Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

Procedure for C2 deuteration of nucleic acids and determination of A psi 31 pseudouridine conformation by nuclear Overhauser effect in yeast tRNAPhe

Nuclear Overhauser effect (NOE) combined with semispecific deuteration provides a general strategy for identification of exchangeable protons in nucleic base pairs, and has been extended to NOEs involving purine C2 protons in tRNA. Deuterated tri-ethyl orthoformate was condensed with 5(4)-amino imidazole 4(5)-carboxamide to yield C2 deuterated hypoxanthine. C2 deuterated hypoxanthine was fed to a purine requiring mutant of yeast and C2 deuterated yeast tRNAPhe was isolated. This C2 deuterated tRNAPhe was used to identify A psi 31 and U8-A14. A psi 31 was found to be bonded through N1H. The utility of C2 deuteration in nucleic acid NMR is thus demonstrated.     

Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases

Hydrogen/deuterium exchange coupled to mass spectrometry has been used to investigate the structure and dynamics of native dimeric cytosolic muscle creatine kinase. The protein was incubated in D₂O for various time. After H/D exchange and rapid quenching of the reaction, the partially deuterated protein was cleaved in parallel by two different proteases (pepsin or type XIII protease from Aspergillus saitoi) to increase the sequence coverage and spatial resolution of deuterium incorporation. The resulting peptides were analyzed by liquid chromatography coupled to mass spectrometry. In comparison with the 3D structure of MM-CK, the analysis of the two independent proteolysis deuteration patterns allowed us to get new insights into CK local dynamics as compared to a previous study using pepsin [Mazon et al. Protein Science 13 (2004) 476-486]. In particular, we obtained more information on the kinetics and extent of deuterium exchange in the N- and C-terminal extremities represented by the 1-22 and 362-380 pepsin peptides. Indeed, we observed a very different behaviour of the 1-12 and 13-22 type XIII protease peptides, and similarly for the 362-373 and 374-380 peptides. Moreover, comparison of the deuteration patterns of type XIII protease segments of the large 90-126 pepsin peptide led us to identify a small relatively dynamic region (108-114).     

Photoluminescent porous alginate hybrid materials containing lanthanide ions

The photoluminescence features of Eu(3+)-, Tb(3+)-, Tb(3+)/Eu(3+)-alginate aerogel (hydrogel and alcogel) and Eu(3+)-alginate xerogel hybrids were investigated. The Eu(3+)-alginate aerogel and alcogel exhibit the highest (5)D0 quantum efficiencies (9.9 and 8.2%, respectively), while the hydrogel and xerogel have lower values (5.2 and 5.6%, respectively). The Tb(3+)/Eu(3+) hybrids are multiwavelength emitters in which the emission color can be tuned across the chromaticity diagram from the red toward the yellowish-green spectral regions, crossing the white area by selecting the excitation wavelength.     

4-vinyl and 2,4-divinyl deuteration effects on the low frequency resonance Raman bands of myoglobin: correlation with the structure of vinyl group

Resonance Raman spectra of myoglobin (Mb) reconstituted with 4-vinyl and 2,4-divinyl deuterated protoheme IX were studied in several oxidation, spin, and ligation states (deoxyMb, MbO2, MbCO, metMbH2O, and metMbCN-) with special attention to the low frequency vibrations. Frequency shifts observed on deuteration enabled us to assign some Raman bands to vibrations specifically involving the 2- or 4-vinyl group. The most significant deuteration effect was found for a band around 410 cm-1 in the ferrous state, which loses intensity on 4-vinyl deuteration and is ascribed to a porphyrin in-plane vibration strongly coupled with the skeletal bend of the vinyl group at position 4. Such strong coupling implies that the vinyl group lies in the same plane as the pyrrole II ring, as in the crystalline state. Thus, frequency shifts on vinyl deuteration may be useful as a probe of the orientation of the vinyl group. Resonance Raman spectra of Mb coordinated with various sizes of isocyanides are interpreted in terms of vinyl orientational changes induced by ligation.     

