Layer-by-layer adsorption of biopolyelectrolytes as a universal approach to fabrication of protein-loaded microparticles

This study was aimed at examination of microparticles formed via the layer-by-layer adsorption of dextran sulfate and chitosan onto the insoluble complexes of various proteins with polyanions. Microparticles with all tested proteins were stable at pH values of 1–5. At pH > 6 the mucoadhesivity of the microparticles changed and the encapsulated proteins were released. Microparticles were able to protect the proteins from proteases. Proteinous protease inhibitors encapsulated as well (2–3%) completely prevented protein proteolysis. The pharmacological effect of microencapsulated insulin was studied in vivo using the model of chronic diabetes in rats, which were treated by oral administration.     

Protein delivery based on uncoated and chitosan-coated mesoporous silicon microparticles

Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60-90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems.     

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH-sensitive drug release property

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH sensitivity were developed for oral delivery of protein drugs, using bovine serum albumin (BSA) as a model drug. The composite drug-loaded microparticles with a mean particle size less than 200 μm were prepared by a convenient shredding method. Since the microparticles were formed by tripolyphosphate cross-linking, electrostatic complexation by alginate and/or pectin, as well as ionotropic gelation with calcium ions, the microparticles exhibited an improved pH-sensitive drug release property. The in vitro drug release behaviors of the microparticles were studied in simulated gastric (pH 1.2 and pH 5.0), intestinal (pH 7.4) and colonic (pH 6.0 and pH 6.8 with enzyme) media. For the composite microparticles with suitable compositions, the releases of BSA at pH 1.2 and pH 5.0 could be effectively sustained, while the releases at pH 7.4, pH 6.8 and pH 6.0 increased significantly, especially in the presence of pectinase. These results clearly suggested that the microparticles had potential for site-specific protein drug delivery through oral administration.     

离子凝聚法制备负载流感疫苗的壳聚糖微球

采用三聚磷酸钠(TPP)作为离子交联剂, 应用离子凝聚法制备负载流感疫苗的壳聚糖微球. 筛选出壳聚糖起始质量分数为1%. TPP的浓度对壳聚糖微球的制备影响较大, 采用低浓度的TPP(200 μg/mL)制备的微球放置过夜均出现沉淀现象, 高浓度的TPP(800 μg/mL)在制备过程中出现絮状沉淀. 固化比影响微球的释放行为, 固化比为1∶1的微球爆炸式释放率达到90％, 固化比为1∶3的微球6 h后逐步释放, 12 h后释放率达到95％. 固化比为1∶5的微球6 h后没有明显的释放行为. 壳聚糖溶液的pH对微球的制备和释放没有显著的影响. 通过对负载流感疫苗的壳聚糖微球的制备条件和释放行为的研究结果表明, pH=5.6的壳聚糖溶液, 固化比为1∶3, TPP的质量浓度为400 μg/mL是较理想的流感疫苗壳聚糖微球的制备条件.     

Hydroxypropyl chitosan bearing beta-cyclodextrin cavities: synthesis and slow release of its inclusion complex with a model hydrophobic drug

Hydroxypropyl chitosan-graft-carboxymethyl beta-cyclodextrin (HPCH-g-CM beta-CD) was synthesized by grafting CM beta-CD onto HPCH using water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the condensing agent. Due to the presence of hydrophobic beta-CD rings onto the HPCH backbone, this polymer can be used as a matrix for controlled drug release. The adsorption of a hydrophobic model drug, ketoprofen, by HPCH-g-CM beta-CD microparticles (using tripolyphosphate as an ionic crosslinking agent) fitted well in the Langmuir isotherm equation. The drug dissolution profile showed that HPCH-g-CM beta-CD microparticles provided a slower release of the entrapped ketoprofen than chitosan, and the release behavior was influenced by the pH value of the medium. These results suggest that beta-CD grafted with chitosan derivatives may become a potential biodegradable delivery system to control the release of hydrophobic drugs with pH-responsive capability.     

