Cryogel Matrix Research Articles

Development of encapsulation technique for curcumin loaded O/W emulsion using chitosan based cryotropic gelation

Cryogel based encapsulation of curcumin, an herbal extract, was successfully carried out with a ternary system of colloidal chitosan, κ-carrageenan, and carboxymethylcellulose sodium salt. The effects of chitosan concentration, κ-carrageenan/CMC ratio of the polymer suspension and molecular weight of chitosan on the sol–gel formation were investigated. The effects of cooling rate during freeze-drying and oil phase composition on the encapsulation yield and the release behavior of curcumin from the hydrogel were determined. And so were the effects of pH of the phosphate-buffered media and oil phase composition on the swelling of the specimens. The microstructure of the resulting specimens revealed core-shell nanoparticles (i.e. oil droplet for core and cryogel membrane for shell) entrapped in the cryogel matrix. The encapsulation yield for two types of suspensions was in a range of 83.9 to 99.6% when a high-MW chitosan was used. Controlled release of the encapsulated curcumin in an aqueous system could be maintained for 4days, and the releasable amount of curcumin was in a range of 41.1 to 59.9%. The encapsulation yield as well as the released pattern and releasable amount of curcumin were significantly influenced by the cooling protocol used during freezing. Irrespective of the introduced oil phase composition, controlled release of curcumin was achievable when the cooling rate was sufficiently high at −2.0°C/min and, interestingly, either a burst release or a first order release could simply be achieved by changing the freezing condition.

Concanavalin A immobilized poly(ethylene glycol dimethacrylate) based affinity cryogel matrix and usability of invertase immobilization

Concanavalin A (Con A) immobilized supermacroporous poly(ethylene glycol dimethacrylate) [poly(EGDMA)] monolithic cryogel column was prepared by radical cryocopolymerization of EGDMA as a monomer and N,N′-methylene-bisacrylamide as a crosslinker. Bioligand Con A was then immobilized by covalent binding onto poly(EGDMA) cryogel via glutaraldehyde activation [Con A–poly(EGDMA)]. Con A–poly(EGDMA) cryogel was characterized by swelling studies and scanning electron microscopy. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50μm size. The equilibrium swelling degree of the cryogel was 15.01g H2O/g dry cryogel. Con A–poly(EGDMA) cryogel was used in the adsorption/desorption of invertase from aqueous solutions. The maximum amount of invertase adsorption from aqueous solution in acetate buffer was 55.45mg/g polymer at pH 5.0. Con A–poly(EGDMA) cryogels were used for repetitive adsorption/desorption of invertase without noticeable loss in invertase adsorption capacity after 10 cycles.

An improved capillary model for describing the microstructure characteristics, fluid hydrodynamics and breakthrough performance of proteins in cryogel beds

A capillary-based model modified for characterization of monolithic cryogels is presented with key parameters like the pore size distribution, the tortuosity and the skeleton thickness employed for describing the porous structure characteristics of a cryogel matrix. Laminar flow, liquid dispersion and mass transfer in each capillary are considered and the model is solved numerically by the finite difference method. As examples, two poly(hydroxyethyl methacrylate) (pHEMA) based cryogel beds have been prepared by radical cryo-copolymerization of monomers and used to test the model. The axial dispersion behaviors, the pressure drop vs. flow rate performance as well as the non-adsorption breakthrough curves of different proteins, i.e., lysozyme, bovine serum albumin (BSA) and concanavalin A (Con A), at various flow velocities in the cryogel beds are measured experimentally. The lumped parameters in the model are determined by matching the model prediction with the experimental data. The results showed that for a given cryogel column, by using the model based on the physical properties of the cryogel (i.e., diameter, length, porosity, and permeability) together with the protein breakthrough curves one can obtain a reasonable estimate and detailed characterization of the porous structure properties of cryogel matrix, particularly regarding the number of capillaries, the capillary tortuousness, the pore size distribution and the skeleton thickness. The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of proteins at different flow velocities. It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance under a given set of operating conditions.

Encapsulation of Curcumin Loaded Oil Droplets By Cryotropic Gel Formation from O/W Emulsion

Cryogel based encapsulation was attempted to entrap oil phase (containing curcumin) with a ternary system of colloidal chitosan, κ-carrageenan, and carboxy methylcellulose sodium salt (NaCMC). The cryotropic gel formation was investigated by varying the cooling rate during freezing and type of polymer suspension. The microstructure of the resulting curcumin cryogels revealed oil droplets entrapped in the cryogel matrix. The encapsulation yield for two types of suspension was found to vary from 83.89 to 99.37%. Controlled release of the curcumin in an aqueous system could be maintained for 4 days, and the released amount of curcumin was found to vary from 41.1-59.9%. The encapsulation yield as well as the released pattern and amount of curcumin were influenced by the cooling protocol used during freezing. The release patterns were found to be sensitive to the ambient aqueous pH and, interestingly, either a burst release or a first order release was achievable simply by changing the freezing condition. These results suggested that freezing could modify the gel formation of the present cryogel, and the resulting structural modification evidently controlled the oil encapsulation manner. The present ternary system (chitosan, κ- carrageenan, and NaCMC) is an interesting matrix for designing controlled release system in a food system.

