Preparation and spectroscopic characterization of methoxy poly(ethylene glycol)-grafted water-soluble chitosan

The object of this study was to test the solubility of a methoxy poly(ethylene glycol) (MPEG)-grafted chitosan copolymer in organic solvents and aqueous solution. Water-soluble chitosan with low molecular weight (LMWSC) was used in a PEG-graft copolymerization. The MPEG was conjugated to chitosan using 4-dicyclohexylcarbodimide (DCC), and N-hydroxysuccimide (NHS). Introduction of PEG was confirmed by (1)H and (13)C NMR spectroscopy and FT-IR spectroscopy. The degree of substitution (DS) of MPEG into chitosan was calculated from (1)H NMR data and also by estimating the molecular weight (MW) using gel permeation chromatography (GPC). The DS values obtained from (1)H NMR spectroscopy and GPC were similar, indicating that MPEG-grafted LMWSC was synthesized and properly characterized. Furthermore, the introduction of PEG into chitosan increases the solubility in aqueous solutions over a range of pH values (4.0-11.0) and organic solvents such as DMF, DMSO, ethanol, and acetone.     

Synthesis of anionic poly(ethylene glycol) derivative for chitosan surface modification in blood-contacting applications

To improve blood compatibility, chitosan surface was modified by the complexa-tion-interpenetration method using an anionic derivative of poly(ethylene glycol) (PEG). Methoxypoly(ethylene glycol) sulfonate (MPEG sulfonate)-modified chitosan was prepared by allowing the base polymer to swell in an acidic medium, followed by polyelectrolyte complexation and interpenetration of MPEG sulfonate with the chitosan matrix. Addition of a strong base collapsed the base polymer to permanently immobilize the modifying agent on the surface. Electron spectroscopy for chemical analysis (ESCA) confirmed the presence of MPEG sulfonate on chitosan and the high resolution Cls peak showed an increase in -C—O- which is indicative of the ethylene oxide residues. The number of adherent platelets and the extent of platelet activation was significantly reduced on MPEG sulfonate-modified chitosan. Compared to an average of more than 66 fully activated platelets on unmodified chitosan surface, only 3.0 contact-adherent platelets were present on MPEG sulfonate-modified chitosan. Plasma recalcification time, a measure of the intrinsic coagulation reaction, was about 11.5 min in contact with modified chitosan. The results of this study show that chitosan surface can be modified by the complexation-interpenetration method with anionic PEG derivative. Surface-immobilized MPEG sulfonate was effective in preventing plasma protein adsorption and platelet adhesion and activation by the steric repulsion mechanism.     

An investigation on the short-term biodegradability of chitosan with various molecular weights and degrees of deacetylation

Research efforts have been devoted to demonstrating that the pH-sensitive characteristics of poly NIPAAm/chitosan nanoparticles can be applied to targeting tumors. A copolymer of (NIPAAm) and chitosan (4:1, m/m) was synthesized, and its drug release characteristics investigated. The results revealed that drug-loaded nanoparticles which encapsulation and loading efficiencies were 85.7% and 9.6%, respectively, exhibited pH-sensitive responses to tumor pH. The cumulative release rate was significantly enhanced below pH 6.8 and decreased rapidly above pH 6.9 at 36.5 ± 0.5 °C. MTT assay and fluorescence microscopic study showed that drug release was drastically promoted in tumor surroundings while exerting less effect in normal conditions. For mice treated with nanoparticles, the decrease in body weight was limited, and significant tumor regression was observed with complete regression in more than 50% of the mice. The life span of tumor-bearing mice was significantly increased when they were treated with nanoparticles. Thereby, the super pH-sensitive poly NIPAAm/chitosan nanoparticles may provide outstanding advantages for anti-cancer drug delivery.     

Chitosan graft copolymer nanoparticles for oral protein drug delivery: preparation and characterization

Several novel functionalized graft copolymer nanoparticles consisting of chitosan (CS) and the monomer methyl methacrylate (MMA), N-dimethylaminoethyl methacrylate hydrochloride (DMAEMC), and N-trimethylaminoethyl methacrylate chloride (TMAEMC), which show a higher solubility than chitosan in a broader pH range, have been prepared by free radical polymerization. The nanoparticles were characterized in terms of particle size, zeta potential, TEM, and FT-IR. These nanoparticles were 150-280 nm in size and carried obvious positive surface charges. Protein-loaded nanoparticles were prepared, and their maximal encapsulation efficiency was up to 100%. In vitro release showed that these nanoparticles provided an initial burst release followed by a slowly sustained release for more than 24 h. These graft copolymer nanoparticles enhanced the absorption and improved the bioavailability of insulin via the gastrointestinal (GI) tract of normal male Sprague-Dawley (SD) strain rats to a greater extent than that of the phosphate buffer solution (PBS) of insulin.     