Ab initio Hartree-Fock/6-31G** calculation on 9-beta-D-arabinofuranosyladenine-5'-monophosphate molecule: application to the analysis of its IR and Raman spectra

9-beta-D-arabinofuranosyaldenine-5'-monophosphate (5'-ara-AMP) is an arabinonucleotide that has antiviral and antitumor activity. The accurate knowledge of the nature of its vibrational modes is a valuable step for the forthcoming elucidation of drug-nucleotide and drug-enzyme interactions. The FTIR and FT Raman spectra (4000-30 cm(-1)) of 5'ara-AMP and two deuterated derivatives ara-AMP-d(C8) (deuteration in C8) and ara-AMP-d7 (deuteration in C8, amino and hydroxyl groups) are reported. Theoretical vibrational calculations were performed using the Hartree-Fock/6-31G** method. An assignment of the observed spectra is proposed considering the scaled potential energy distribution of the vibrational modes of the 5'ara-AMP molecule and the observed band shifts by deuteration. The scaled ab initio frequencies are in good agreement with the experimental data (<3 cm(-1) SD).     

NMR crystallography: the effect of deuteration on high resolution 13C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by <0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the C delta, C gamma 1, and C gamma 2 Ile 13C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured 13C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. 1) decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power 1H decoupling is required.     

Preparation and properties of highly soluble chitosan–l-glutamic acid aerogel derivative

The objective of this work is to improve the solubility of chitosan at neutral or basic pH using the supercritical carbon dioxide (sc.CO₂). A novel water-soluble chitosan–l-glutamic acid (Cl-GA) aerogel derivative was synthesized by reaction of 85% deacetylated chitosan with l-glutamic acid (l-GA) in aq.AcOH subjected to solvent exchange prior to using sc.CO₂ as a nonsolvent for the polymer. The prepared aerogel derivative and molecular conformation of modified chitosan are characterized by using UV, FTIR, ¹H NMR, and CD techniques. Some physical properties and surface morphology were analyzed by X-ray diffraction, differential scanning calorimetry (DSC), thermogravimetry (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and porosimetry analysis. Overall, the sc.CO₂ assisted chitosan aerogel derivative opens new perspectives in biomedical applications.     

Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies

Deuterium isotope labelling is important for NMR studies of large proteins and complexes. Many eukaryotic proteins are difficult to express in bacteria, but can be efficiently produced in the methylotrophic yeast Pichia pastoris. In order to facilitate NMR studies of the malaria parasite merozoite surface protein-1 (MSP1) complex and its interactions with antibodies, we have investigated production of the MSP1-19 protein in P. pastoris grown in deuterated media. The resulting deuteration patterns were analyzed by NMR and mass spectrometry. We have compared growth characteristics and levels of heterologous protein expression in cells adapted to growth in deuterated media (95% D₂O), compared with expression in non-adapted cells. We have also compared the relative deuteration levels and the distribution pattern of residual protiation in protein from cells grown either in 95% D₂O medium with protiated methanol as carbon source, or in 95% D₂O medium containing deuterated methanol. A high level of uniform C deuteration was demonstrated, and the consequent reduction of backbone amide signal linewidths in [¹H/¹⁵N]-correlation experiments was measured. Residual protiation at different positions in various amino acid residues, including the distribution of methyl isotopomers, was also investigated. The deuteration procedures examined here should facilitate economical expression of ²H/¹³C/¹⁵N-labelled protein samples for NMR studies of the structure and interactions of large proteins and protein complexes.     

Clemmensen reduction of diosgenin and kryptogenin: synthesis of [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol

A new experimental protocol has been established for the Clemmensen reduction of diosgenin and kryptogenin with the aim to prepare deuterated isotopomers of (25R)-26-hydroxycholesterol. Uncontrolled deuteration has been achieved from diosgenin, whereas [16,16,22,22,23,23-(2)H(6)]-(25R)-26-hydroxycholesterol (1) can be synthesized from kryptogenin.     

Low frequency resonance Raman spectra of isolated alpha and beta subunits of hemoglobin and their deuterated analogues

In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm(-1), are reported for the deoxy and ligated (CO and O2) forms of the isolated alpha and beta subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3-protoheme (d12- protoheme), the 1,3-C2H3-protoheme (1,3-d6-protoheme), the 5,8-C2H3-protoheme (5,8-d6-protoheme), and the meso-C2H4-protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm(-1), revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6- and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups.     

