Chitosan nanoparticle as protein delivery carrier--systematic examination of fabrication conditions for efficient loading and release

Chitosan nanoparticles fabricated via different preparation protocols have been in recent years widely studied as carriers for therapeutic proteins and genes with varying degree of effectiveness and drawbacks. This work seeks to further explore the polyionic coacervation fabrication process, and associated processing conditions under which protein encapsulation and subsequent release can be systematically and predictably manipulated so as to obtain desired effectiveness. BSA was used as a model protein which was encapsulated by either incorporation or incubation method, using the polyanion tripolyphosphate (TPP) as the coacervation crosslink agent to form chitosan-BSA-TPP nanoparticles. The BSA-loaded chitosan-TPP nanoparticles were characterized for particle size, morphology, zeta potential, BSA encapsulation efficiency, and subsequent release kinetics, which were found predominantly dependent on the factors of chitosan molecular weight, chitosan concentration, BSA loading concentration, and chitosan/TPP mass ratio. The BSA loaded nanoparticles prepared under varying conditions were in the size range of 200-580nm, and exhibit a high positive zeta potential. Detailed sequential time frame TEM imaging of morphological change of the BSA loaded particles showed a swelling and particle degradation process. Initial burst released due to surface protein desorption and diffusion from sublayers did not relate directly to change of particle size and shape, which was eminently apparent only after 6h. It is also notable that later stage particle degradation and disintegration did not yield a substantial follow-on release, as the remaining protein molecules, with adaptable 3-D conformation, could be tightly bound and entangled with the cationic chitosan chains. In general, this study demonstrated that the polyionic coacervation process for fabricating protein loaded chitosan nanoparticles offers simple preparation conditions and a clear processing window for manipulation of physiochemical properties of the nanoparticles (e.g., size and surface charge), which can be conditioned to exert control over protein encapsulation efficiency and subsequent release profile. The weakness of the chitosan nanoparticle system lies typically with difficulties in controlling initial burst effect in releasing large quantities of protein molecules.     

Preparation of chitosan nanoparticles as carrier for immobilized enzyme

This work investigated the preparation of chitosan nanoparticles used as carriers for immobilized enzyme. The morphologic characterization of chitosan nanoparticles was evaluated by scanning electron microscope. The various preparation methods of chitosan nanoparticles were discussed and chosen. The effect of factors such as molecular weight of chitosan, chitosan concentration, TPP concentration, and solution pH on the size of chitosan nanoparticles was studied. Based on these results, response surface methodology was emploved. The results showed that solution pH, TPP concentration, and chitosan concentration significantly affected the size of chitosan nanoparticles. The adequacy of the predictive model equation for predicting the magnitude orders of the size of chitosan nanoparticles was verified effectively by the validation data. Immobilization conditions were investigated as well. The minimum particles size was about 42±5 nm under the optimized conditions. The optimal conditions of immobilization were as follow: one milligram of neutral proteinase was immobilized on chitosan nanoparticles for about 15 min at 40°C. Under the optimized conditions, the enzyme activity yield was 84.3%.     

Effects of ionic strength on the size and compactness of chitosan nanoparticles

In this work, chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate (TPP). The effects of the ionic strength of the solvent employed in the particle preparation on the average size and compactness of the particles were investigated. In addition, the effects of the chitosan concentration and the crosslinker to polymer ratio on the particle characteristics were studied. The chitosan–TPP nanoparticles were characterized by dynamic light scattering, zeta potential, and turbidity measurements. The compactness of the nanoparticles was estimated with a method based on the size of the nanoparticles and the turbidity of the nanoparticle suspension. All the investigated preparation parameters, i.e., the ionic strength of the solvent, the chitosan concentration, and the TPP to chitosan ratio, affected the particle characteristics. For instance, smaller and more compact particles were formed in saline solvents, compared to particles formed in pure water. Further, the addition of monovalent salt rendered it possible to prepare particles in the nanometer size range at a higher polymer concentration. Solvent salinity is thus an important parameter to address in the preparation of chitosan nanoparticles crosslinked with TPP.     

