Chitosan-based polyelectrolyte complexes: A review

This review focuses on the formation of polyelectrolyte chitosan complexes with biologically active compounds and the prospects of use thereof. The possibility of obtaining low-molecular-weight, water-soluble batches of chitosan, which differ in their degree of acetylation, is discussed, with emphasis on their use for binding nucleic acids into complexes.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 9–16.Original Russian Text Copyright © 2005 by Ilina, Varlamov.     

Mechanism of cell transfection with plasmid/chitosan complexes

Chitosan is useful as a non-viral vector for gene delivery. Although there are several reports supporting the use of chitosan for gene delivery, studies regarding effects on transfection and the chitosan-specific transfection mechanism remain insufficient. In this report, the level of expression with plasmid/chitosan was observed to be no less than that with plasmid/lipofectin complexes in SOJ cells. The transfection mechanism of plasmid/chitosan complexes as well as the relationship between transfection activity and cell uptake was analyzed by using fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. In regard to effects on transfection, there were several factors to affect transfection activity and cell uptake, for example: the molecular mass of chitosan, stoichiometry of complex, as well as serum concentration and pH of transfection medium. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, ratio of chitosan nitrogen to DNA phosphate (N/P ratio) was 5, and transfection medium contained 10% serum at pH 7.0. We also investigated the transfection mechanism, and found that plasmid/chitosan complexes most likely condense to form large aggregates (5-8 microm), which absorb to the cell surface. After this, plasmid/chitosan complexes are endocytosed, and possibly released from endosomes due to swelling of lysosomal in addition to swelling of plasmid/chitosan complex, causing the endosome to rupture. Finally, complexes were also observed to accumulate in the nucleus using a confocal laser scanning microscope.     

Interaction of bacterial endotoxins with chitosan. Effect of endotoxin structure, chitosan molecular mass, and ionic strength of the solution on the formation of the complex

The interaction of endotoxins of different structure (lipopolysaccharides (LPS) and lipopolysaccharide-protein complexes (LPPC)) with chitosan has been studied. It was shown that the mechanism of interaction is rather complicated and depends on the macromolecular organization of endotoxin as well as on the degree of polymerization of the chitosan. Chitosan with molecular mass of 20 kD reveals higher affinity to LPS than chitosan with molecular mass of 140 kD. Endotoxins with long O-specific chains can bind completely with chitosan with the formation of LPS-chitosan and LPPC-chitosan complexes with weight ratios between the original components of 1:1 and 1:5. When endotoxins with higher degree of hydrophobicity and short O-specific chains were mixed with chitosan, a part of the LPS remained unbound. The stability of the complexes formed depends on ionic strength. It was shown that, in addition to electrostatic forces, other types of forces take part in the formation of the complexes. A decrease in acute toxicity of various LPSs is observed on their binding with chitosans.     

Synthesis of diamine maleyl chitosans, and in vitro transfection studies

Three novel diamine-modified chitosan derivatives were synthesized from N-maleyl chitosan via Michael addition reaction with 1,2-diaminoethane, 1,4-diaminobutane, and 1,6-diaminohexane, respectively. These chitosan derivatives exhibited well binding ability of condensing plasmid DNA to form complexes with size ranging from 150 to 500 nm when the chitosan derivative/DNA weight ratios were above 10. The complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The cytotoxicity of the chitosan derivatives presented a dependence on their side-chain structures. The gene transfection experiments were evaluated in 293 T and HeLa cells. The data obtained demonstrated that the gene transfection efficiencies of these chitosan derivatives were better than that of chitosan, suggesting these chitosan derivatives as potential gene vectors in vitro.     

Chitosan-Mediated siRNA Delivery <Emphasis Type="Italic">In Vitro</Emphasis>: Effect of Polymer Molecular Weight,Concentration and Salt Forms

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270–373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors.     