Immobilization of lipase on methyl-modified silica aerogels by physical adsorption

In this work, methyl-modified silica aerogels, a new kind of macro-porous material with high porosity, were used as carriers to immobilize lipase by adsorption. SEM, TEM, nitrogen adsorption device, and thermogravimetric analysis were used to characterize the properties of modified aerogels. The surface area was 395.6 m(2)/g, and the average pore diameter was 68.72 nm. The contact angle of aerogel particles increased from 20.9 degrees to 99.2 degrees after methyl modification. Reaction characteristics of the material after enzyme loading were also discussed. The results showed that adsorption capacity could reach 67.42 mg/g; and the maximal enzyme activity was 19.87 micromol min(-1)mg(-1) (protein), and activity retention could reach 56.44%. It is worth mentioning that the amount of modified aerogels added had significant effects on the diameter of droplets and the mass transfer behavior of substrates in the reaction emulsion. Online microscope was used to visualize the droplets in the emulsion, where the aerogel particles were observed locating at the interface of oil and water. The average diameter of droplets reached the minimum when 0.06 g of modified aerogels was added into the reaction emulsion which contained 10 ml of oil and 10 ml of phosphate buffer solution. The phenomenon was resulted from the wettability of methyl-modified silica aerogels.     

Deuteration effects on the in vivo EPR spectrum of the reduced secondary photosystem I electron acceptor A1 in cyanobacteria

The photoreduction of the secondary PSI electron acceptor A1 in vivo has recently been detected via X-band EPR spectroscopy in intact spinach chloroplasts and in marine cyanobacteria Synechococcus PCC 7002 [Klughammer, C., and Pace, R. J. (1997) Biochim. Biophys. Acta 1318, 133-144]. A further study of the A1- EPR spectrum of Synechococcus PCC 7002 at room temperature with higher-field resolution revealed partially resolved hyperfine structure which was dominated by 0.4 mT splittings of three equivalent protons. The hyperfine splitting was not significantly affected by incubation of the cyanobacteria in 2H2O medium for 20 h, but was absent in fully deuterated cyanobacteria that were grown in 2H2O medium. Anisotropic g-factors consistent with a phylloquinone radical were derived by spectra simulation. Biosynthetic protonation of quinones via the CH3 donor L-methionine in deuterated cells maintained hyperfine structure in the A1- spectrum, indicating the incorporation of CH3 groups in 60% of the deuterated, photoactive A1 molecules. Conversely, biosynthetic quinone deuteration via L-[methyl-d3]methionine in protonated cells led to the loss of the 0. 4 mT splittings in 54% of the A1 molecules. These observations confirm the conclusion of Heathcote et al. [(1996) Biochemistry 35, 6644-6650] of the identity of EPR-detected, photoreduced A1- in vivo with a phylloquinone (vitamin K1) radical in PSI. The partially resolved hyperfine structure of the A1- spectrum indicates an altered spin distribution in the bound vitamin K1- radical in vivo compared to that of unbound vitamin K1- in vitro.     

Deuterium labeling of diethylstilbestrol and analogues

E-2,2,3',3″,5,5,5',5″-octadeuteriodiethylstilbestrol (DES-d8) and Z-2,3',3″,4,5,5,5',5″-octadeuterio-3,4-bis(p-hydroxyphenyl)-2-hexene (ψ-DES-d8) were synthesized from E-diethylstilbestrol (DES) by hydrogen/ deuterium exchange in a mixture of methanol-d and deuterium chloride in deuterium oxide. The structures, isotopic purity, and positions of up-take of deuterium were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Additional confirmation of the positions of deuterium exchange in stilbestrols was obtained from an analysis of the oxidation of DES-d8 to Z,Z-2,3',3″,5,5',5″-hexadeuteriodienestrol (β-DIES-d6) and of the hydrogen/deuterium exchange reaction of hexestrol (HEX) to 3',3″,5',5″-hexestrol (HEX-d4). Structural analysis and the determination of isotopic purity of the latter two compounds were also carried out by NMR and MS. The uptake of eight deuterium atoms by DES is postulated to proceed via two different reactions occurring simultaneously: 1. acid catalyzed deuteration of all four phenolic ortho-positions (3',3″,5',5″); 2. acid catalyzed deuteration of the olefin bridge with subsequent formation of deuterated ψ-DES (3 or 4). Due to the equilibration between DES, ψ-DES, and Z-diethylstilbestrol (cis-DES) in the acidic reaction mixture at 85°C, the deuterated ψ-DES is thought to rapidly rearrange to deuterated DES. Repeated deuteration will eventually form DES-d8 fully labeled in the 2,2,5,5 methylene positions.     

