of S-layer proteins resulted in decreases in the electric potential and aggregation ability of some strains. Therefore, xylan-mediated aggregation of L. brevis was considered to be caused by an electrostatic effect between the cells and xylan. L. brevis also aggregated in the presence of mucin, and the strengths of aggregation among the strains were similar to that induced by xylan. Thus, xylan- and mucin-mediated L. brevis aggregation was supposed to be caused by a similar mechanism.     

Development of a chitosan-based nanoparticle formulation for delivery of a hydrophilic hexapeptide, dalargin

Nanoparticle based delivery systems can offer opportunities for targeting, controlled release, and enhanced stability of their drug, protein, or gene therapy payload. This study investigated the use of chitosan in combination with the ionic additives sulfobutyl-ether-7-beta-cyclodextrin (SB-CD) or SB-CD/dextran sulfate (SB-CD/DS) mixture in comparison with chitosan: DS in the formulation of nanoparticles incorporating the hexapeptide dalargin. The physical characteristics (particle size, zeta potential), entrapment and loading efficiency, and release of dalargin were quantified. It was demonstrated that anionic cyclodextrin, SB-CD, can be used in complex coacervation with chitosan, with and without the presence of DS, to form nanoparticles. The presence of SB-CD or DS in the nanoparticle formulation and the weight ratio of chitosan to anionic additive(s) influenced the physical properties of the nanoparticles and their ability to carry dalargin. In addition, the particle size of nanoparticles was also affected by the molecular weight of chitosan and DS. The use of either DS or SB-CD/DS mixture produced chitosan nanoparticles with small particle size, high dalargin entrapment efficiency, enhanced peptide stability, and sustained release characteristics.     

Preparation and antibacterial activity of chitosan nanoparticles

Chitosan nanoparticles, such as those prepared in this study, may exhibit potential antibacterial activity as their unique character. The purpose of this study was to evaluate the in vitro antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against various microorganisms. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. Copper ions were adsorbed onto the chitosan nanoparticles mainly by ion-exchange resins and surface chelation to form copper-loaded nanoparticles. The physicochemical properties of the nanoparticles were determined by size and zeta potential analysis, atomic force microscopy (AFM), FTIR analysis, and XRD pattern. The antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, S. choleraesuis, S. typhimurium, and S. aureus was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results show that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 microg/mL, and the MBC values of nanoparticles reached 1 microg/mL. AFM revealed that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm.     

Quaternized chitosan (QCS)/poly (aspartic acid) nanoparticles as a protein drug-delivery system

The aim of this study was to generate a new type of nanoparticles made of quaternized chitosan (QCS) and poly (aspartic acid) and to evaluate their potential for the association and delivery of protein drugs. QCS and poly (aspartic acid) were processed to nanoparticles via the ionotropic gelation technique. The size, polydispersity, zeta potential, and morphology of the nanoparticles were characterized. Entrapment studies of the nanoparticles were conducted using bovine serum albumin (BSA) as a model protein. The effects of the pH value of nanoparticles with different QCS/poly (aspartic acid) ratios, QCS molecular weight (MW), poly (aspartic acid) concentration, and BSA concentration on the nanoparticle size, the nanoparticle yield, and BSA encapsulation were studied in detail. Suitably pH value of nanoparticles with different QCS/poly (aspartic acid) ratios, moderate QCS MW, optimal concentration ratio of poly (aspartic acid), and QCS favored more nanoparticles formed and higher BSA encapsulation efficiency. The release of BSA from nanoparticles was pH-dependent. Fast release occurred in 0.1 M phosphate buffer solution (PBS, pH 7.4), while the release was slow in 0.1 M HCl (pH 1.2). The results showed that the new QCS/poly (aspartic acid) nanoparticles have a promising potential in protein delivery system.     

Preparation,Statistical Optimization,and <Emphasis Type="Italic">In vitro</Emphasis> Characterization of Insulin Nanoparticles Composed of Quaternized Aromatic Derivatives of Chitosan

