Separation of lysozyme using superparamagnetic carboxymethyl chitosan nanoparticles

Functionalized Fe(3)O(4) nanoparticles conjugated with polyethylene glycol (PEG) and carboxymethyl chitosan (CM-CTS) were developed and used as a novel magnetic absorbing carrier for the separation and purification of lysozyme from the aqueous solution and chicken egg white, respectively. The morphology of magnetic CM-CTS nanoparticles was observed by transmission electron microscope (TEM). It was found that the diameter of superparamagnetic carboxymethyl chitosan nanoparticles (Fe(3)O(4) (PEG+CM-CTS)) was about 15 nm, and could easily aggregate by a magnet when suspending in the aqueous solution. The adsorption capacity of lysozyme onto the superparamagnetic Fe(3)O(4) (PEG+CM-CTS) nanoparticles was determined by changing the medium pH, temperature, ionic strength and the concentration of lysozyme. The maximum adsorption loading reached 256.4 mg/g. Due to the small diameter, the adsorption equilibrium of lysozyme onto the nanoparticles reached very quickly within 20 min. The adsorption equilibrium of lysozyme onto the superparamagnetic nanoparticles fitted well with the Langmuir model. The nanoparticles were stable when subjected to six repeated adsorption-elution cycles. Separation and purification were monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The lysozyme was purified from chicken egg white in a single step had higher purity, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Considering that the superparamagnetic nanoparticles possess the advantages of high efficiency, cost-effectiveness and excellent binding of a larger amount of lysozyme and easier separation from the reaction system, thus this type of superparamagnetic nanoparticles would bring advantages to the conventional separation techniques of lysozyme from chicken egg white.     

Designing chitosan-dextran sulfate nanoparticles using charge ratios

The purpose of this study was to examine the effect of charge ratio on the formation and properties of the chitosan (CS)-dextran sulfate (DS) nanoparticles developed for the delivery of water-soluble small and large molecules, including proteins. Rhodamine 6G (R6G) and bovine serum albumin (BSA) were chosen as model molecules. CS-DS nanoparticles were formulated by a complex coacervation process under mild conditions. The influence of formulation and process variables, including the charge ratio of the 2 ionic polymers, on particle size, zeta potential, and nanoparticle entrapment of R6G and BSA was studied. The in vitro release of R6G and BSA was also evaluated, and the integrity of BSA in the release fraction was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Depending on the concentration and charge ratio of DS and CS, nanoparticles with varied size (>or=244 nm) and zeta potential (-47.1-60 mV) were obtained. High entrapment efficiency (98%) was achieved for both R6G and BSA when the charge ratio of the 2 ionic polymers was greater than 1.12. The release of both R6G and BSA from nanoparticles was based on the ion-exchange mechanism. BSA showed much slower continuous release for up to 7 days while still maintaining its integrity for an extended period. The CS-DS nanoparticles developed based on the modulation of charge ratio show promise as a system for controlled delivery of both small and large molecules, including proteins.     

Adsorption of humic acid from aqueous solution onto irradiation-crosslinked carboxymethylchitosan

This paper presents the adsorption of humic acid from aqueous solution onto crosslinked chitosan derivative (carboxymethylchitosan), formed by additionless irradiation technique. The surface charge and swelling properties of crosslinked samples were investigated. The adsorption of humic acid onto crosslinked carboxymethylchitosan was carried out by the batch method at room temperature, and it was found to be strongly pH-dependent. Maximum amount of humic acid was adsorbed under acidic conditions at the optimum pH value of 3.5. Adsorption kinetic studies indicated the adsorption process was transport-limited at the same pH. The adsorption isotherm analysis data under various initial humic acid concentrations confirms that experimental data fitted well into the Langmuir equation. X-ray photoelectron spectroscopy (XPS) revealed that the amino groups of carboxymethylated chitosan were protonated, suggesting the formation of organic complex between the protonated amino groups and humic acid. From these preliminary evaluations, it was concluded that crosslinked carboxymethylated chitosan derivatives have a great potential in water treatment for the removal of humic acid and other polarized or electrically charged species.     