Development and characterization of new insulin containing polysaccharide nanoparticles

A nanoparticle insulin delivery system was prepared by complexation of dextran sulfate and chitosan in aqueous solution. Parameters of the formulation such as the final mass of polysaccharides, the mass ratio of the two polysaccharides, pH of polysaccharides solution, and insulin theorical loading were identified as the modulating factors of nanoparticle physical properties. Particles with a mean diameter of 500 nm and a zeta potential of approximately −15 mV were produced under optimal conditions of DS:chitosan mass ratio of 1.5:1 at pH 4.8. Nanoparticles showed spherical shape, uniform size and good shelf-life stability. Polysaccharides complexation was confirmed by differential scanning calorimetry and Fourier transformed infra-red spectroscopy. An association efficiency of 85% was obtained. Insulin release at pH below 5.2 was almost prevented up to 24 h and at pH 6.8 the release was characterized by a controlled profile. This suggests that release of insulin is ruled by a dissociation mechanism and DS/chitosan nanoparticles are pH-sensitive delivery systems. Furthermore, the released insulin entirely maintained its immunogenic bioactivity evaluated by ELISA, confirming that this new formulation shows promising properties towards the development of an oral delivery system for insulin.     

Development and characterization of new insulin containing polysaccharide nanoparticles

A nanoparticle insulin delivery system was prepared by complexation of dextran sulfate and chitosan in aqueous solution. Parameters of the formulation such as the final mass of polysaccharides, the mass ratio of the two polysaccharides, pH of polysaccharides solution, and insulin theorical loading were identified as the modulating factors of nanoparticle physical properties. Particles with a mean diameter of 500 nm and a zeta potential of approximately −15 mV were produced under optimal conditions of DS:chitosan mass ratio of 1.5:1 at pH 4.8. Nanoparticles showed spherical shape, uniform size and good shelf-life stability. Polysaccharides complexation was confirmed by differential scanning calorimetry and Fourier transformed infra-red spectroscopy. An association efficiency of 85% was obtained. Insulin release at pH below 5.2 was almost prevented up to 24 h and at pH 6.8 the release was characterized by a controlled profile. This suggests that release of insulin is ruled by a dissociation mechanism and DS/chitosan nanoparticles are pH-sensitive delivery systems. Furthermore, the released insulin entirely maintained its immunogenic bioactivity evaluated by ELISA, confirming that this new formulation shows promising properties towards the development of an oral delivery system for insulin.     

Caffeine loading into micro- and nanoparticles of mesoporous silicate materials: in vitro release kinetics

Caffeine was loaded into microparticles and nanoparticles of MCM-41 and MCM-48 to study its release into simulated body fluid at pH 7.4 and 37°C. The silicate systems were characterized by X-Rar Diffraction (XRD), scanning electron microscopy, and nitrogen adsorption/desorption isotherms. Loading of caffeine was confirmed by thermal gravimetric analysis and FTIR and the loading capacity was determined by high performance liquid chromatography (HPLC). The loading capacities for the microparticles are higher than that for the nanoparticles for both silicate systems, with the highest (56.8%) for the microparticles of MCM-48. However, for each system, the release from nanoparticle is faster than from microparticles. For all systems, diffusion is the major dissolution mechanism.     

去质子化调控的肝素/壳聚糖抗凝血多层膜

本文研究了环境pH值变化对多层膜表面性能的影响, 并且评价了组装pH值与测试pH值的差异对多层膜血液相容性的影响.     

Evaluation of retention and release processes of two antibiotics from the biocompatible core-shell microparticles

By dropwise addition of a chitosan solution into different non-solvent, such as: 1 N and 2 N NaOH as well as 1 N NaOH: C₂H₅OH mixture (2:1, v/v) at temperature of 25 °C and 50 °C under stirring, the spherical pure chitosan microparticles were performed. As solvents for chitosan was used 0.1 N acetic acid or 0.1 N HCl. The immersion of the pure chitosan microparticles in hyaluronan solution led to complex microparticles, namely chitosan microparticles covered by a hyaluronan layer. For all the microparticles performed the behaviours in the retention process of two antibiotics: chloramphenicol succinate sodium salt and cefotaxime sodium salt were analyzed. Also, the study shows the release behaviour of cefotaxime sodium salt by the microparticles loaded with this drug. Among the microparticles performed a type of complex microparticles can be considered a suitable drug delivery system for cefotaxime. These microparticles were performed by dropwise addition of chitosan solution in 0.1 N acetic acid into the 1 N NaOH: C₂H₅OH (2:1, v/v) non-solvent at 20 °C for 3 h, followed by their washing up to alkalinity loss and the immersion in hyaluronan solution of 10 g/L concentration for 24 h.     