Synthesis and characterization of a novel MPEG–chitosan diblock copolymer and self-assembly of nanoparticles

In this paper, a simple and novel method based on free-radical polymerization initiated by potassium persulfate (KPS) was developed to synthesize the MPEG–chitosan diblock copolymer (MPEG–CS). The obtained MPEG–CS diblock copolymer was characterized by Fourier transform infrared (FTIR), ¹H nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC), respectively. The MPEG–CS copolymer could self-assemble into nanoparticles in aqueous solution. A typical TEM photography indicated that the well-spherical nanoparticles with diameter at about 200 nm were obtained. In vitro cell culture assay indicated that MPEG–CS nanoparticles are non-toxic and cell-compatible as the polymer concentration was smaller than 0.6 mg/ml. In conclusion, the obtained MPEG–CS nanoparticles might have great potential application in drug-delivery system.     

Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

Real-time evidence of surface modification at polystyrene lattices by poloxamine 908 in the presence of serum: in vivo conversion of macrophage-prone nanoparticles to stealth entities by poloxamine 908

Intravenously injected polystyrene nanoparticles, which are prone to rapid sequestration by professional phagocytes, are converted to stealth entities by prior bolus intravenous injection of poloxamine 908. This behaviour is not due to alteration in macrophage phagocytic activity. Laser Doppler anemometry and surface plasmon resonance were used to unravel the mechanisms fundamental to generation of such stealth entities in vivo by poloxamine 908. Electrophoretic mobility of poloxamine pre-coated monodisperse polystyrene nanoparticles in serum, which behave as stealth entities in vivo, was similar to that of uncoated nanoparticles incubated in poloxamine pre-treated serum. This observation supported the notion that poloxamine in serum can modify the surface of nanoparticles with similar topography to that of stealth poloxamine pre-coated particles, i.e. with polyoxyethylene chains projected from the surface. Surface plasmon resonance optical phenomenon was used for real-time monitoring of protein-poloxamine interactions and adsorption at the polystyrene interface. It was found that poloxamine can not only adsorb to a serum-modified surface but in addition poloxamine in serum can form macromolecular complexes with high affinity for adsorption to a polystyrene lattice. A role for serum albumin in surface modification of nanoparticles by poloxamine 908 is also identified. Hence, our biophysical observations correlate precisely with the in vivo longevity of uncoated polystyrene nanoparticles in poloxamine pre-treated rats. This rational and sensitive biophysical approach has unravelled the probable mechanism fundamental to generation of stealth entities in vivo and therefore has application in the design and nano-engineering of stealth colloidal carriers for optimal biological performance.     

Preparation and characterizations of self-assembled PEGylated gelatin nanoparticles

The PEGylated gelatin nanoparticles were prepared by self-assembling method and characterized. The gelatin drug carrier was proposed as a targeting drug delivery system with the hypothesis that the gelatin carrier could be degraded by the matrix metalloprotease (MMP) and release the anticancer drug loaded inside carriers around the cancer site. The gelatin nanoparticles proposed in this study were composed of deoxycholic acid (DOCA), monomethoxy polyethylene glycol (MPEG), and gelatin. The carboxyl groups of DOCA and carboxylated MPEG were coupled with amine group of gelatin by dichlorohexylcarbodiimide (DCC) method. One molecule of gelatin coupled with 205 molecules of MPEG and 275 molecules of DOCA. The synthesized gelatin/DOCA/MPEG conjugates (GDM) were ultrasonicated to produce self-assembled nanoparticles. DOCA acted as the hydrophobic core, thereby aggregating gelatin molecules and hydrophilic MPEG chains located at the surface of the nanoparticles. The concentration of GDM, intensity of sonication, sonication time and temperature, all affected to control the particle size in the ultrasonication. The optimum condition was obtained as 1.0 mg/mL of GDM, 28 W for sonication intensity, 3 min of sonication time, and room temperature. The size distribution of particle was found to be 100–1000 nm in this condition. The particles which had a broad size distribution were filtered by 0.2 μm membrane. The product yield of particles having below 200 nm of size was about 30%. After filtration, an average diameter of GDM nanoparticle was 176 nm (155–200 nm).     

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.     