Preparation of chitosan microspheres by ionotropic gelation under a high voltage electrostatic field for protein delivery

Biodegradable microspheres have been widely used in drug/protein delivery system. In this paper, a modified ionotropic gelation method combined with a high voltage electrostatic field was developed to prepare protein-loaded chitosan microspheres. Bovine serum albumin (BSA) was chosen as a model protein. The preparation process and major parameters were discussed and optimized. The morphology, particle size, encapsulation efficiency and in vitro release behavior of the prepared microspheres were investigated. The results revealed that the microspheres exhibited good sphericity and dispersity when the mixture of sodium tripolyphosphate (TPP) and ethanol was applied as coagulation solution. Higher encapsulation efficiency (>90%) was achieved for the weight ratio of BSA to chitosan below 5%. 35% of BSA was released from the microspheres cured in 3% coagulation solution, and more than 50% of BSA was released from the microspheres cured in 1% coagulation solution at pH 8.8. However, only 15% of BSA was released from the microspheres cured in 1% coagulation solution at pH 4. The results suggested that ionotropic gelation method combined with a high voltage electrostatic field will be an effective method for fabricating chitosan microspheres for sustained delivery of protein.     

Preparation and properties of ionically cross‐linked chitosan nanoparticles

Chitosan nanoparticles were fabricated by a method of tripolyphosphate (TPP) cross‐linking. The influence of fabrication conditions on the physical properties and drug loading and release properties was investigated by transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV–vis spectroscopy. The nanoparticles could be prepared only within a zone of appropriate chitosan and TPP concentrations. The particle size and surface zeta potential can be manipulated by variation of the fabrication conditions such as chitosan/TPP ratio and concentration, solution pH and salt addition. TEM observation revealed a core–shell structure for the as‐prepared nanoparticles, but a filled structure for the ciprofloxacin (CH) loaded particles. Results show that the chitosan nanoparticles were rather stable and no cytotoxicity of the chitosan nanoparticles was found in an in vitro cell culture experiment. Loading and release of CH can be modulated by the environmental factors such as solution pH and medium quality. Copyright © 2008 John Wiley & Sons, Ltd.     

Novel nanoparticles composed of chitosan and β-cyclodextrin derivatives as potential insoluble drug carrier

This research was aim to develop novel cyclodextrin/chitosan(CD/CS) nanocarriers for insoluble drug delivery through the mild ionic gelation method previously developed by our lab. A series of different bcyclodextrin(β-CD) derivatives were incorporated into CS nanoparticles including hydroxypropyl-bcyclodextrin(HP-β-CD), sulphobutylether-β-cyclodextrin(SB-β-CD), and 2,6-di-O-methy-β-cyclodextrin(DM-β-CD). Various process parameters for nanoparticle preparation and their effects on physicochemical properties of CD/CS nanoparticles were investigated, such as the type of CD derivatives,CD and CS concentrations, the mass ratio of CS to TPP(CS/TPP), and p H values. In the optimal condition,CD/CS nanoparticles were obtained in the size range of 215–276 nm and with the zeta potential from30.22 m V to 35.79 m V. Moreover, the stability study showed that the incorporation of CD rendered the CD/CS nanocarriers more stable than CS nanoparticles in PBS buffer at p H 6.8. For their easy preparation and adjustable parameters in nanoparticle formation as well as the diversified hydrophobic core of CD derivatives, the novel CD/CS nanoparticles developed herein might represent an interesting and versatile drug delivery platform for a variety of poorly water-soluble drugs with different physicochemical properties.     