Chitosan and lactic acid-grafted chitosan nanoparticles as carriers for prolonged drug delivery

Nanoparticles of approximately 10nm in diameter made with chitosan or lactic acid-grafted chitosan were developed for high drug loading and prolonged drug release. A drug encapsulation efficiency of 92% and a release rate of 28% from chitosan nanoparticles over a 4-week period were demonstrated with bovine serum protein. To further increase drug encapsulation, prolong drug release, and increase chitosan solubility in solution of neutral pH, chitosan was modified with lactic acid by grafting D,L-lactic acid onto amino groups in chitosan without using a catalyst. The lactic acid-grafted chitosan nanoparticles demonstrated a drug encapsulation efficiency of 96% and a protein release rate of 15% over 4 weeks. With increased protein concentration, the drug encapsulation efficiency decreased and drug release rate increased. Unlike chitosan, which is generally soluble only in acid solution, the chitosan modified with lactic acid can be prepared from solutions of neutral pH, offering an additional advantage of allowing proteins or drugs to be uniformly incorporated in the matrix structure with minimal or no denaturization.     

壳聚糖-有机累托石/海藻酸钠复合纳米粒的制备

目的:以BSA作为模型药物,制备壳聚糖季铵盐-OREC复合物纳米微粒,建立一种安全有效的药物控释传递系统。方法:超声条件下,制备不同质量比的具有壳聚糖硅酸盐插层结构的复合物纳米微粒,观察其形态学特征、进行红外光谱分析。同时,测定OREC对BSA包封率和载药量的影响。结果:成功制备了不同质量比的OREC-HTCC纳米粒子。电镜结果显示纳米粒呈圆球形,均匀,平均粒径约为30nm。红外图谱分析证实,HTCC插入了OREC插层中,BSA成功地包裹入HTCC-ALG/OREC混合材料制备的纳米微粒。加入OREC后,纳米粒子的包封率及载药量均明显提高,但随着加入量的增加,包封率及载药量逐渐减少。结论:OREC-HTCC纳米粒子是良好的蛋白药物载体,具有粒径小、包封率高、缓释效果好等优点,为CS-OREC作为潜在的药物给药系统的进一步应用提供科学依据。     

Surface modification of iron oxide nanoparticles with polyarginine as a highly positively charged magnetic nano-adsorbent for fast and effective recovery of acid proteins

Polyarginine has been successfully bound onto iron oxide nanoparticles via carbodiimide activation as a highly positively charged magnetic nano-adsorbent for protein separation. They were nearly superparamagnetic with a mean diameter of 10.3 ± 2.36 nm, and the binding process did not change the spinel structure of iron oxide. From the analyses of FTIR spectra and zeta potential, the binding of polyarginine on the surface of iron oxide was confirmed and the resultant polyarginine-coated magnetic nanoparticles (PA-MNPs) were positively charged even up to pH 11. By thermogravimetric analysis, the typical product contained about 7.1 wt% of polyarginine. From the adsorption of the proteins with different pI values, the resultant PA-MNPs were found to be quite efficient for the fast and effective adsorption of acid proteins. For the typical acid protein, bovine serum albumin (BSA), the adsorption equilibrium was achieved within few minutes and obeyed the Langmuir isotherm equation. At pH 7 and 25 °C, the maximum adsorption capacity and equilibrium constant were 67.6 mg/g and 0.0623 L/mg, respectively. Moreover, by SDS–polyacrylamide gel electrophoresis, the capability of PA-MNPs for the separation of BSA-lysozyme mixture and egg white was further confirmed. Accordingly, the PA-MNPs were useful for the fast and effective magnetic recovery of acid proteins.     

