Kjell M. Vårum

Chitosan as a nonviral gene delivery system. Structure–property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo

Chitosan is a natural cationic linear polymer that has recently emerged as an alternative nonviral gene delivery system. We have established the relationships between the structure and the properties of chitosan-pDNA polyplexes in vitro. Further, we have compared polyplexes of ultrapure chitosan (UPC) of preferred molecular structure with those of optimised polyethylenimine (PEI) polyplexes in vitro and after intratracheal administration to mice in vivo. Chitosans in which over two out of three monomer units carried a primary amino group formed stable colloidal polyplexes with pDNA. Optimized UPC and PEI polyplexes protected the pDNA from serum degradation to approximately the same degree, and they gave a comparable maximal transgene expression in 293 cells. In contrast to PEI, UPC was non toxic at escalating doses. After intratracheal administration, both polyplexes distributed to the mid-airways, where transgene expression was observed in virtually every epithelial cell, using a sensitive pLacZ reporter containing a translational enhancer element. However, the kinetics of gene expression differed – PEI polyplexes induced a more rapid onset of gene expression than UPC. This was attributed to a more rapid endosomal escape of the PEI polyplexes. Although this resulted in a more efficient gene expression with PEI polyplexes, UPC had an efficiency comparable to that of commonly used cationic lipids. In conclusion, this study provides insights into the use of chitosan as a gene delivery system. It emphasises that chitosan is a nontoxic alternative to other cationic polymers and it forms a platform for further studies of chitosan-based gene delivery systems.

Chitosans as absorption enhancers for poorly absorbable drugs. 1: Influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells.

AbstractPurpose. Chitosan has recently been demonstrated to effectively enhance the absorption of hydrophilic drugs such as peptides and proteins across nasal and intestinal epithelia (1–3). In this study, the effect of the chemical composition and molecular weight of chitosans on epithelial permeability and toxicity was investigated using monolayers of human intestinal epithelial Caco-2 cells as a model epithelium.nMethods. Eight chitosans varying in degree of acetylation (DA) and molecular weight were studied. The incompletely absorbed hydrophilic marker molecule 14C-mannitol was used as a model drug to assess absorption enhancement. Changes in intracellular dehydrogenase activity and cellular morphology were used to assess toxicity.nResults. Chitosans with a low DA (1 and 15%) were active as absorption enhancers at low and high molecular weights. However, these chitosans displayed a clear dose-dependent toxicity. Chitosans with DAs of 35 and 49% enhanced the transport of 14C-mannitol at high molecular weights only, with low toxicity. One chitosan (DA = 35%; MW = 170kD) was found to have especially advantageous properties such as an early onset of action, very low toxicity, and a flat dose-absorption enhancement response relationship.nConclusions. The structural features of chitosans determining absorption enhancement are not correlated with those determining toxicity, which makes it possible to select chitosans with maximal effect on absorption and minimal toxicity.

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy☆

The composition and sequence of 2-acetamido-2-deoxy-beta-D-glucose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogeneous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degree of acetylation of 50% were soluble at neutral pH.

Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers

Nonviral gene delivery systems based on conventional high-molecular-weight chitosans are efficient after lung administration in vivo, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow dissociation and release of plasmid DNA, resulting in a slow onset of action. We therefore developed highly effective nonviral gene delivery systems with improved physical properties from a series of chitosan oligomers, ranging in molecular weight from 1.2 to 10u2009kDa. First, we established structure–property relationships with regard to polyplex formation and in vivo efficiency after lung administration to mice. In a second step, we isolated chitosan oligomers from a preferred oligomer fraction to obtain fractions, ranging from 10 to 50-mers, of more homogeneous size distributions with polydispersities ranging from 1.01 to 1.09. Polyplexes based on chitosan oligomers dissociated more easily than those of a high-molecular-weight ultrapure chitosan (UPC, approximately a 1000-mer), and released pDNA in the presence of anionic heparin. The more easily dissociated polyplexes mediated a faster onset of action and gave a higher gene expression both in 293 cells in vitro and after lung administration in vivo as compared to the more stable UPC polyplexes. Already 24u2009h after intratracheal administration, a 120- to 260-fold higher luciferase gene expression was observed compared to UPC in the mouse lung in vivo. The gene expression in the lung was comparable to that of PEI (respective AUCs of 2756±710 and 3320±871u2009pg luciferase × days/mg of total lung protein). In conclusion, a major improvement of chitosan-mediated nonviral gene delivery to the lung was obtained by using polyplexes of well-defined chitosan oligomers. Polyplexes of oligomer fractions also had superior physicochemical properties to commonly used high-molecular-weight UPC.

