In vitro antifungal activity of metal complexes of a quaternized chitosan derivative with copper ions

Antifungal activity of synthetic metal complexes of quaternized N-(propyl) chitosan derivatives with Сu(II) against yeastlike (Saccharomyces cereviseae, Rodothorula rubra, and Candida albicans) and mycelial fungi (Fusarium oxysporum, Alternaria alternata, Cladosporium herbarum) was studied. In vitro application (at 250?500 μg/mL) of the metal complex of quaternized N-(propyl) derivative of low-molecular chitosan with 53% substitution and 1.3% copper ions proved efficient against F. оxysporum, one of ten most common fungal plant pathogens. Water-soluble quaternized N-(propyl) chitosan derivatives with 40?58% degree of substitution were synthesized using glycidyltrimethylammonium chloride under optimally adjusted conditions. Metal complexes of the chitosan derivative with 53% degree of substitution with Сu(II) ions were obtained by dialysis. The quantity of copper ions in the metal complexes was determined by atomic emission spectrometry. The structure of chitosan derivatives was confirmed by spectral analysis (IR, ¹H NMR).     

Antibacterial characteristics and activity of acid-soluble chitosan

The antibacterial activity of chitosan was investigated by assessing the mortality rates of Escherichia coli and Staphylococcus aureus based on the extent of damaged or missing cell walls and the degree of leakage of enzymes and nucleotides from different cellular locations. Chitosan was found to react with both the cell wall and the cell membrane, but not simultaneously, indicating that the inactivation of E. coli by chitosan occurs via a two-step sequential mechanism: an initial separation of the cell wall from its cell membrane, followed by destruction of the cell membrane. The similarity between the antibacterial profiles and patterns of chitosan and those of two control substances, polymyxin and EDTA, verified this mechanism. The antibacterial activity of chitosan could be altered by blocking the amino functionality through coupling of the chitosan to active agarose derivatives. These results verify the status of chitosan as a natural bactericide.     

壳寡糖对大肠杆菌抑菌活性研究

分析壳寡糖对大肠杆菌抑菌效果的影响因素.采用摇瓶法和ELISA板法对不同浓度的壳寡糖进行抑菌试验;比较不同pH、不同脱乙酰度的壳寡糖对大肠杆菌抑菌效果的差异;比较不同聚合度的单一聚合度壳寡糖抑菌效果的差异.壳寡糖浓度大于5 mg/mL时抑菌效果与同浓度苯甲酸钠相近;pH为4时,0.156 mg/mL的壳寡糖溶液抑菌活性即能超过90%;pH为7时,5 mg/mL的壳寡糖才能达到90%抑菌活性.脱乙酰度为95%时,5 mg/mL的壳寡糖溶液抑菌活性能超过97%;脱乙酰度为45%时,40 mg/mL的壳寡糖溶液抑菌活性仅有56%;聚合度大于4的单一聚合度壳寡糖40 mg/mL时抑菌活性能达到99%.结果表明:提高壳寡糖溶液浓度、降低pH、提高脱乙酰度,能提高壳寡糖的抑菌活性,单一聚合度壳寡糖聚合度越高,对大肠杆菌的抑制作用越强.此外,采用ELISA板的方法进行实验,即节省试药又方便快捷.     

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.     

Antibacterial testing of metal ions using a chemically defined medium

Data from in-vitro tests on potential germicides can be greatly influenced by the culture medium. The bioavailability and biochemical reactivity of the biocides can be influenced by chemical interference with media components (Spooner & Sykes 1972). Bird et al . (1985) showed that metal ions are particularly prone to chemical interferences. A chemically defined solid medium has been developed to monitor the antibacterial activity of metal ions. The minimum inhibitory concentrations of zinc and silver have been determined against a range of bacteria using this medium.     