Cryotropic Gel Formation for Food Nutrients Encapsulation - a Controllable Processing of Hydrogel By Freezing

Considering severe security regulations for food and pharmaceutical ingredients, it would be ideal to make various types of encapsulants from a widely approved single substance. Use of cryogels for encapsulation would be an interesting idea. Cryogel is defined as a hydrogel where sol-gel transitions are induced by localized concentration increase of the substrate due to ice formation during freezing. It would thus be expected that gel network formations can be controlled by a freezing process, and the controlled polymeric structures are advantageous for release control. In this work, chitosan based cryogels were prepared for investigating encapsulation characteristics of a model ingredient. It was found that the release behaviour of the ingredient from the prepared cryogel matrix could be controlled by the freezing condition for the gel preparation, indicating that a freezing operation controlled the gel network formation that determined consequent release characteristics from the hydrogel matrix. The differences of gel network structures formed via the different freezing conditions were illuminated by SAXS analysis. A clear structural anisotropy appeared when the chitosan suspension was rapidly frozen. On the other hand, this strong anisotropy disappeared when the cooling rate was decreased. It is a clear evidence of polymeric structural modifications appeared in cryogels prepared via different cryo-processing. This cryogel based encapsulation would be a potential technique for realizing a structural design of the encapsulants simply by tuning the freezing conditions.

Preparation of Supermacroporous Composite Cryogel Embedded with SiO 2 Nanoparticles

Supermacroporous composite cryogels embedded with SiO 2 nanoparticles were prepared by radical cryogenic copolymerization of the reactive monomer mixture of acrylamide (AAm) and N, N-methylene-bis-acrylamide (MBAAm) containing SiO 2 nanoparticles (mass ratios of nanoparticles to the monomer AAm from 0.01 to 0.08) under the freezing-temperature variation condition in glass columns. The properties of these composite cryogels were measured. The height equivalent to theoretical plate (HETP) of the cryogel beds at different liquid flow rates was determined by residence time distribution (RTD) using tracer pulse-response method. The composite cryogel matrix embedded with the mass fraction of SiO 2 nanoparticles of 0.02 presented the best properties and was employed in the following graft polymerization. Chromatographic process of lysozyme in the composite cryogel grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) was carried out to evaluate the protein breakthrough and elution characteristics. The chromatography can be carried out at relatively high superficial velocity, i.e., 15 cm·min −1, indicating the satisfactory mechanical strength due to the embedded nanoparticles.

Proliferation of Myoblast Skeletal Cells on Three-Dimensional Supermacroporous Cryogels

Cardiac and skeletal muscle tissue engineering provides a smart approach to overcome problems associated with organ transplantation and cardiac tissue and also lays a platform for superior alternative approaches in muscle regeneration. The aim of the study was to demonstrate cryogel scaffold potential in the field of skeletal muscle and cardiac tissue engineering. Poly-hydroxyethyl methacrylate (pHEMA)-gelatin cryogel scaffold was synthesized using cryogelation technique and such a designed material is being reported first time. Rheology study of the pHEMA-gelatin (HG) suggested that the cryogel scaffolds were stable at different temperatures and phase angle remained constant in both dry and wet state. HG cryogel was able to bear increased stress without leading to deformation. Monitoring the hydration of HG scaffold showed shift from a stiff to a more pliable material and upon continuing hydration, shear modulus remained constant with no further change observed. However, the change in phase angle <0.24 masculine indicates a gradual increase in stiffness of the material over time. Scaffold synthesised using such polymer combinations gave cells a native environment for proliferation and surface stiffness have shown to help in differentiation of the cells. Myoskeletal cell lines were cultured on these scaffolds to check the biocompatibility and cell proliferation. Alamar blue assay performed over a period of 3 weeks analysed the metabolic activity of cells which showed more than 60% increase in the total cellular activity. DNA content of cells was found to be directly related to number of cells present at a given time point and this was found to have increased by more than 50% in 3 weeks. Since in 3-D scaffold the surface area is more in comparison to 2-D, hence better cell proliferation is observed. Hoechst and DAPI staining showed tubular structure and alignment of the cells during formation of the tubules shows promising cellular response to the cryogel matrix. The mechanical strength, stiffness and elastic measurements of the scaffold indicated potential application of these materials for skeletal and cardiac tissue engineering.

Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange

Extraction and purification of high-value minor proteins directly from milk without pre-treatment is a challenge for the dairy industry. Pre-treatment of milk before extraction of proteins by conventional packed-bed chromatography is usually necessary to prevent column blockage but it requires several steps that result in significant loss of yield and activity for many minor proteins. In this paper, we demonstrate that it is possible to pass 40-50 column volumes of various milk samples (raw whole milk, homogenized milk, skim milk and acid whey) through a 5 mL cryogel chromatographic column at 550 cm/h without exceeding its pressure limits if the processing temperature is maintained above 35 degrees C. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg/mL) was similar to that of the binding capacity (2.01 mg/mL) at equilibrium with 0.1 mg/mL of lactoferrin in the feed samples. The cryogel column selectively binds lactoferrin and lactoperoxidase with only minor leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of over 85% and a purity of more than 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed-bed chromatography for direct capture of proteins from milk.

One-step isolation of adenosine triphosphate from crude fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel

Adenosine triphosphate (ATP) is an important high-energy compound widely used in biological and therapeutic fields. It can be produced by phosphorylation of adenosine monophosphate (AMP) with microbial cells in industrial scale and the effective isolation of ATP from microbial fermentation broth is a challenging work. In this work, we develop a novel one-step method to directly separate ATP from fermentation broth of Saccharomyces cerevisiae by anion-exchange chromatography using supermacroporous cryogel. The cryogel bed with tertiary amine groups was prepared by grafting N, N-dimethylaminoethyl methacrylate (DMAEMA) monomer chains onto the matrix of a polyacrylamide-based cryogel in a glass column and its properties of liquid dispersion, water permeability, porosity as well as the ligand density were measured. Chromatographic separation of ATP from the fermentation broth by the cryogel was carried out using deionised water and 0.01 M HCl as running buffer, respectively. The breakthrough characteristics and elution performance in the cryogel bed were revealed and analyzed. The purities of the obtained ATP were analyzed quantitatively by high performance liquid chromatography (HPLC). The maximal purity of ATP by the one-step separation method was 95.5% using 0.01 M HCl as running buffer in this work. The corresponding chromatographic behaviors were investigated and analyzed.

Monoclonal Antibody Production Using a New Supermacroporous Cryogel Bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.

Ion‐exchange macroporous hydrophilic gel monolith with grafted polymer brushes

The grafting of functional polymers to the pore surface of macroporous monolithic polyacrylamide cryogels was found to be an efficient and convenient method for the preparation of macroporous polyacrylamide gels, so-called cryogels (pAAm cryogels), with both controlled extent of functional group incorporated and with tailored surface chemistries. Anion-exchange polymer chains of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA) and poly([2-(methacryloyloxy)ethyl]-trimethylammonium chloride) (pMETA), and cation-exchange polymer chains of polyacrylate have been grafted onto pAAm cryogels using potassium diperiodatocuprate as initiator. It was possible to achieve the ion-exchange capacity up to 0.2-0.5 mmol/ml. The graft polymerization did not alter the macroporous structure of the pAAm cryogel, however the flow rate of solutes through the cryogel matrix decreased with increase in the density of polymer grafted. The sorption of low-molecular-weight (metal ion, dye) and high-molecular-weight (protein) substances on the grafted monolithic pAAm column has been studied. The results indicate that a 'tentacle'-type binding of protein to grafted polymer depended on the architecture of the grafted polymer layer and took place after a certain degree of grafting has been reached. The binding of proteins by tentacle-like polymer chains allowed for increasing the binding capacity for proteins on the grafted pAAm cryogels up to 6-12 mg/ml.

Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices

An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days.

Supermacroporous cryogel matrix for integrated protein isolation: Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.

Affinity fractionation of lymphocytes using a monolithic cryogel

A new type of continuous, supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing specific fractionation and separation of human peripheral blood lymphocytes in a chromatographic format. The affinity adsorbent was used to design a novel cell separation strategy, which was based on the interaction of protein A from Staphylococcus aureus with cells bearing IgG antibodies on the surface. After treating lymphocytes with goat anti-human IgG(H+L), the IgG-positive B-lymphocytes were efficiently separated from T-lymphocytes. Protein A covalently coupled to epoxy activated dimethylacrylamide (DMAA) cryogel matrix specifically bound IgG-bearing B-lymphocytes through the Fc region, while non-bound T-lymphocytes passed through the column. More than 90% of the B-lymphocytes were retained in the column while the cells in the breakthrough fraction were enriched in T-lymphocytes (81%). The viability of the T-lymphocytes isolated was greater than 90%. The bound lymphocytes released by human or dog IgG recovered 60–70% of the B-cells without significantly impairing the cell viability. The technique can be applied in general to cell separation systems where IgG antibodies against specific cell surface markers are available.