Preparation, characterization, and in vitro drug release behavior of biodegradable chitosan-graft-poly(1, 4-dioxan-2-one) copolymer

A novel copolymer of chitosan-g-poly(p-dioxanone) (CGP) was synthesized in bulk by ring-opening polymerization of p-dioxanone (PDO) initiated by the hydroxyl group or amino group of chitosan using SnOct₂ as catalyst. The chemical structure was determined by ¹H NMR. It was found that the feed ratio of chitosan to PDO had a great effect on the degree of polymerization (DP) and the substitution (DS) of PDO. The thermal stability and crystallization behavior of graft copolymer CGP were closely related to the values of DP and DS. When the resulting copolymer was used as Ibuprofen carrier, the release rate of Ibuprofen decreased compared with that of pure chitosan carrier. The drug release behavior was also influenced by the structure of graft copolymers.     

A Novel Method for Preparing Surface-Modified Fluocinolone Acetonide Loaded PLGA Nanoparticles for Ocular Use: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluations

Our objective was to prepare nanoparticulate system using a simple yet attractive innovated method as an ophthalmic delivery system for fluocinolone acetonide to improve its ocular bioavailability. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared by adopting thin film hydration method using PLGA/poloxamer 407 in weight ratios of 1:5 and 1:10. PLGA was used in 75/25 and 50/50 copolymer molar ratio of DL-lactide/glycolide. Results revealed that using PLGA with lower glycolic acid monomer ratio exhibited high particle size (PS), zeta potential (ZP) and drug encapsulation efficiency (EE) values with slow drug release pattern. Also, doubling the drug concentration during nanoparticles preparation ameliorated its EE to reach almost 100%. Furthermore, studies for separating the un-entrapped drug in nanoparticles using centrifugation method at 20,000 rpm for 30 min showed that the separated clear supernatant contained nanoparticles encapsulating an important drug amount. Therefore, separation of un-entrapped drug was carried out by filtrating the preparation using 20–25 μm pore size filter paper to avoid drug loss. Aiming to increase the PLGA nanoparticles mucoadhesion ability, surface modification of selected formulation was done using different amount of stearylamine and chitosan HCl. Nanoparticles coated with 0.1% w/v chitosan HCl attained most suitable results of PS, ZP and EE values as well as high drug release properties. Transmission electron microphotographs illustrated the deposition of chitosan molecules on the nanoparticles surfaces. Pharmacokinetic studies on Albino rabbit’s eyes using HPLC indicated that the prepared novel chitosan-coated PLGA nanoparticles subjected to separation by filtration showed rapid and extended drug delivery to the eye.     

PEGylated degradable composite nanoparticles based on mixtures of PEG-b-poly(γ-benzyl L-glutamate) and poly(γ-benzyl L-glutamate)

In the present work, the possibility to obtain PEGylated nanoparticles from two PBLG derivatives, PEG-b-poly(γ-benzyl L-glutamate), PBLG-PEG-60, and poly(γ-benzyl L-glutamate), PBLG-Bnz-50, by nanoprecipitation has been investigated. Particles were prepared not only from one polymer (PBLG-PEG-60 or PBLG-Bnz-50), but also from mixtures of two PBLG derivatives, PBLG-PEG-60 and PBLG-Bnz-50, in different ratios (90/10, 77/23, and 40/60 wt %). Because of the presence of PEG chains, hydrophilic particles were obtained, which was confirmed by ζ potential measurements (ζ from -13 mV and -21 mV) and by isothermal titration microcalorimetry (ITC). This last technique has shown no heat exchange when BSA was added to PEGylated nanoparticles. Further, complement activation has been evaluated by crossed immuno-electrophoresis demonstrating that the introduction of 77 wt % of PEGylated PBLG chains in the particles was enough to ensure a low complement activation activity. This effect was strongly correlated to the ζ potential of the particles, which decreased with an increase of PEG chains content. Interestingly, such properties are of interest for the preparation of degradable stealth nanocarriers. Moreover, it is suggested that the introduction of a reasonable amount (up to 20 wt %) of a second copolymer in the particle composition can be possible without modifying their stealth character. Moreover, the presence of this second copolymer would help to introduce a second functionality to the particles.     

Cytotoxic activities of chitosan nanoparticles and copper-loaded nanoparticles

Chitosan nanoparticles and copper(II)-loaded chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions and copper ion sorption. In this study, the cytotoxic activities of the chitosan nanoparticles and copper(II)-loaded chitosan nanoparticles was investigated and a relationship between physiochemical properties and activity is suggested. The chitosan nanoparticles and copper(II)-loaded chitosan nanoparticles elicited dose-dependent inhibitory effects on the proliferation of tumor cell lines.     