Reprogramming virus nanoparticles to bind metal ions upon activation with heat

We have reprogrammed the stimulus-responsive conformational change property of a virus nanoparticle (VNP) to enable the surface exposure of metal binding motifs upon activation with heat. The VNP is based on the widely investigated adeno-associated virus (AAV). An intrinsic bioactive functionality of AAV was genetically replaced with a hexahistidine (His) tag. The peptide domain with the inserted His tag is normally inaccessible. Upon external stimulation with heat, the VNP undergoes a conformational change, resulting in externalization of His tag-containing domains and the conferred ability to bind metal. We show that beyond this newfound functionality of the capsid, the VNPs maintain many of the wild-type capsid properties. Our work lays the groundwork for developing stimulus-responsive VNPs that can be used as "smart" building blocks for the creation of higher order structures.     

Sorption efficiency of chitosan nanofibers toward metal ions at low concentrations

Chitosan fibers showing narrow diameter distribution with a mean of 42 nm were produced by electrospinning and utilized for the sorption of Fe(III), Cu(II), Ag(I), and Cd(II) ions from aqueous solutions. The ion concentrations in the supernatant solutions were determined using inductively coupled plasma-mass spectrometry (ICP-MS). The filtration efficiency of the fibers toward these ions was studied by both batch and microcolumn methods. High efficiency in sorption of the metal ions was obtained in the both methods. The effects of sorbent amount (0.10-0.50 mg), shaking time (15-120 min), initial metal ion concentration (10.0-1000.0 μg·L(-1)), and temperature (25 and 50 °C) on the extent of sorption were examined. The sorbent amount did not significantly alter the efficiency of sorption; however, shaking time, temperature, and metal ion concentration were found to have a strong influence on sorption. By virtue of its mechanical integrity, the applicability of the chitosan mat in solid phase extraction under continuous flow looks promising.     

Interactions of metal ions with a 2,4-diaminopyrimidine derivative (trimethoprim). Antibacterial studies

The interaction of copper (II), zinc(II) and cadmium(II) with Trimethoprim (2,4-diamino-5-(3',4',5'-trimethoxybenzyl) pyrimidine) has been studied. The crystal structures of [Zn(Trim)2Cl2] (2) and [Cd(Trim)Cl2(CH3OH)]n (4) are reported. Compound (2) exhibits a distorted tetrahedral environment around the metal center and crystallizes in the triclinic space group P1 with a=10.2397(6), b=10.4500(6), c=16.3336(16) A, alpha=96.141(8), beta=106.085(5), gamma=96.551(5) degrees and Z=2. In complex (4), the Cd(II) centers are bridged sequentially by two chlorine ions to form infinite chains and present a six-coordinated environment; the compound crystallizes in the monoclinic P2(1)/C space group with a=13.958(5), b=7.532(2), c=18.390(2) A, alpha=90, beta=97.32(5), gamma=90 degrees and Z=4. In both structures the Trimethoprim acts as a monodentate ligand through the pyrimidinic nitrogen N(1) atom. The characterization of the Cu(Trim)2(CH3O)(ClO4) complex through EPR and magnetic measurements suggests a binuclear or polinuclear nature, with bridging methoxo groups. The complexes were screened for their activity against several bacteria, showing activity similar to that of trimethoprim.     

The controlled release of insulin-mimetic metal ions by the multifunction of chitosan

Vanadium, which is an insulin-mimetic metal ion, was efficiently adsorbed on chitosan (CS). The adsorption of vanadium on CS was affected by the vanadium/CS ratio and the initial concentration of vanadium in preparative medium under constant pH condition. The vanadium-CS complex was able to control vanadium release. Moreover, a consistent control of vanadium release was achieved by incorporation of the vanadium-CS complex into a CS gel. After implantation of the CS gel retaining the vanadium-CS complex into diabetic mice, insulin-mimetic efficacy was confirmed by observation of a steady reduction in blood glucose levels. The sustained vanadium release also contributed to minimization of the side-effects. Thus, CS gel retaining the vanadium-CS complex appears promising as a vehicle for vanadium with long-term action and a low toxicity leading to its clinical use.     

Self-cleavage activity of the genomic HDV ribozyme in the presence of various divalent metal ions.