The aim of this study was the preparation, optimization, and in vitro characterization of insulin nanoparticles composed of methylated N-(4-N,N-dimethylaminobenzyl), methylated N-(4-pyridinyl), and methylated N-(benzyl) chitosan. Three types of derivatives were synthesized by the Schiff base reaction followed by quaternization. Nanoparticles were prepared by the polyelectrolyte complexation method. Experimental design D-optimal response surface methodology was used for the optimization of the nanoparticles. Independent variables were pH of polymer solution, concentration ratio of polymer/insulin, and also polymer type. Dependent variables include size, zeta potential, polydispersity index (PdI), and entrapment efficiency (EE%). Optimized nanoparticles were studied morphologically by transmission electron microscopy (TEM), and in vitro release of insulin from nanoparticles were determined under phosphate buffer (pH = 6.8) condition. Although a quadratic model has been chosen to fit the responses for size, PdI, and EE%, the zeta potential of the particles has been best fitted to 2-FI model. The optimized nanoparticles were characterized. The size of the particles were found to be 346, 318, and 289 nm; zeta potentials were 28.5, 27.7, and 22.2 mV; PdI of particles were 0.305, 0.333, and 0.437; and calculated EE% were 70.3%, 84.5%, and 69.2%, for methylated (aminobenzyl), methylated (pyridinyl), and methylated (benzyl) chitosan nanoparticles, respectively. TEM images show separated and non-aggregated nanoparticles with sub-spherical shapes and smooth surfaces. An in vitro release study of the prepared nanoparticles showed that the cumulative percentage of insulin released from the nanoparticles were 47.1%, 38%, and 68.7% for (aminobenzyl), (pyridinyl), and (benzyl) chitosan, respectively, within 300 min.     

Antioxidant,Cytotoxic and Antimicrobial Activity of Chitosan Preparations Extracted from Ganoderma Lucidum Mushroom

Two chitosan extracts were prepared by chemical and enzymatic treatment of Ganoderma lucidum mushroom, as an alternative source to crustacean shells. The molecular weight of the enzymatic extract was lower than that of the chemical one and of shrimp chitosan, as determined by viscosity measurements. Characteristic signals were identified in the ¹H‐NMR spectra and high deacetylation degree indicated good physico‐chemical properties for both mushroom chitosan extracts. The scavenging capacity of mushroom chitosan extracts was moderate against the synthetic radicals of 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), but higher values were observed for the enzymatic extract, compared to the chemical extract and shrimp chitosan. In vitro cytotoxicity was evaluated in L929 mouse fibroblast cell lines and the results of MTT assay showed good cytocompatibility in the tested range of concentrations. The growth of Gram‐positive bacteria was inhibited more than Gram‐negative bacteria in the presence of mushroom chitosan extracts, in particular by the chemical one, indicating their efficiency as antimicrobial agents. All these results strengthen the evidence of mushroom polysaccharide preparations availability for biomedical applications.     

Preparation and characterization of chitosan and trimethyl-chitosanmodified poly-(ε-caprolactone) nanoparticles as DNA carriers

The purpose of this research was to prepare poly-(ε-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA. Published: August 10, 2005     

Photoacoustically‐guided photothermal killing of mosquitoes targeted by nanoparticles

In biomedical applications, nanoparticles have demonstrated the potential to eradicate abnormal cells in small localized pathological zones associated with cancer or infections. Here, we introduce a method for nanotechnology‐based photothermal (PT) killing of whole organisms considered harmful to humans or the environment. We demonstrate that laser‐induced thermal, and accompanying nano‐ and microbubble phenomena, can injure or kill C. elegans and mosquitoes fed carbon nanotubes, gold nanospheres, gold nanoshells, or magnetic nanoparticles at laser energies that are safe for humans. In addition, a photoacoustic (PA) effect was used to control nanoparticle delivery. Through the integration of this technique with molecular targeting, nanoparticle clustering, magnetic capturing and spectral sharpening of PA and PT plasmonic resonances, our laser‐based PA‐PT nano‐theranostic platform can be applied to detection and the physical destruction of small organisms and carriers of pathogens, such as malaria vectors, spiders, bed bugs, fleas, ants, locusts, grasshoppers, phytophagous mites, or other arthropod pests, irrespective of their resistance to conventional treatments. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Prolonged stimuli alter the bacterial chemosensory clusters

The clustering of membrane‐bound receptors plays an essential role in various biological systems. A notable model system for studying this phenomenon is the bacterial chemosensory cluster that allows motile bacteria to navigate along chemical gradients in their environment. While the basic structure of these chemosensory clusters is becoming clear, their dynamic nature and operation are not yet understood. By measuring the fluorescence polarization of tagged receptor clusters in live Escherichia coli cells, we provide evidence for stimulus‐induced dynamics in these sensory clusters. We find that when a stimulus is applied, the packing of the receptors slowly decreases and that the process reverses when the stimulus is removed. Consistent with these physical changes we find that the effective cooperativity of the kinase response slowly evolves in the presence of a stimulus. Time‐lapse fluorescence imaging indicates that, despite these changes, the receptor clusters do not generally dissociate upon ligand binding. These data reveal stimulus‐dependent plasticity in chemoreceptor clusters.