Formulation of Quaternized Aminated Chitosan Nanoparticles for Efficient Encapsulation and Slow Release of Curcumin

An effective drug nanocarrier was developed on the basis of a quaternized aminated chitosan (Q-AmCs) derivative for the efficient encapsulation and slow release of the curcumin (Cur)-drug. A simple ionic gelation method was conducted to formulate Q-AmCs nanoparticles (NPs), using different ratios of sodium tripolyphosphate (TPP) as an ionic crosslinker. Various characterization tools were employed to investigate the structure, surface morphology, and thermal properties of the formulated nanoparticles. The formulated Q-AmCs NPs displayed a smaller particle size of 162 ± 9.10 nm, and higher surface positive charges, with a maximum potential of +48.3 mV, compared to native aminated chitosan (AmCs) NPs (231 ± 7.14 nm, +32.8 mV). The Cur-drug encapsulation efficiency was greatly improved and reached a maximum value of 94.4 ± 0.91%, compared to 75.0 ± 1.13% for AmCs NPs. Moreover, the in vitro Cur-release profile was investigated under the conditions of simulated gastric fluid [SGF; pH 1.2] and simulated colon fluid [SCF; pH 7.4]. For Q-AmCs NPs, the Cur-release rate was meaningfully decreased, and recorded a cumulative release value of 54.0% at pH 7.4, compared to 73.0% for AmCs NPs. The formulated nanoparticles exhibited acceptable biocompatibility and biodegradability. These findings emphasize that Q-AmCs NPs have an outstanding potential for the delivery and slow release of anticancer drugs.     

Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery

The development of targeted vehicles for systemic drug delivery relies on optimizing both the cell-targeting ligand and the physicochemical characteristics of the nanoparticle carrier. A versatile platform based on modification of gold nanoparticles with thiolated polymers is presented in which design parameters can be varied independently and systematically. Nanoparticle formulations of varying particle size, surface charge, surface hydrophilicity, and galactose ligand density were prepared by conjugation of PEG-thiol and galactose-PEG-thiol to gold colloids. This platform was applied to screen for nanoparticle formulations that demonstrate hepatocyte-targeted delivery in vivo. Nanoparticle size and the presence of galactose ligands were found to significantly impact the targeting efficiency. Thus, this platform can be readily applied to determine design parameters for targeted drug delivery systems.Modified gold nanoparticles are a suitable model for nanoparticle-based gene carriers.     

Physical and chemical characterization insulin-loaded chitosan-TPP nanoparticles

The purpose of this study was to develop and characterize insulin nanoparticles systems using chitosan. Insulin-loaded nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The interactions between insulin and chitosan were evaluated by differential scanning calorimetry (DSC), thermogravimetry/derivative thermogravimetry (TG/DTG), and Fourier-transform infrared (FTIR) spectroscopy. Besides, particle size distribution, polydispersity index (PDI), zeta potential, and association efficiency (AE%) of the nanoparticles were evaluated. In general, inert nanoparticles and insulin-loaded nanoparticles showed an average size of 260.56 nm (PDI 0.502) and 312.80 nm (PDI 0.481), respectively. Both nanoparticles showed positive charge, but after insulin incorporation the zeta potential was reduced, evidencing its incorporation. Nanoparticles obtained also showed AE% around 70%, measured by high-performance liquid chromatography (HPLC). The results of FTIR, DSC, and TG/DTG corroborated the data presented suggesting that insulin was successfully encapsulated. However, drug incorporation seems to be related not only to electrostatic interactions, but also to physical process and/or adsorption phenomena.     

Microwave Assisted Synthesis of Chitosan Nanoparticles

Chitosan nanoparticles (CHN) were prepared based on ionotropic gelation between low moleculer weight chitosan and sodium tripolyphosphate (TPP) under microwave irradiation. Particle size, zeta potential, and FT-IR techniques were used for characterization of CHN. The influence of reaction time on the nanoparticle size distribution was investigated, and the results showed that the microwave irradiation method evidently decreases the reaction times and particle size over the conventional method. It was determined by the results of the zeta potential measurements that synthesized CHN under microwave irradiation clearly exhibits more homogeneous and stable dispersion.     