Optimization of PEGylation Conditions for BSA Nanoparticles Using Response Surface Methodology

Chemical coupling of polyethylene glycol (PEG) to proteins or particles (PEGylation), prolongs their circulation half-life by greater than 50-fold, reduces their immunogenicity, and also promotes their accumulation in tumors due to enhanced permeability and retention effect. Herein, phase separation method was used to prepare bovine serum albumin (BSA) nanoparticles. PEGylation of BSA nanoparticles was performed by SPA activated mPEG through their free amino groups. Effect of process variables on PEGylation efficiency of BSA nanoparticles was investigated and optimized through response surface methodology with the amount of free amino groups as response. Optimum conditions was found to be 32.5 g/l of PEG concentration, PEG-nanoparticle incubation time of 10 min, incubation temperature of 27°C, and pH of 7 for 5 mg of BSA nanoparticles in 1 mL phosphate buffer. Analysis of data showed that PEG concentration had the most noticeable effect on the amount of PEGylated amino groups, but pH had the least. Mean diameter and zeta potential of PEGylated nanoparticles under these conditions were 217 nm and −14 mV, respectively. In conclusion, PEGylated nanoparticles demonstrated reduction of the negative surface charge compared to the non modified particles with the zeta potential of −31.7 mV. Drug release from PEGylated nanoparticles was almost slower than non-PEGylated ones, probably due to existence of a PEG layer around PEGylated particles which makes an extra resistance in opposition to drug diffusion.     

Protein interactions with nanosized hydrotalcites of different composition

Nanosized hydrotalcite-like compounds (HTlc) with different chemical composition were prepared and used to study protein adsorption. Two soft proteins, myoglobin (Mb) and bovine serum albumin (BSA), were chosen to investigate the nature of the forces controlling the adsorption and how these depend on the chemical composition of the support. Both proteins strongly interact with HTlc exhibiting in most cases a Langmuir-type adsorption. Mb showed a higher affinity for Nickel Chromium (NiCr-HTlc) than for Nickel Aluminum (NiAl-HTlc), while for BSA no significant differences between supports were found. Adsorption experiments in the presence of additives showed that proteins exhibited different types of interactions onto the same HTlc surface and that the adsorption was strongly suppressed by the addition of disodium hydrogen phosphate (Na₂HPO₄). Atomic force microscopy images showed that the adsorption of both proteins onto nanoparticles was followed by the aggregation of biocomposites, with a more disordered structure for BSA. Fluorescence measurements for adsorbed Mb showed that the inorganic nanoparticles induced conformational changes in the biomolecules; in particular, the interactions with HTlc surface quenched the tryptophan fluorescence and this process was particularly efficient for NiCr-HTlc. The adsorption of BSA onto the HTlc nanoparticles induced a selective quenching of the exposed fluorescent residues, as indicated by the blue-shift of the emission spectra of tryptophan residues and by the shortening of the fluorescence decay times.     

Polyelectrolyte nanoparticles based on water-soluble chitosan-poly(l-aspartic acid)-polyethylene glycol for controlled protein release

Water-soluble chitosan (WSC)-poly(l-aspartic acid) (PASP)-polyethylene glycol (PEG) nanoparticles (CPP nanoparticles) were prepared spontaneously under quite mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and their physicochemical properties were characterized by turbidity, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and zeta potential. PEG was chosen to modify WSC-PASP nanoparticles to make a protein-protective agent. Investigation on the encapsulation efficiency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted. Encapsulation efficiency was obviously decreased with the increase of initial BSA concentration. Furthermore, its in vitro release characteristics were evaluated at pH 1.2, 2.5, and 7.4. In vitro release showed that these nanoparticles provided an initial burst release, followed by a slowly sustained release for more than 24 h. The BSA released from CPP nanoparticles showed no significant conformational change compared with native BSA, which is superior to the BSA released from nanoparticles without PEG. A cell viability study suggested that the nanoparticles had good biocompatibility. This nanoparticle system was considered promising as an advanced drug delivery system for the peptide and protein drug delivery.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

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.     