Solution properties of chitosans: conformation and chain stiffness of chitosans with different degrees of N-acetylation

Chitosans with degrees of N-acetylation, FA, ranging from 0 to 0·6 were randomly degraded to different molecular weights and characterized by measuring intrinsic viscosities and osmotic pressure. In order to determine the stiffness parameter, B, and the Mark-Houwink-Kuhn-Sakurada (MHKS) equations at different ionic strengths, intrinsic viscosities were measured at various ionic strengths. The Kuhn lengths at θ-conditions, Amθ, were estimated from an interpolation utilizing the obtained stiffness parameters and a published relation between B and Amθ for different polyelectrolytes. These values were then used to compute the characteristic ratios, C∞, which are defined at θ-conditions. From the Flory-Fox viscosity relationship and a theory of Bloomfield and Zimm, the radii of gyration, RG, and also the Kuhn lengths at different ionic strengths, AmI, were calculated. In order to investigate the influence of the N-acetyl groups in contrast to the polyelectrolyte effect of the NH3+- group on the overall conformation, the intrinsic viscosity data were extrapolated to infinite ionic strength. and the resulting MHKS equations, the RG∞, and the Am∞ were estimated.

Water-solubility of partially N-acetylated chitosans as a function of pH: effect of chemical composition and depolymerisation

Abstract The solubility of four partially N-acetylated chitosans with fraction of acetylated units (FA) of 0.01, 0.17, 0.37 and 0.60 as a function of pH was investigated. The chitosan with FA=0.60 was soluble at all pH-values between 4 and 9. The solubility versus pH curve of the other chitosans, showed that all chitosans precipitated between pH 6 and 7.5, but with increasing solubility at higher pH-values with increasing FA. Such solubility differences may have profound effects on enzyme accessability and biological effects of chitosans. The three chitosans with the lowest FA values were depolymerised by treatment with nitrous acid, and the fraction of water-soluble material at pH 7.5 was determined. The almost fully deacetylated chitosan was completely insoluble at pH 7.5 in the depolymerisation range investigated, while the most acetylated chitosan (FA=0.60) was fully soluble at all pH-values. However, the two chitosans with FA=0.17 and 0.37 could be fractionated into a neutral-soluble and a neutral-insoluble fraction. The amount of neutral-soluble material increased with decreasing depolymerisation. The neutral-soluble and the neutral-insoluble fraction differed in both chemical composition and degree of polymerisation. Generally, the neutral-soluble fraction had a higher fraction of acetylated units and a lower degree of depolymerisation than the neutral-insoluble fraction. This compositional heterogeneity of the degraded chitosans was shown to be consistent with what is expected from the theoretical random degradation of chitosans with a Bernoullian (random) distribution of acetylated and deacetylated units.

Degradation of fully water-soluble, partially N-acetylated chitosans with lysozyme☆

Chitosans, prepared by homogeneous N-deacetylation of chitin, with degrees of N-acetylation ranging from 4 to 60% (FA = 0·04 to 0·60) exhibiting full water solubility and known random distribution of acetyl groups, were degraded with lysozyme. Initial degradation rates (r) were determined from plots of the viscosity decrease (Δ1/[η]) against time of degradation. The time course of degradation of chitosans with lysozyme were non-linear, while the time course of degradation of chitosans with an oxidative-reductive depolymerization reaction (using H2O2) showed the expected linear relationship for a first-order, random depolymerization reaction, independent of the chemical composition of the chitosan. n nThe effect of lysozyme concentration and substrate concentration on the initial degradation rates were determined, showing that this lysozyme-chitosan system obeys Michaelis-Menten kinetics. n nThe initial degradation rates of chitosan with lysozyme increased strongly with increasing fraction of acetylated units (FA). From a Michaelis-Menten analysis of the degradation data that assumes different catalytic activities of lysozyme for the different hexameric substrates in the polysaccharide chain, it is concluded that the hexameric substrates that contain three-four or more acetylated units contribute mostly to the initial degradation rate when lysozyme degrades partially N-acetylated chitosans. n nA chitosan with a very low fraction of acetylated units (FA = 0·010) was studied as an enzyme inhibitor. Initial degradation rates of chitosan (with different FA values) decreased as the inhibitor concentration increased, while the relative rates stayed constant, indicating that the ratio between initial reaction rates for productive sites (hexamers containing three-four or more N-acetylated units) are unaffected by non-productive sites, as deduced from the theory of competing substrates.