Comparative study of electrokinetic potentials and binding affinity of lipopolysaccharides-chitosan complexes

Electrokinetic properties of complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5, Yersinia pseudotuberculosis 1B 598, and Proteus vulgaris O25 (48/57) and their size distribution were investigated using zeta-potential distribution assay and quasi-elastic light scattering. The interaction of LPS from different microorganisms with chitosan at the same w/w ratio of components (1:1) resulted in the formation of complexes in which the negative charge of LPS was neutralized (LPS from E. coli) or overcompensated (Y. pseudotuberculosis and P. vulgaris). The changing in size of the endotoxin aggregates during binding with chitosan was observed. The binding constants of chitosan with LPSs were determined by a method with using the anionic dye Orange II. The LPS from E. coli possess higher affinity to chitosan in comparison with the two others samples of endotoxin.     

Gene Transfer by DNA/mannosylated Chitosan Complexes into Mouse Peritoneal Macrophages

Chitosan is a biodegradable and biocompatible polymer and is useful as a non-viral vector for gene delivery. In order to deliver pDNA/chitosan complex into macrophages expressing a mannose receptor, mannose-modified chitosan (man-chitosan) was employed. The cellular uptake of pDNA/man-chitosan complexes through mannose recognition was then observed. The pDNA/man-chitosan complexes showed no significant cytotoxicity in mouse peritoneal macrophages, while pDNA/man-PEI complexes showed strong cytotoxicity. The pDNA/man-chitosan complexes showed much higher transfection efficiency than pDNA/chitosan complexes in mouse peritoneal macrophages. Observation with a confocal laser microscope suggested differences in the cellular uptake mechanism between pDNA/chitosan complexes and pDNA/man-chitosan complexes. Mannose receptor-mediated gene transfer thus enhances the transfection efficiency of pDNA/chitosan complexes.     

Interaction of chitosans and their N-acylated derivatives with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the natural polycation chitosan and its derivatives--high molecular weight chitosans (80 kD) with different degree of acetylation, low molecular weight chitosan (15 kD), acylated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose, and tetraose)--were studied using ligand-enzyme solid-phase assay. The LPS-binding activity of chitosans (80 kD) decreased with increase in acetylation degree. Affinity of LPS interaction with chitosans increased after introduction of a fatty acid residue at the reducing end of chitosan. Activity of N-monoacylated chitooligosaccharides decreased in the order: oligochitosan --> tetra- > tri- --> disaccharides. The three-dimensional structures of complexes of R-LPS and chitosans with different degree of acetylation, chitooligosaccharides, and their N-monoacylated derivatives were generated by molecular modeling. The number of bonds stabilizing the complexes and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was shown that binding sites of chitooligosaccharides on R-LPS overlapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. The input of the hydrophobic fragment in complex formation energy is most prominent for complexes in water phase and is due to the hydrophobic interaction of chitooligosaccharide acyl fragment with fatty acid residues of LPS.     

Effect of Oppositely Charged Polymer and Dissolution Media on Rheology of Spray-Dried Ionic Complexes

The purpose of this research was to address the utility of rheological study in understanding the influence of oppositely charged polymers on release of naproxen sodium encapsulated in chitosan particles. The interaction between oppositely charged κ-carrageenan (κ-Ca) and chitosan leads to relatively higher gel strength, which is proportional to the ability to retard the drug release at acidic pH. The oscillatory tests within the linear viscoelastic range where the stress is proportional to the applied strain were performed on the hydrated sample matrices containing chitosan-naproxen sodium spray-dried complexes and k-Ca or hydroxypropyl methylcellulose (HPMC) in various ratios. It was observed that the effect of pH change on the dynamic moduli in spray-dried complexes containing κ-Ca was much stronger than that with HPMC reflecting presence of strong ionic interaction between κ-Ca and chitosan. The combination of oppositely charged polymers in different ratios proved to be useful in modulating the rheological properties of the hydrated formulations and their release-retarding properties. Dynamic moduli can be used to measure gel strength and are significant for the interpretation of oral sustained release spray-dried complexes.     