Tripolyphosphate-responsive release property of monoolein cubic phase containing sodium dodecyl sulfate and oligo chitosan

Tripolyphosphate (TPP)-responsive MO cubic phase was prepared by immobilizing oligo chitosan in the water channel through its electrostatic attraction with sodium dodecyl sulfate (SDS). The phase transition temperature (PTT) increased with increasing the content of SDS. The PTT of cubic phase whose SDS content was 0%, 0.21%, 0.42%, 0.84%, and 1.68%, determined by polarized microscopy, was about 69.5°C, 72°C, 75°C, 80.5°C, and 95°C, respectively. The PTT did not markedly deviate from that determined by differential scanning calorimetry. The release degree for 72 h of dye (i.e., amaranth and methylene blue) was dependent on the pH value of release medium (pH 3.0 and pH 7.0). Moreover, the release degree significantly increased when the TPP concentration in the release medium increased to 0.4% (w/v). Oligo chitosan was electrostatically complexed with TPP and the complexation took place extensively at the oligo chitosan/TTP mass ratio of 1:0.125 and 1:0.25 and at the oligo chitosan concentration of 1.6% (w/v), evidenced by optical spectroscopy and scanning electron microscopy. It was thought that the complexation was responsible for the TPP concentration-dependent release.     

Improved Antimicrobial Potency through Synergistic Action of Chitosan Microparticles and Low Electric Field

Techniques to inhibit gram-negative bacteria such as Shiga toxin-producing Escherichia coli are valuable as the prevalence of large-scale industrial food preparation increases the likelihood of contamination. Chitosan, the deacetylated derivative of chitin, has been demonstrated to inhibit bacteria growth in acidic environments, but is significantly less effective in preventing bacteria grown at pH?>7.0. Pulsed electric fields, constituting another method of bacteria inhibition, are difficult to generate at sufficient strength due to the high electric potentials required. This study utilizes adsorption of particulate chitosan in a very low electric field for an increased inhibition of gram-negative bacteria in neutral or alkaline pH conditions. Chitosan microparticles are demonstrated to flocculate E. coli, inhibit growth, and exhibit increased efficacy when combined with a low voltage electric field applied over 2-min intervals. Using sustained pulses of approximately 100?V/cm, it is demonstrated that bacteria viability is reduced by several orders of magnitude. The degree of bacterial inhibition is increased when chitosan microparticles are introduced to the system prior to imposing a small electric field.     

壳聚糖/乙酰半胱氨酸纳米粒子的性质及体外释药性

制备了一种基于壳聚糖/乙酰半胱氨酸偶合物(CS-NAC)的新型巯基纳米粒子并进行了结构表征, 同时对纳米粒子的黏附性、溶胀性和药物释放进行了测试. 结果表明, 纳米粒子具有较小的粒径(140~210 nm)和正的表面电位(19.5~31.7 mV), 胰岛素的载药量达到13%~42%. 这些性质随着巯基含量的变化而变化. 与壳聚糖纳米粒子相比, 巯基壳聚糖纳米粒子表现出了更强更快的黏附性质. 体外释放研究结果表明, 巯基壳聚糖纳米粒子的胰岛素释放具有pH响应性. 在pH=6.8时, 15 min即能释放58.6 %的胰岛素; 而在pH=5.4时, 24 h内仅有不到40%的胰岛素被释放. 因此, CS-NAC纳米粒子用于胰岛素的黏膜给药体系具有很好的应用前景.     

Adsorption of chromium from aqueous solution using chitosan beads

A basic investigation on the removal of Cr(III) and Cr(VI) ions from aqueous solution by chitosan beads was conducted in a batch adsorption system. The chitosan beads were prepared by casting an acidic chitosan solution into an alkaline solution. The influence of different experimental parameters; pH, agitation period and different concentration of Cr(III) and Cr(VI) ions was evaluated. A pH 5.0 was found to be an optimum pH for Cr(III) adsorption, and meanwhile pH 3.0 was the optimum pH for the adsorption of Cr(VI) onto chitosan beads. The Langmuir and Freundlich adsorption isotherm models were applied to describe the isotherms and isotherm constants for the adsorption of Cr(III) and Cr(VI) onto chitosan beads. Results indicated that Cr(III) and Cr(VI) uptake could be described by the Langmuir adsorption model. The maximum adsorption capacities of Cr(III) and Cr(VI) ions onto chitosan beads were 30.03 and 76.92 mg g⁻¹, respectively. Results showed that chitosan beads are favourable adsorbents. The Cr(III) and Cr(VI) ions can be removed from the chitosan beads by treatment with an aqueous EDTA solution.     