Compartmentation in the poly(vinyl alcohol) cryogels. 1H NMR self-diffusion study

The self-diffusion of the poly(vinyl alcohol) (PVA) cryogels prepared by freezing–thawing the aqueous and water/DMSO solutions of PVA was studied with the NMR FT-PGSE method. The temperature dependencies of the self-diffusion coefficients, D s, for the PVA chains have a maximum at 45°C due to the syneresis of cryogels. They are quite different from the monotonous increase of D s for the aqueous solutions of PVA. Evaluated apparent activation energies, E a, of the self-diffusion for the PVA chains in the PVA solutions and cryogels in D 2O are practically the same and equal 22–24 kJ mol −1 below the crucial point. The proton spin-lattice relaxation times, T 1, of the PVA chain also coincide with one another for solutions and cryogels. It means that molecular packing in cryogels depends mainly on the dimensions of the ice and polymer microcrystallites formed by freezing the solution. Above the crucial point polymer compartments become firmer, and the chain mobility somewhat reduces. The strength of cryogels also increases along with growing the DMSO contents and decreases by the BSA addition. For an estimation of the cryogel morphology, effects of the restricted diffusion of both the water and PVA in a q-space have been taken into account. By introducing DMSO to cryogels, the solvent filled pores become smaller, and channels become much shorter. The diameter of the PVA filaments is similar to those for all the cryogels, but the length of filaments with D 2O is twice those for cryogels with a mixed solvent. Entrapment of BSA in the cryogel matrix by preparation leads to the increase of an average diameter of the water filled pores, and destroys molecular packing the cryogel.

1H NMR Self-Diffusion Study of Morphology and Structure of Polyvinyl Alcohol Cryogels

The multicomponent self-diffusion of the polyvinyl alcohol (PVA) cryogels prepared by a freezing–thawing treatment of aqueous and water–DMSO solutions of PVA has been studied with the NMR FT-PGSE method. The temperature dependencies of the self-diffusion coefficients,Ds, for the PVA chains have a maximum at 45°C due to the syneresis of cryogels. They are quite different from the monotonous increase ofDsfor the aqueous solutions of PVA. Evaluated apparent activation energies,Ea, of the self-diffusion for the PVA chains in the PVA solutions and cryogels in D2O are practically the same and equal 22–24 kJ/mol below the crucial point. The proton spin–lattice relaxation times,T1, of the PVA chain also coincide with one another for solutions and cryogels. This means that molecular packing in cryogels depends mainly on the dimensions of the ice and polymer microcrystallites formed by freezing the solution. Above the crucial point polymer compartments become firmer, and the chain mobility somewhat reduces. The strength of cryogels also increases along with growing the DMSO contents and decreases by the BSA addition. For estimation of the cryogel morphology, effects of the restricted diffusion of both the water and PVA in aq-space have been taken into account. By the introduction of DMSO to cryogels the solvent filled pores become smaller, and channels become much shorter. The diameter of the PVA filaments is similar to those for all the cryogels, but the length of filaments with D2O is twice that for cryogels with a mixed solvent. Entrapment of BSA in the cryogel matrix by preparation leads to the increase of an average diameter of the water filled pores and destroys molecular packing the cryogel.

Study of cryostructurization of polymer systems. IX. Poly(vinyl alcohol) cryogels filled with particles of crosslinked dextran gel

AbstractThe influence of deformable disperse filler on rheological and thermal properties of poly(vinyl alcohol) cryogels (obtained by freezing–thawing of concentrated aqueous solutions of the polymer) was studied. Spherical particles of crosslinked dextran gel—the Sephadex beads of two types: G‐25 and G‐100—were used as filler. It was found that an increase in the filler concentration from 0 to 20 wt% produced and increase in the composite cryogel strength; the greater reinforcement effect was observed with large‐pore Sephadex beads into the cryogel matrix. An increase in the fraction of disperse phase also leads to an increase in fusion temperatures of these composites, such effect being determined mainly by the filler type. The filling of cryogels with G‐100 beads improves the thermostability of composite samples to a higher extent as compared with the use of G‐25 particles. Several possible reasons for the trends observed in rheological and thermal properties of Sephadexfilled PVA cryogels were revealed.