13C-N.m.r. studies of the acetylation sequences in partially N-deacetylated chitins (chitosans)

Chitosans obtained under homogeneous conditions of N-deacetylation, with degrees of N-deacetylation between 46% and 94%, were depolymerised and their 125-MHz 13C-n.m.r. spectra have been interpreted. The sequence of 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN) residues influenced the chemical shifts, and the diad and triad frequencies have been calculated. Chitosans that were N-deacetylated under homogeneous and heterogeneous conditions gave values for the diad and triad frequencies that were consistent with a random arrangement of GlcN and GlcNAc residues.

Compositional heterogeneity of heterogeneously deacetylated chitosans

Abstract Commercial chitosans prepared by heterogeneous alkaline deacetylation with different FA values were fractionated into acid-soluble and acid-insoluble fractions. The amount of acid-soluble fractions increased with increasing time of deacetylation. The acid-soluble fractions were characterized with respect to fraction of acetylated units (FA), diad frequencies, intrinsic viscosities, molecular weights and molecular weight distributions. FA values for the acid-insoluble fractions were obtained using CPMAS 13C NMR spectroscopy. As a control, the FA values for the acid-soluble fractions determined from CPMAS 13C NMR spectroscopy were compared and found to be consistent with the FA values from 1H NMR spectroscopy. The diad frequencies indicated that the acetyl groups in the acid-soluble fractions of the commercially heterogeneously deacetylated chitosans were randomly distributed. Determination of [η], Mw and Mw/Mn indicated negligible depolymerization of the acid-soluble fractions when deacetylation was performed under a nitrogen purge at 75 °C. Chemical characterization revealed compositional heterogeneity in the chitosans, with chitin-like acid-insoluble fractions with FA values between 0.88 and 0.95, and acid-soluble fractions with FA values from 0.20 to 0.52.

Aggregates in acidic solutions of chitosans detected by static laser light scattering

Abstract Chitosans having degrees of N -acetylation, F A , ranging from 0 to 0·6, were randomly degraded to different molecular weights and studied by multi angle static laser light scattering (LLS). Under the given experimental conditions, negative second virial coefficients of the solutions, A ′ 2 , revealed the presence of concentration dependent aggregates. Attempts to remove the aggregates, or to influence the aggregation behavior, were made by ultracentrifugation and extensive filtering of the solutions. Modification of the solvent conditions such as pH, ionic strength and temperature were carried out, and chitosan solutions were digested with an acidic proteinase. Non-degraded samples and chitosans prepared by both heterogeneous and homogeneous N -deacetylation of chitin were also studied. In all cases, the negative A ′ 2 remained. However, it was observed that ultracentrifugation and filtering of the solutions decreased the measured molecular weights and radii of gyration, indicating that some of the material of high molecular weight and size could be removed by ultracentrifugation and filtration. The chemical nature of the physical basis of the molecular association was not revealed. Nevertheless, by the use of gel permeation chromatography coupled to an on-line low angle laser light scattering instrument and a differential refractive index concentration detector (HPSEC-LALLS-RI), a bimodal molecular weight distribution was observed in which about 5% of the sample had a very high molecular weight. These results coupled with the positive virial coefficients obtained earlier from osmotic pressure measurements suggest that a small fraction of the chitosan is aggregated to high molecular weight material, probably following a closed association model. Electron microscopy revealed the presence of some supramolecular structures. The positive second virial coefficients obtained earlier from osmometry are in harmony with these findings. The results demonstrate the occurrence of reversible aggregation in chitosan solutions. Static laser light scattering therefore cannot readily be used to determine molecular weights and sizes of chitosans under these conditions. It was not possible to correlate the extent of aggregation with the chemical composition of the chitosans.