Glycosaminoglycan destabilization of DNA–chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA–chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA–chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from ∼10 to ∼1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Chitosan-g-PEG/DNA complexes deliver gene to the rat liver via intrabiliary and intraportal infusions

BACKGROUND: Chitosan has been shown to be a non-toxic and efficient vector for in vitro gene transfection and in vivo gene delivery through pulmonary and oral administrations. Recently, we have shown that chitosan/DNA nanoparticles could mediate high levels of gene expression following intrabiliary infusion 1. In this study, we have examined the possibility of using polyethylene glycol (PEG)-grafted chitosan/DNA complexes to deliver genes to the liver through bile duct and portal vein infusions. METHODS: PEG (Mw: 5 kDa) was grafted onto chitosan (Mw: 47 kDa, deacetylation degree: 94%) with grafting degrees of 3.6% and 9.6% (molar percentage of chitosan monosaccharide units grafted with PEG). The stability of chitosan-g-PEG/DNA complexes was studied by measuring the change in particle size and by agarose gel electrophoresis against bile or serum challenge. The influence of PEG grafting on gene transfection efficiency was evaluated in HepG2 cells using luciferase reporter gene. Chitosan and chitosan-g-PEG/DNA complexes were delivered to the liver through bile duct and portal vein infusions with a syringe pump. Gene expression in the liver and the distribution of gene expression in other organs were evaluated. The acute liver toxicity of chitosan and chitosan-g-PEG/DNA complexes was examined by measuring serum alanine aminotranferase (ALT) and aspartate aminotransferase (AST) activities as a function of time. RESULTS: Both chitosan and chitosan-g-PEG displayed comparable gene transfection efficiency in HepG2 cells. After challenge with serum and bile, chitosan-g-PEG/DNA complexes, especially those prepared with chitosan-g-PEG (GD = 9.6%), did not form large aggregates like chitosan/DNA complexes but remained stable for up to 30 min. In addition, chitosan-g-PEG prevented the degradation of DNA in the presence of serum and bile. On day 3 after bile duct infusion, chitosan-g-PEG (GD = 9.6%)/DNA complexes mediated three times higher gene expression in the liver than chitosan/DNA complexes and yielded background levels of gene expression in other organs. On day 1 following portal vein infusion, gene expression level induced by chitosan/DNA complexes was hardly detectable but chitosan-g-PEG (GD = 9.6%) mediated significant transgene expression. Interestingly, transgene expression by chitosan-g-PEG/DNA complexes in other organs after portal vein infusion increased with increasing grafting degree of PEG. The ALT and AST assays indicated that grafting of PEG to chitosan reduced the acute liver toxicity towards the complexes. CONCLUSION: This study demonstrated the potential of chitosan-g-PEG as a safe and more stable gene carrier to the liver.     

Glycosaminoglycan destabilization of DNA-chitosan polyplexes for gene delivery depends on chitosan chain length and GAG properties

Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA-chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA-chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from approximately 10 to approximately 1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.     

Alginate/chitosan hydrogels as perspective transport systems for cefotaxime

This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer–Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime–chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan–cefotaxime complexation considering the influence of a solvent.     

Generation of stable cell line by using chitosan as gene delivery system

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.     

A morphological study of heavy metal complexes of chitosan and crosslinked chitosans by SEM and WAXRD

Metal complexes of salts of Hg, Cu, Cd, Pb, Zn, and Mn with chitosan and crosslinked chitosans were prepared, and their morphologies were studied using scanning electron microscopy and wide angle X-ray diffraction. The metal ions which were specifically and strongly complexed to the amino functions of chitosans, like Hg, showed smooth surface morphology inspite of large number of ions complexed (372 mg/g of chitosan). The presence of metal ions on the surface of the chitosans could be detected with decrease in metal ion binding, in the following sequence Hg > Cu > Cd > Zn > Pb > Mn. Particularly in the case of Pb ions, the presence of these ions is clearly seen on the surface of the polymer by SEM. The number of ions of Mn complexed on the polymers was too few (5 mg/g of chitosan) to be visible. SEM of Hg and Cu complexes do not show the “holes” observed in the crosslinked polymers as they bind specifically to amino groups of chitosan, but for Cd, Zn, Mn, and Pb complexes, these “holes” are clearly visible. These results are also in agreement with the morphologies studied by WAXRD. The metal complexation data for each of these metal ions was also in the same sequence.     