To identify the divalent metal ions that can support the self-cleavage activity of the genomic ribozyme of human hepatitis delta virus (HDV), we tested the activity of various divalent metal ions in the ribozyme reactions catalyzed by HDV88 (683-770 nt) and 88DI3 (HDV88 with the sequence from 740-752 nt deleted). Among various metal ions tested, Mg2+, Mn2+, Ca2+ and Sr2+ efficiently supported the self-cleavage reactions of the HDV88 and 88DI3 ribozymes. In the case of the 88DI3 ribozyme, other divalent metal ions, such as Cd2+, Ba2+, Co2+, Pb2+ and Zn2+, were also able to support the self-cleavage reaction to some extent (< 10%). In the presence of spermidine (0.5 mM), the cleavage reaction was promoted at lower concentrations of effective divalent metal ions. The HDV ribozyme represents the only example of ribozyme to date of a ribozyme that catalyzes the self-cleavage reaction in the presence of Ca2+ ions as efficiently as it does in the presence of Mg2+ ions.     

Design of deformable chitosan microspheres loaded with superparamagnetic iron oxide nanoparticles for embolotherapy detectable by magnetic resonance imaging

The purpose of this study was to design chitosan microspheres (MS) loaded with superparamagnetic iron oxide nanoparticles (SPIO) suitable for anti-cancer embolotherapy detectable by MRI. Deformable chitosan MS loaded with varying SPIO concentrations (SPIO-chitosan MS) were prepared by ionotropic gelation and a porogenic technique using polyethylene glycol, followed by genipin crosslinking. Adding SPIO nanoparticles to chitosan MS did not significantly affect the chitosan MS morphology. An in vitro phantom study led to selecting SPIO-chitosan MS prepared with 1.0mM SPIO for an in vivo MR traceability study. SPIO-chitosan MS could be identified following embolization in the renal artery by MRI at 18weeks. Histological and pathological evidence also showed that SPIO-chitosan MS blocked and remained in the target vessels. Therefore, deformable SPIO-chitosan MS is MR-detectable embolic material with a possible application for anti-cancer embolotherapy.     

Preparation of aspirin and probucol in combination loaded chitosan nanoparticles and in vitro release study

We design and develop chitosan nanoparticles which load two different drugs simultaneously. Aspirin (acetylsalicylic acid, ASA), a hydrophilic drug and probucol (PRO), a hydrophobic drug, are chosen as typical drugs, which are widely used to treat restenosis. The drug loaded chitosan nanoparticles are prepared by gelation of chitosan with tripolyphosphate (TPP) by ionic cross-linking. The physicochemical properties of nanoparticles are investigated by FTIR, transmission electron microscope (TEM), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The images show that these particles are spherical in shape with ASA being in the amorphous phase, while PRO is crystalline. The properties of chitosan nanoparticles such as encapsulation capacity and controlled release behaviors of ASA and PRO are evaluated. Experimental results indicate that the loading capacity (LC), encapsulation efficiency (EE) and ASA and PRO release behaviors are affected by several factors including pH, concentration of TPP, chitosan molecular weight (MW) and ASA initial concentration as well as PRO. In vitro release shows that the nanoparticles provide a continuous release. Entrapped ASA is released for more than 24 h and PRO lasts longer for 120 h.     

Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli

The antibacterial activity and acting mechanism of silver nanoparticles (SNPs) on Escherichia coli ATCC 8739 were investigated in this study by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with SNPs. The experimental results indicated 10 μg/ml SNPs could completely inhibit the growth of 10⁷ cfu/ml E. coli cells in liquid Mueller–Hinton medium. Meanwhile, SNPs resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that SNPs were able to destroy the permeability of the bacterial membranes. When the cells of E. coli were exposed to 50 μg/ml SNPs, many pits and gaps were observed in bacterial cells by transmission electron microscopy and scanning electron microscopy, and the cell membrane was fragmentary, indicating the bacterial cells were damaged severely. After being exposed to 10 μg/ml SNPs, the membrane vesicles were dissolved and dispersed, and their membrane components became disorganized and scattered from their original ordered and close arrangement based on TEM observation. In conclusion, the combined results suggested that SNPs may damage the structure of bacterial cell membrane and depress the activity of some membranous enzymes, which cause E. coli bacteria to die eventually.     