Regenerated keratin proteins as potential biomaterial for drug delivery

This work aims to preliminarily evaluate the reliability of regenerated keratins (RKs) in the design of microparticulate drug delivery systems by studying their processability and cytotoxicity. RKs were extracted by sulfitolysis from wool waste. A 4.5% w/w RK solution was spray‐dried, and microparticles were sterilized by steam vapor under pressure. Scanning electron microscope, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and Fourier transform infrared spectroscopy were used to characterize RKs and microparticles thereof. The in vitro cytotoxicity was determined by assessing the release of lactate dehydrogenase in the human monocytic cell line Tamm–Horsfall glycoprotein‐1. RK‐based microparticles with a narrow and unimodal particle size distribution (~6 µm) were obtained. They had a raisin‐like structure with a smooth surface. Both microparticle morphology and RK molecular weight were well‐preserved after sterilization. The curve fitting of the amide I bands showed that RK in the microparticles was prevalently present in the disordered/α‐helix secondary structures which made the protein soluble in water. To promote crystallization in the β‐sheet secondary structure and, therefore, water insolubility, RK‐based microparticles were immersed in an aqueous solution of acetic acid at pH 3.5 overnight. RK did not induce any appreciable cellular cytotoxicity at any of the concentrations (from 1 up to 1000 µg sterile microparticles in 1 ml cell culture medium) or time‐points (24–72 h) tested. These preliminary data suggest the feasibility of producing RK biocompatible microparticles using waste wool as starting material. Copyright © 2013 John Wiley & Sons, Ltd.     

Kinetics of adsorption of Saccharomyces cerevisiae mandelated dehydrogenase on magnetic Fe3O4–chitosan nanoparticles

The adsorption of Saccharomyces cerevisiae mandelated dehydrogenase (SCMD) protein on the surface-modified magnetic nanoparticles coated with chitosan was studied in a batch adsorption system. Functionalization of surface-modified magnetic particles was performed by the covalent binding of chitosan onto the surface of magnetic Fe₃O₄ nanoparticles. Characterization of these particles was carried out using FTIR spectra, transmission electron micrography (TEM), X-ray diffraction (XRD) and vibrating sample magnetometry (VSM). Magnetic measurement revealed that the magnetic Fe₃O₄–chitosan nanoparticles were superparamagnetic and the saturation magnetization was about 37.3 emu g⁻¹. The adsorption capacities and rates of SCMD protein onto the magnetic Fe₃O₄–chitosan nanoparticles were evaluated. The adsorption capacity was influenced by pH, and it reached a maximum value around pH 8.0. The adsorption capacity increased with the increase in temperature. The adsorption isothermal data could be well interpreted by the Freundlich isotherm model. The kinetic experimental data properly correlated with the first-order kinetic model, which indicated that the reaction is the adsorption control step. The apparent adsorption activation energy was 27.62 kJ mol⁻¹ and the first-order constant for SCMD protein was 0.01254 min⁻¹ at 293 K.     

Stabilization of Liposomes by Polyelectrolytes: Mechanism of Interaction and Role of Experimental Conditions

ζ-potential measurements on LUVs allow to evidence the influence of pH, ionic salt concentration, and polyelectrolyte charge on the interaction between polyelectrolyte (chitosan and hyaluronan) and zwitterionic lipid membrane. First, chitosan adsorption is studied: adsorption is independent on the chitosan molecular weight and corresponds to a maximum degree of decoration of 40% in surface coverage. From the dependence with pH and independence with MW, it is concluded that electrostatic interactions are responsible of chitosan adsorption which occurs flat on the external surface of the liposomes. The vesicles become positively charged in the presence of around two repeat units of chitosan added per lipid accessible polar head in acid medium down to pH = 7.2. Direct optical microscopy observations of GUVs shows a stabilization of the composite liposomes under different external stresses (pH and salt shocks) which confirms the strong electrostatic interaction between the chitosan and the lipid membrane. It is also demonstrated that the liposomes are stabilized by chitosan adsorption in a very wide range of pH (2.0 < pH < 12.0). Then, hyaluronan (HA), a negatively charged polyelectrolyte, is added to vesicles; the vesicles turn rapidly negatively charged in presence of adsorbed HA Finally, we demonstrated that hyaluronan adsorbs on positively charged chitosan-decorated liposomes at pH < 7.0 leading to charge inversion in the liposome decorated by the chitosan-hyaluronan bilayer. Our results demonstrate the adsorption of positive and/or negative polyelectrolyte at the surface of lipidic vesicles as well as their role on vesicle stabilization and charge control.     