Preparation of chitosan derivative with polyethylene glycol side chains for porous structure without specific processing technique

The graft copolymer, chitosan-g-polyethylene glycol (PEG), was prepared through graft polymerization of PEG chains to chitosan due to the esterification reaction between PEG and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA). The graft copolymer with porous structure was observed from scanning electron micrographs. It is a potential method to combine chitosan with the hydrophilic synthetic polymers. The graft reaction was carried out in homogeneous system and yielded copolymers with high grafting content. FTIR, NMR, XRD, DSC, spectrofluorophotometer and SEM were detected to characterize the copolymer.     

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.     

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.     

Preparation of gold nanopowders and nanoparticles using chitosan suspensions

Simple methods for preparation of gold nanopowders and nanoparticles are reported. Gold/chitosan nanoparticles were prepared by using basic chitosan suspension as a dispersant and as a reductant. The resulting nanoparticles were processed by pyrolysis and thus obtain black gold nanopowder. The FESEM images indicate that most diameters of the nanopowder prepared were in the range of 50 and 200 nm. Hydrolysis is another quick decomposition method for chitosan. Acetic acid was adopted to implement the hydrolysis. The AEM images of the auberginic suspension show that the average gold nanoparticle diameter was less than 40 nm with good dispersion. Use of chitosan suspensions can produce gold nanopowder as well as gold nanoparticle without using toxic organic chemicals.     

基于海藻酸钠羟基改性的MPEG原位共价修饰微胶囊及抗蛋白性能

目的:通过对海藻酸钠链段羟基位点改性制备甲氧基聚乙二醇(MPEG)原位共价修饰的海藻酸钠/壳聚糖(AC)微胶囊,在保证MPEG修饰微胶囊机械强度不受影响的基础上,有效提高表面MPEG修饰密度,实现兼具良好机械稳定性及抗蛋白性能的微胶囊制备方法。方法:利用溴化氰对海藻酸钠羟基进行活化并将末端氨基的点击化学linker(BAT)接枝在主链上进而制备MPEG原位共价修饰微囊A_(B(OH))CP_N,用球磨法表征微囊机械强度,用Ig G和Fgn为模型考察微囊表面抗蛋白吸附性能,以L929细胞在其二维模拟平板膜上的黏附情况作为衡量指标,考察MPEG修饰微胶囊表面细胞粘附情况,并最终通过体内移植考察MPEG修饰微囊的生物相容性。结果:基于海藻酸钠羟基位点的MPEG原位共价修饰微胶囊能够实现与常规条件制备的微胶囊接近的机械强度;同时与对照组相比Ig G吸附量降低87.4%,Fgn吸附量降低75.5%,实现了良好的抗蛋白吸附性能;二维模拟平板膜表面L929细胞粘附情况显著改善,细胞粘附数与对照组相比降低了76.9%;体内移植结果证明MPEG修饰微囊细胞粘附极少,微囊与纤维层分离明显。结论:基于海藻酸钠羟基位点的MPEG原位修饰能够实现兼具良好机械稳定性及抗蛋白吸附性能的微胶囊。     

Preparation of biocompatible chitosan grafted poly(lactic acid) nanoparticles

Chitosan grafted poly(lactic acid) (CS-g-PLA) copolymer was synthesized and characterized by FT-IR and elemental analysis. The degree of poly(lactic acid) substitution on chitosan was 1.90 ± 0.04%. The critical aggregation concentration of CS-g-PLA in distilled water was 0.17 mg/ml. Three methods of preparing CS-g-PLA nanoparticles (diafiltration method, ultrasonication method and diafiltration combined with ultrasonication method) were investigated and their effect was compared. Of the three methods, diafiltration combined with ultrasonication method produced nanoparticles with optimal property in terms of size and morphology, with size ranging from 133 to 352 nm and zeta potential from 36 to 43 mV. Also, the hemolytic activity and cytotoxicity of the CS-g-PLA based nanoparticles was tested, and results showed low hemolysis rate (<5%) and no significant cytotoxicity effect of these nanoparticles.     

Preparation and pharmacodynamics of low-molecular-weight chitosan nanoparticles containing insulin

Low-molecular-weight chitosan (LMWC) was obtained by enzymatic degradation and ultrafiltration separation. LMWC nanoparticles with LMWC having 20 kDa weight average molecular weight (M_w) were then prepared by solvent evaporation method. The resultant nanoparticles were spherical with a narrow particle size distribution. LMWC nanoparticles loaded with insulin as a model drug were prepared. The average entrapment efficiency of insulin could reach up to 95.54%. The in vitro drug release profiles from the nanoparticles showed an initial burst of release in the first 2 h, followed by zero order release kinetics. In vivo pharmacodynamics of chitosan nanoparticles containing insulin showed that the nanoparticles showed some hypoglycemic activity. Compared with an insulin solution, a relative bioavailability of 0.737 was observed for four times the dosage of insulin in the chitosan nanoparticles after pulmonary administration.