Infrared absorption spectrum of keratin. II. Deuteration studies

A number of new bands have been found in the spectra of deuterated α- and β-keratin. In particular, the deuteration difference spectrum has been useful for the determination of frequencies of previously unsuspected bands. Thus it is found that the amide A and II frequencies of the nonhelical component in α-keratin occur at 3310–3320 and 1520 cm.^?1, respectively, and that both bands exhibit dichroism consistent with polypeptide chains which have a measure of alignment parallel to the fiber axis. The parallel dichroism of the amide II′ band of this phase at about 1435 cm.^?l also indicates some alignment. A nondichroic residual band at 1513 cm.^?1 in highly deuterated α-keratin is assigned to the tyrosine residue, as a sharp band near this frequency is found in the spectrum of polytyrosine. The ν‖(o) component of the α-helix is weak or absent in α-keratin, and the relatively sharp band observed near this frequency is thought to be due to the tyrosine residue, while its dichroism is caused by the presence of dichroic nonhelical material. A band near 1575 cm.^?1 in deuterated α- and β-keratin is tentatively assigned to the deuterated guanidinium group of arginine. This band becomes progressively more prominent during deuteration, which indicates that some arginine side chains arc slow to exchange, possibly because their environment prevents interaction with D₂O. The deuteration difference spectrum also shows that, contrary to earlier views, helical material in α-keratin exchanges significantly during the early stages of deuteration, although at a slower rate than the nonhelical material, while part of the nonhelical phase does not exchange as rapidly as had been thought and makes a contribution even after many hours or days.     

NMR crystallography: The effect of deuteration on high resolution C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the ¹³C linewidths of U-¹³C, ¹⁵N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in ²H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by < 0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ₁, and Cγ₂ Ile ¹³C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured ¹³C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. ¹H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power ¹H decoupling is required.     

Structural study of rhodopsin in detergent micelles by small-angle neutron scattering

Monodisperse solutions of bovine rhodopsin monomers, devoid of lipid, associated with a linear polyoxyethylene alcohol detergent have been prepared. The composition and homogeneity of these complexes have been determined by hydrodynamic characterisation. Each rhodopsin molecule is associated with about 110 monomers of the detergent. These rhodopsin-detergent complexes have been studied by small-angle neutron scattering. Partial or total deuteration of the detergent, as well as variation of the ²H₂O/H₂O ratio in the solvent, were used to eliminate the detergent—solvent contrast at various protein—solvent contrasts. The size and shape of the detergent micelle and of the rhodopsin-detergent complexes were shown to be independent of solvent or detergent deuteration. Mixture of selectively deuterated detergent molecules allowed us to obtain an homogeneous scattering density for the detergent part of the micelles and therefore to eliminate totally its contribution to the scattering when it is contrast matched. Neutron scattering from rhodopsin alone was then measured even in highly deuterated solvents, with low incoherent background, as for a water-soluble protein. Supplementary neutron scattering measurements on rhodopsin-dodecyl dimethylamine oxide micelles confirmed essentially the results reported by Yeager (1975). Analysis of the neutron scattering data indicates that most of the hydrophobic residues of rhodopsin form a compact region which has zero hydration, this probably being the part which is embedded in the disc membrane, and that the unhydrated rhodopsin molecule is asymmetrically arranged with respect to the membrane. Comparison with the results of a small-angle X-ray scattering study (Sardet et al., 1976) implies that the peripheral regions on both sides of the membrane are highly hydrated. Several schematic models are discussed.     

Biosynthesis and characterization of a series of deuterated cis,cis-octadeca-6,9-dienoic acids

[4,4-2H2]-, [5,5-2H2)-, [6-2H]-, [7-2H]-, [8,8-2H2)-, [11,11-2H2]-, [14,14-2H2]- and [18,18,18-2H3]-cis,cis-octadeca-6,9-dienoic (isolinoleic) acid were synthesized by supplementing cultures of the protozoan Tetrahymena with the corresponding deuterated cis-octadeca-9-enoic (oleic) acids. The cultures were harvested, the deuterated isolinoleic acids isolated and analyzed for purity by GC and TLC, and the structure and the level and position of deuteration of each fatty acid determined by 13C-NMR spectroscopy. The 13C resonances of all 18 carbons were also assigned based upon alpha-carbon deuterium isotope shifts and by comparison of the spectra to those of other polyunsaturated fatty acids. The results illustrate the utility of a biological approach for the synthesis of deuterated polyunsaturated fatty acids in yields suitable for 2H-NMR studies of membranes and possibly human metabolism.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler¹ in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m²/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO₂ conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO₂ drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm³), high specific surface area (350 - 500 m²/g) and pore volume (3 - 7 cm³/g for pore sizes less than 150 nm).