Antibacterial activity of ZnO nanoparticles prepared via non-hydrolytic solution route

The antibacterial activity of ZnO nanoparticles has been investigated and presented in this paper. Nanoparticles were prepared via non-hydrolytic solution process using zinc acetate di-hydrate (Zn(CH₃COO)₂·2H₂O) and aniline (C₆H₅NH₂) in 6 h refluxing at ∼65 °C. In the presence of four pathogens such as Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Klebsiella pneumoniae, the antibacterial study of zinc oxide nanoparticles were observed. The antibacterial activity of ZnO nanoparticles (ZnO-NPs) were studied by spectroscopic method taking different concentrations (5–45 μg/ml) of ZnO-NPs. Our investigation reveals that the lowest concentration of ZnO-NPs solution inhibiting the growth of microbial strain is found to be 5 μg/ml for K. pneumoniae, whereas for E. coli, S. aureus, and S. typhimurium, it was calculated to be 15 μg/ml. The diameter of each ZnO-NPs lies between “20 and 30 nm” as observed from FESEM and transmission electron microscopy images. The composition of synthesized material was analyzed by the Fourier transform infrared spectroscopy, and it shows the band of ZnO at 441 cm⁻¹. Additionally, on the basis of morphological and chemical observations, the chemical reaction mechanism of ZnO-NPs was also proposed.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment

Background

Evaluation of the combinatorial anticancer effects of curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) on colon cancer cells and the analysis of pharmacokinetics and biodistribution of CRC-TCS-NPs/5-FU-TCS-NPs in a mouse model.

Methods

CRC-TCS-NPs/5-FU-TCS-NPs were developed by ionic cross-linking. The in vitro combinatorial anticancer effect of the nanomedicine was proven by different assays. Further the pharmacokinetics and biodistribution analyses were performed in Swiss Albino mouse using HPLC.

Results

The 5-FU-TCS-NPs (size: 150 ± 40 nm, zeta potential: + 48.2 ± 5 mV) and CRC-TCS-NPs (size: 150 ± 20 nm, zeta potential: + 35.7 ± 3 mV) were proven to be compatible with blood. The in vitro drug release studies at pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days, where both the systems exhibited a higher release in acidic pH. The in vitro combinatorial anticancer effects in colon cancer (HT29) cells using MTT, live/dead, mitochondrial membrane potential and cell cycle analysis measurements confirmed the enhanced anticancer effects (2.5 to 3 fold). The pharmacokinetic studies confirmed the improved plasma concentrations of 5-FU and CRC up to 72 h, unlike bare CRC and 5-FU.

Conclusions

To conclude, the combination of 5-FU-TCS-NPs and CRC-TCS-NPs showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo.

General significance

The enhanced anticancer effects of combinatorial nanomedicine are advantageous in terms of reduction in the dosage of 5-FU, thereby improving the chemotherapeutic efficacy and patient compliance of colorectal cancer cases.     

Improved stability and catalytic activity of graphene oxide/chitosan hybrid beads loaded with porcine liver esterase

Graphene oxide/chitosan and reduced graphene oxide/chitosan (GO/CS and RGO/CS) beads were prepared by precipitation with NaOH. Porcine liver esterase was immobilized on these beads to give GO/CS/E and RGO/CS/E beads. The optimum conditions for the maximum activity of RGO/CS/E beads were pH 8 and 50°C. The stability of the enzyme immobilized on GO/CS/E and RGO/CS/E was high in the pH range of 5–8. The GO/CS/E beads showed superior stability compared to that of the free enzyme and CS/E beads between 20 and 50°C. Kinetic analysis showed that GO/CS/E was a better catalyst than the RGO/CS/E beads with a lower K_m value of 0.9?mM. The hybrid beads also retained more than 95% activity after 10 consecutive cycles. The GO/CS/E and RGO/CS/E beads retained 84% and 87% activity after 40 days at 4°C. The GO/CS/E beads were used for the successful hydrolysis of methyl 4-hydroxy benzoate.