Physicochemical characterization of chitosan nanoparticles: electrokinetic and stability behavior

Some physical properties of nanogel particles formed by chitosan ionically cross-linked by tripolyphosphate (TPP) have been studied. Electrokinetic properties and colloidal stability were analyzed as a function of pH and ionic strength of the medium. Chitosan particles showed volume phase transitions (swelling/shrinking processes) when the physicochemical conditions of the medium were changed. Experimental data were mainly obtained by electrophoretic mobility measurements and by photon correlation spectroscopy and static light scattering techniques. Chitosan chains possess glucosamine groups that can be deprotonated if the pH increases. Therefore, modification of pH from acid to basic values caused a deswelling process based on a reduction of the intramolecular electric repulsions inside the particle mesh. Electrophoretic mobility data helped to corroborate the above electrical mechanism as responsible for the size changes. Additionally, at those pH values around the isoelectric point of the chitosan-TPP particles, the system became colloidally unstable. Ionic strength variations also induced important structural changes. In this case, the presence of KCl at low and moderate concentrations provoked swelling, which rapidly turned on particle disintegration due to the weakness of chitosan-TPP ionic interactions. These last results were in good agreement with the predictions of gel swelling theory by salt in partially ionized networks.     

Experimental and model study of the formation of chitosan-tripolyphosphate-siRNA nanoparticles

Chitosan-tripolyphosphate (TPP) nanoparticles have received great interest as a drug delivery system due to the simple and mild procedure of ionic gelation and the biocompatibility of chitosan. We have studied the formation of chitosan nano- and microparticles through ionic gelation with TPP in the absence and presence of NaCl, by measuring the kinetics of formation, particle size, and zeta potential. Depending on the experimental conditions (concentrations of chitosan and TPP and the presence or absence of NaCl), particle formation displays an exponential or a sigmoidal time dependency. In order to explain the kinetics measurements, we have set up a simple kinetics model involving four different species. The model is constructed on the basis of previously proposed mechanisms of particle formation and our measurements of particle size and kinetics of formation. The model can simulate all the different time dependencies of particle formation. We also determined the effect of small interfering RNA (siRNA) on the rate of particle formation, but apparently siRNA has little or no influence on particle formation when TPP is present.     

Monovalent salt enhances colloidal stability during the formation of chitosan/tripolyphosphate microgels

Chitosan micro- and nanoparticles are routinely prepared through ionotropic gelation, where sodium tripolyphosphate (TPP) is added as a cross-linker to dilute chitosan solutions. Despite the wide use of these gel-like particles, their preparation currently relies on trial and error. To address this, we used isothermal titration calorimetry (ITC), dynamic light scattering (DLS), transmission electron microscopy (TEM), and ζ-potential measurements to investigate how the formation, structure, and colloidal stability of chitosan microgels are linked to the molecular interactions that underlie their self-assembly. The strength of the chitosan/TPP interactions was systematically varied through the addition of monovalent salt (NaCl). Remarkably, and contrary to other colloidal systems, this revealed that moderate amounts of NaCl (e.g., 150 mM) enhance the colloidal stability of chitosan/TPP microgels during their formation. This stems from the weakened chitosan/TPP binding, which apparently inhibits the bridging of the newly formed microgels by TPP. The enhanced colloidal stability during the ionic cross-linking process yields microgels with dramatically narrower size distributions, which hitherto have typically required the deacetylation and fractionation of the parent chitosan. Conversely, at high ionic strengths (ca. 500 mM) the chitosan/TPP binding is weakened to the point that the microgels cease to form, thus suggesting the existence of an optimal ionic strength for ionotropic microgel preparation.     

Formation of positively charged poly(butyl cyanoacrylate) nanoparticles stabilized with chitosan

To investigate the formation of positively charged nanoparticles (NP) stabilized with chitosan, positively charged poly(butyl cyanoacrylate) (PBCA) NP were prepared by emulsion polymerization in the presence of chitosan as a polymeric stabilizer at low pH. The effect of physicochemical factors such as the pH, the concentration and the volume of the chitosan solution, the chitosan molecular weight and the temperature on the mean particle size and the turbidity of PBCA-NP was investigated. Particle size was determined using a transmission electron microscope. The chemical interaction between chitosan and PBCA was identified by Fourier transform infrared (FT-IR) spectroscopy and the grafting percentage at various pH values was determined. The zeta potential of PBCA-NP coated with chitosan was determined from the electrophoretic mobility in 10 mM NaCl. The pH, the concentration and the volume of the chitosan solution and the molecular weight of chitosan were shown to be important factors in controlling the mean particle size of NP in the range 10–100 nm. FT-IR spectra indicated that chitosan was covalently linked to PBCA and the maximum grafting percentage reached about 120% w/w at pH 2.0. Nimodipine as a model drug was successfully incorporated into chitosan-stabilized PBCA-NP with a mean particle diameter of 31.6 nm. PBCA-NP coated with chitosan carried a positive charge. The results indicate that positively charged NP may be produced in the presence of cationic polysaccharide chitosan and might increase their potential use as a targeting drug delivery system. Received: 17 March 1999/Accepted in revised form: 4 October 1999     