Biodegradable Electrostatic Complexes of Chitosan Cationic Microparticles and Anionic Liposomes

Using the electrostatic adsorption of anionic liposomes on the surface of cationic microparticles of ion-crosslinked chitosan, complexes in which each microparticle can bind up to 110 intact (undestroyed) liposomes are prepared. The saturated complex 350–400 nm in diameter does not dissociate to initial components in aqueous solutions with pH 7 and a NaCl concentration of 0.15 mol/L, but decomposes to 10-nm particles in the presence of proteolytic enzymes. The chitosan–liposome complex and its biodegradation products are characterized by a low cytotoxicity. The described technique may be used to obtain biodegradable multiliposomal containers for the encapsulation and delivery of drugs.     

可生物降解肝素钠/两性壳聚糖复合物用于蛋白药物pH响应释放研究

制备了一类可生物降解肝素钠两性壳聚糖复合物(HPACS),并探索将其用于蛋白药物pH响应释放.两性壳聚糖由壳聚糖与丙烯酸加成反应得到,丙烯酸取代度可通过丙烯酸壳聚糖投料比调控;用胶体与pH浊度滴定研究了肝素钠与两性壳聚糖的复合作用,发现两组分在一定pH范围内能通过静电相互作用形成复合物,复合转变临界pH(pHΦ)与两性壳聚糖中丙烯酸取代度有关,取代度越低,pHΦ值越高.以牛血清白蛋白(BSA)为模型,测定了其在复合物中包埋及不同pH介质中的释药行为.结果表明,BSA可以在非常温和条件下有效包埋于复合物中,包埋率接近100%;BSA从复合物中释放具有很高的pH响应性,释放转变在很窄的pH范围内(<0.4pH单位)完成,释放转变临界pH(pH′Φ)可由两性壳聚糖中丙烯酸取代度调控.复合物形成和蛋白质释放在对pH依赖性上存在很好的相关性.同时还发现,在中性介质中(pH7.4),复合物对BSA具有很好的缓释作用,BSA持续释放时间可达15天左右.     

Facile synthesis of hydrous zirconia-impregnated chitosan beads as a filter medium for efficient removal of phosphate from water

The removal and recovery of phosphate from water using adsorption technology require that the adsorbent material is easily separable from treated water. Continuous efforts are still awaited to develop additional efficient phosphate adsorbents that are economical to fabricate. In this study, hydrous zirconia-impregnated chitosan beads (HZCB) containing different Zr/chitosan ratios were synthesized using a facile scheme. We found that HZCB with a Zr/amine molar ratio of?~?1 (HZCB-1) possessed excellent stability and phosphate removal performance. This optimized material was characterized with XRD, SEM, FTIR, XPS, specific surface area and point of zero charge measurements. The maximum adsorption capacity was 42.02 mg/g (at pH?~?6.7). The adsorption kinetics were best described by a pseudosecond-order model, and the rate constant of HZCB-1 was much lower than that of its powder but was similar to the commercial bead product Ferrolox. The removal of phosphate depended substantially upon pH and was enhanced by lowering the pH. Good selectivity of HZCB-1 for phosphate was observed, although the coexistence of sulfate produced a significant negative effect. Direct coordination of phosphate to Zr atoms by replacing hydroxyls was the dominant adsorption mechanism (~?85%), while chitosan also contributed to phosphate removal (~?15%). Adsorbed phosphate was successfully eluted by an NaOH solution, and the material obtained after desorption and regeneration was able to be repeatedly used. The results of column studies indicated that this material could be implemented in long-term application.