Preparation and properties of hybrid monodispersed magnetic α-Fe2O3 based chitosan nanocomposite film for industrial and biomedical applications

In this study, hydrothermally prepared magnetic α-Fe2O3 nanoparticles were dispersed in chitosan (CH) solution to fabricate nanocomposite film. X-ray diffraction (XRD) patterns indicated that the α-Fe2O3 nanoparticles were pure α-Fe2O3 with rhombohedral structures, and the fabrication of CH did not result in a phase change. The scanning electron microscopy (SEM) and transmission electron microscope (TEM) results showed that the hexagonal and spherical monodispersed α-Fe2O3 nanoparticles were encapsulated into the spherical dumb shaped CH-α-Fe2O3 nanocomposite film with a mean diameter of ～87 and ～110 nm respectively. The α-Fe2O3 nanoparticles and CH-α-Fe2O3 nanocomposite film were also characterized by Fourier transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and vibrating sample magnetometer (VSM). Magnetic measurements revealed that the saturated magnetization (Ms) and remanent magnetization (Mr) of the pure α-Fe2O3 nanoparticles reached 0.573 emu/g and 0.100 emu/g respectively and the nanoparticles showed the characteristics of weak ferromagnetic before and after coating with CH.     

Spectroscopic studies of interaction between CuO nanoparticles and bovine serum albumin

Recently, the great interests in manufacturing and application of metal oxide nanoparticles in commercial and industrial products have led to focus on the potential impact of these particles on biomacromolecules. In the present study, the interaction of copper oxide (CuO) nanoparticles with bovine serum albumin (BSA) was studied by spectroscopic techniques. The zeta potential value for BSA and CuO nanoparticles with average diameter of around 50 nm at concentration of 10 μM in the deionized (DI) water were ?5.8 and ?22.5 mV, respectively. Circular dichroism studies did not show any changes in the content of secondary structure of the protein after CuO nanoparticles interaction. Fluorescence data revealed that the fluorescence quenching of BSA by CuO nanoparticles was the result of the formed complex of CuO nanoparticles – BSA. Binding constants and other thermodynamic parameters were determined at three different temperatures. The hydrogen bond interactions are the predominant intermolecular forces to stabilize the CuO nanoparticle – BSA complex. This study provides important insight into the interaction of CuO nanoparticles with proteins, which may be of importance for further application of these nanoparticles in biomedical applications.     

Purification of 6×His-tagged phycobiliprotein using zinc-decorated silica-coated magnetic nanoparticles

Zinc-decorated silica-coated magnetic nanoparticles (ZnSiMNPs) were prepared by adsorbing zinc onto colloidal silica. These nanoparticles were used for the rapid purification of 6×His-tagged recombinant phycobiliprotein. The surface changes in the magnetic nanoparticles after zinc adsorption were characterized by transmission electron microscopy. The adsorption of the 6×His-tagged phycobiliprotein onto ZnSiMNPs in 10 mM PBS at 25°C was found to follow the Langmuir isotherm. ZnSiMNPs could be used to extract 6×His-tagged phycobiliprotein from lysate to single-band purity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No spectral variation was observed in the purified phycobiliprotein. Thus, ZnSiMNPs served as a useful tool for the magnetic separation and delivery of the 6×His-tagged phycobiliprotein.     

Analysis of protein adsorption characteristics to nano-pore silica particles by using confocal laser scanning microscopy

The effect of average pore size of nano-pore silica particles on protein adsorption characteristics was determined experimentally by the dissociation constant and the adsorption capacity determined from the Langmuir equation. As the average pore size was increased from 2.2 to 45 nm, the BSA adsorption capacity increased from 16.8 to 84.3 mg/g-silica so as the equilibrium constant (from 2.6 to 9.4 mg/ml). Using confocal microscopy with fluorescence labeling, we could visualize the protein adsorption in situ and determine the minimum pore size required for efficient intraparticle adsorption. The confocal microscopy analysis revealed that BSA was adsorbed mainly on the surface of the particles with a smaller pore size, but diffused further into the interstitial surface when it was sufficiently large. It was concluded that for BSA whose Stoke's diameter is ca. 3.55 nm the minimum pore size of about 45 nm or larger was required for a sufficient adsorption capacity.     