Controlled association in suspensions of charged nanoparticles with a weak polyelectrolyte

The properties of high-pH suspensions of mixtures of silica with low-molecular-weight samples of the water-soluble polymer polyethylenimine (PEI) have been studied. At pH > 10 and low ionic strength, silica nanoparticles are stabilized by a negative surface charge, and PEI has only a very low positive charge. The adsorption of PEI induces a localized positive charge on the segments of polymer closest to the silica surface. The parts of the molecule furthest away from the surface have little charge because of the high pH of the medium. The polymer-covered particle remains negatively charged, imparting some electrostatic stabilization. Suspensions of silica and low-molecular-weight PEI are low-viscosity fluids immediately after mixing, but aggregation occurs leading to the eventual gelation (or sedimentation at lower concentrations) of these mixtures, indicating colloidal instability. The gelation time passes through a minimum with increasing surface coverage. The rate of gelation increases exponentially with molecular weight: for molecular weight > or = 10,000 Da PEI, the instability is so severe that uniform suspensions cannot be produced using simple mixing techniques. The gelation rates increase rapidly with temperature, ionic strength, and reduction in pH. The rate of gelation increases with increasing particle concentration at low surface coverage but decreases at high coverage as a consequence of a small increase in pH. Gels are broken by application of high shear into aggregates that re-gel more rapidly than the original discrete coated particles.     

Preparation of PMMA/acid-modified chitosan core-shell nanoparticles and their potential as gene carriers

The core-shell nanoparticles consisting of poly(methyl methacrylate) (PMMA) cores surrounded by various acid-modified chitosan shells were synthesized using a surfactant-free emulsion copolymerization, induced by a tert-butylhydroperoxide (TBHP) solution. Methyl methacrylate (MMA) was grafted onto four acid-modified chitosans (hydrochloric, lactic, aspartic, and glutamic acids) with MMA conversions up to 64%. The prepared nanoparticles had diameter ranging from 100 to 300 nm characterized by atomic force microscopy and displayed highly positive surface charges up to +77 mV. Transmission electron microscopic images clearly revealed well-defined core-shell morphology of the nanoparticles where PMMA cores were coated with acid-modified chitosan shells. The effect of acid-modified chitosans on particle size, intensity of surface charge, morphology, and thermal stability were determined systematically. The plasmid DNA/nanoparticles complexes were investigated with ζ-potential measurement. The results suggested that these nanoparticles can effectively complex with plasmid DNAs via electrostatic interaction and could be used as gene carriers.

Preparation of chitosan nanoparticles by TPP ionic gelation combined with spray drying,and the antibacterial activity of chitosan nanoparticles and a chitosan nanoparticle–amoxicillin complex

Chitosan nanoparticles were prepared from chitosan with various molecular weights by tripolyphosphate (TPP) ionic gelation combined with a spray drying method. The morphologies and characteristics of chitosan nanoparticles were determined by TEM, FE-SEM and from their mean sizes and zeta potentials. The effect of chitosan molecular weight (130, 276, 760 and 1200 cPs) and size of spray dryer nozzle (4.0, 5.5 and 7.0 µm) on mean size, size distribution and zeta potential values of chitosan nanoparticles was investigated. The results showed that the mean size of chitosan nanoparticles was in the range of 166–1230 nm and the zeta potential value ranged from 34.9 to 59 mV, depending on the molecular weight of chitosan and size of the spray dryer nozzles. The lower the molecular weight of chitosan, the smaller the size of the chitosan nanoparticles and the higher the zeta potential. A test for the antibacterial activity of chitosan nanoparticles (only) and a chitosan nanoparticle–amoxicillin complex against Streptococcus pneumoniae was also conducted. The results indicated that a smaller chitosan nanoparticle and higher zeta potential showed higher antibacterial activity. The chitosan nanoparticle–amoxicillin complex resulted in improved antibacterial activity as compared to amoxicillin and chitosan nanopaticles alone. Using a chitosan nanoparticle–amoxicillin complex could reduce by three times the dosage of amoxicillin while still completely inhibiting S. pneumoniae.     

Development and Optimization of Chitosan Nanoparticle-Based Intranasal Vaccine Carrier

Chitosan is a natural polysaccharide, mainly derived from the shell of marine organisms. At present, chitosan has been widely used in the field of biomedicine due to its special characteristics of low toxicity, biocompatibility, biodegradation and low immunogenicity. Chitosan nanoparticles can be easily prepared. Chitosan nanoparticles with positive charge can enhance the adhesion of antigens in nasal mucosa and promote its absorption, which is expected to be used for intranasal vaccine delivery. In this study, we prepared chitosan nanoparticles by a gelation method, and modified the chitosan nanoparticles with mannose by hybridization. Bovine serum albumin (BSA) was used as the model antigen for development of an intranasal vaccine. The preparation technology of the chitosan nanoparticle-based intranasal vaccine delivery system was optimized by design of experiment (DoE). The DoE results showed that mannose-modified chitosan nanoparticles (Man-BSA-CS-NPs) had high modification tolerance and the mean particle size and the surface charge with optimized Man-BSA-CS-NPs were 156 nm and +33.5 mV. FTIR and DSC results confirmed the presence of Man in Man-BSA-CS-NPs. The BSA released from Man-BSA-CS-NPs had no irreversible aggregation or degradation. In addition, the analysis of fluorescence spectroscopy of BSA confirmed an appropriate binding constant between CS and BSA in this study, which could improve the stability of BSA. The cell study in vitro demonstrated the low toxicity and biocompatibility of Man-BSA-CS-NPs. Confocal results showed that the Man-modified BSA-FITC-CS-NPs promote the endocytosis and internalization of BSA-FITC in DC2.4 cells. In vivo studies of mice, Man-BSA-CS-NPs intranasally immunized showed a significantly improvement of BSA-specific serum IgG response and the highest level of BSA-specific IgA expression in nasal lavage fluid. Overall, our study provides a promising method to modify BSA-loaded CS-NPs with mannose, which is worthy of further study.     

壳聚糖纳米粒子荧光探针的制备和表征

通过低分子量的壳聚糖(LCS)聚阳离子与三聚磷酸钠(TPP)的静电作用制备纳米级壳聚糖微球,并利用壳聚糖链上丰富的氨基与荧光素异硫氰酸酯(FITC)反应从而制备纳米壳聚糖微球荧光探针(NFCS)。结果表明,当壳聚糖分子量为60000,LCS与TPP的质量比为6∶1时,可得到粒度均一的球形纳米粒子,平均粒径为40±3 nm。荧光倒置显微镜观察证实FITC结合到壳聚糖微球上。荧光光谱分析显示NFCS的最大激发波长、最大发射波长与游离态FITC无显著差异。光漂白实验证实NFCS的稳定性比游离态FITC有显著提高。     

Chitosan nanoparticles crosslinked by glycidoxypropyltrimethoxysilane for pH triggered release of protein

pH-responsive-chitosan nanoparticles for the control release of protein drug were prepared by combining two-step crosslinking method,in which chitosan was subsequently crosslinked by sodium tripolyphosphate(TPP)and glycidoxypropyltrimethoxysilane (GPTMS).Compared with TPP crosslinked chitosan particles,the two-step crosslinked nanoparticles were not only pH-responsive but also more stable in wide pH range.Fluorescein isothiocyanate(FITC)labeled anti-human-IgG antibody was used as a model protein drug for...