脂质体、免疫刺激复合物和壳聚糖作为黏膜免疫载体的研究现状

查阅国内外文献,采用整理归纳、引用和分析的方法,概述了鼻黏膜免疫载体材料脂质体、免疫刺激复合物和壳聚糖的生物学特性及其在气溶胶疫苗领域的应用。脂质体、免疫刺激复合物和壳聚糖作为气溶胶疫苗鼻黏膜免疫的载体,能够增强疫苗的免疫原性,提高机体的体液免疫和细胞免疫水平,因此,可作为疫苗抗原激发特异免疫应答的生物材料。     

Polyelectrolyte complexes between (cross-linked) N-carboxyethylchitosan and (quaternized) poly[2-(dimethylamino)ethyl methacrylate]: preparation, characterization, and antibacterial properties

Novel polyelectrolyte complexes (PECs) between N-carboxyethylchitosan (CECh) and well-defined (quaternized) poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) have been obtained. The modification of chitosan into CECh allows the preparation of PECs in a pH range in which chitosan cannot form complexes. The CECh/PDMAEMA complex is formed in a narrow pH range around 7. The quaternization of the tertiary amino groups of PDMAEMA enables complex formation with CECh both in neutral and in alkaline medium. Cross-linked CECh is also capable of forming complexes with (quaternized) PDMAEMA. The antibacterial activity of (cross-linked) CECh, (quaternized) PDMAEMA, and their complexes against Escherichia coli has been evaluated. In contrast to (quaternized) PDMAEMA, (cross-linked) CECh exhibits no antibacterial activity. The complex formation between cross-linked CECh and (quaternized) PDMAEMA results in a loss of the inherent antibacterial activity of the latter in neutral medium. In acidic medium, the complexes exhibit strong antibacterial activity due to complex disintegration and release of (quaternized) PDMAEMA.     

Study of lipoprotein sorption by some sulfoderivatives of chitosan

The possibility of deposition of lipoproteins from blood plasma by different chitosan sulfoderivatives has been demonstrated. The influence of structure, substitution degree and molecular weight of chitosan sulfates on lipoprotein sorption was studied as well as the influence of composition of chitosan interpolymer complexes with dextransulfate. Affinic sorbents based on the silica matrixes and sulfoderivatives of chitosan as a ligand can reveal high specificity in relation to low density lipoprotein, but do not cause essential changes in blood count during contact with blood.     

Chitosan-cholesterol and chitosan-stearic acid interactions at the air-water interface

We report in this work the isotherms of cholesterol and stearic acid at the air-water interface modified by different chitosans (chitosan chloride, hydrophobic modified chitosan, and medium and high molecular weight chitosans) in the aqueous subphase. The Langmuir-Blodgett films of the complexes cholesterol-chitosan and stearic acid-chitosan are analyzed by atomic force microscopy (AFM), and a molecular simulation was performed to visualize the chitosan-lipid interactions. Strong modifications are obtained in the isotherms as a result of the chitosan interactions with cholesterol and stearic acid at the air-water interface. These modifications were dependent on the type and concentration of chitosan. Severe modifications of all phases were noticed with larger molecular areas, and the observed changes in the compressional modulus were dependent on the type of chitosan used. The complexes of chitosan-stearic acid were more flexible than the ones of chitosan-cholesterol. The AFM images demonstrated that chitosan was disaggregated by the cholesterol and stearic acid interactions producing more homogeneous surfaces in some cases. The hydrophobic chitosan showed more affinity with stearic acid, while both medium and high molecular weight chitosans produced homogeneous surfaces with cholesterol. The simulated chitosan chains interacting with cholesterol and stearic acid demonstrated the possibility of specific sites of electrostatic bonds between these molecules. Adsorption of cholesterol on the different powdered chitosans, performed by HPLC, showed that the medium and high molecular weight chitosans could retain higher proportions of cholesterol compared with the other analyzed samples.     

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.