不同pH值与电位耦合对牛血清白蛋白包被酒石酸长春瑞滨纳米粒制备工艺的影响

在反溶剂法制备纳米粒过程中,pH值在一定程度上会对其产生影响。本文通过在不同酸碱环境下运用反溶剂法制备牛血清白蛋白包被酒石酸长春瑞滨纳米粒,进而借助于电位耦合作用来研究纳米粒制备工艺。研究结果表明:当pH=4.5至7.5时,酒石酸长春瑞滨和牛血清白蛋白带有异种电荷,而当pH=2.5,3.5,8.5,9.5时它们均带有同种电荷。当pH=7.5时,牛血清白蛋白带有负电荷即-8.52 mV,酒石酸长春瑞滨带有正电荷即+4.48mV。此时得到牛血清白蛋白包被酒石酸长春瑞滨纳米粒粒径为193.3 nm,Zeta电位为-30.86 mV,而且在该pH下对纳米粒制备工艺进行了优化,最终它的载药量和包封率达到了45.6%和90.6%。     

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.     

5-Fluorouracil-Loaded BSA Nanoparticles: Formulation Optimization and In Vitro Release Study

Over the past few decades, there has been considerable interest in developing protein nanoparticles as drug delivery devices. The underlying rationale is their exceptional characteristics, namely biodegradability and nonantigenicity. Herein, phase separation method was used to prepare 5-fluorouracil-loaded bovine serum albumin (BSA) nanoparticles. Drug release was tracked by continuous flow dialysis technique. Effect of process variables on loading efficiency of 5-fluorouracil was investigated and optimized through Taguchi’s M16 design with the amount of entrapped drug as response. Optimum condition was found to be 2 mg/mL of 5-fluorouracil, 3.7 mL of added ethanol, 176 μL of glutaraldehyde, drug–protein incubation time of 30 min, and pH of 8.4 for 200 mg of BSA in 2 mL drug solution. pH had the most noticeable effect on the amount of entrapped drug, but glutaraldehyde had the least. Mean diameter and zeta potential of fabricated nanoparticles under these conditions were 210 nm and −31.7 mV, respectively. Drug-loaded BSA nanoparticles suspension maintained constant release of drug for 20 h under experimental conditions, so this colloidal drug carrier is capable of releasing drug in a sustained manner.     

Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles

A novel and efficient immobilization of β-d-galactosidase from Aspergillus oryzae has been developed by using magnetic Fe₃O₄–chitosan (Fe₃O₄–CS) nanoparticles as support. The magnetic Fe₃O₄–CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe₃O₄ nanoparticles made through co-precipitation of Fe²⁺ and Fe³⁺. The resultant material was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry and thermogravimetric analysis. β-d-Galactosidase was covalently immobilized onto the nanocomposites using glutaraldehyde as activating agent. The immobilization process was optimized by examining immobilized time, cross-linking time, enzyme concentration, glutaraldehyde concentration, the initial pH values of glutaraldehyde and the enzyme solution. As a result, the immobilized enzyme presented a higher storage, pH and thermal stability than the soluble enzyme. Galactooligosaccharide was formed with lactose as substrate by using the immobilized enzyme as biocatalyst, and a maximum yield of 15.5% (w/v) was achieved when about 50% lactose was hydrolyzed. Hence, the magnetic Fe₃O₄–chitosan nanoparticles are proved to be an effective support for the immobilization of β-d-galactosidase.     

Protein adsorption at polymer-grafted surfaces: comparison between a mixture of saliva proteins and some well-defined model proteins

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1 h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, beta-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface.