Patent Application: US-1112608-A

Abstract:
an immobilized catalytic system comprising a carrier layer containing a catalytic entity and a permeable screening layer for providing controlled access between the immobilizing catalytic entity and the surrounding environment and methods of making such systems are disclosed . the carrier layer includes the catalytic entity mixed with a neutral or anionic carrier polymer , which may or may not be cross - linked with a cross - linking agent . the screening layer over the carrier layer includes a matrix of a cationic polymer that is permeable to molecules processed by , produced by or acted upon by the catalytic entity but is not permeable to the catalytic entity itself . any counter ion to the neutral or anionic carrier polymer cannot be the same as the cationic polymer of the screening layer , and any counter ion to the cationic polymer cannot be the same as the neutral or anionic carrier polymer .

Description:
alginate - chitosan core - shell microcapsules according to the invention have been prepared as novel biocompatible matrix systems for enzyme immobilization , where the catalyst is confined to the core and the transport properties of the substrate and product are dictated by the permeability of the shell . alginate as the primary core component provides several advantages . if ca 2 + or ba 2 + ions are used for crosslinking alginate , microcapsules with liquid or solid cores , respectively , can be prepared . with β - galactosidase as a model enzyme , the system of the invention achieved 60 % loading efficiency with a ca 2 + - alginate liquid core and 100 % loading efficiency with a ba 2 + - alginate solid core . the enzymatic activity of β - galactosidase in the immobilized system was determined using onpg as a substrate . the v max values for the ca 2 + - alginate - and ba 2 + - alginate - chitosan core - shell microcapsules were significantly lower than that of the free enzyme due to the additional layer necessary for the influx of the substrate and outflux of the product . the solid core microcapsules , prepared with ba 2 + - alginate , however , did improve the stability of the enzyme at 37 ° c . as compared to the liquid core ca 2 + - alginate microcapsules and the free enzyme . chitosan was selected as a material for the microcapsule shell for several reasons . first , as described earlier , it is an abundant cationic biopolymer with intra - and intermolecular hydrogen bonding ability . various geometries such as spheres , capsules , membranes and fibers can easily be formed from chitosan ( hirano et al ., 1987 ; hann et al ., 2002 ). catalytic systems according to the invention configured in a variety of geometries are shown in fig2 . second , we have developed a unique approach to modifying the surface of chitosan by complexation - interpenetration of anions ( e . g ., heparin , dextran sulfate , and poly ( ethylene glycol )- sulfonate ) to improve biocompatibility ( lesney , 2001 ; sun et al ., 1984 ; hann et al ., 2002 ). surface - modified chitosan can resist protein adsorption and cell adhesion in the biological milieu . lastly , we have also developed the technology to create chitosan membranes with controlled pore size and density , such that molecules of specific sizes can permeate through ( amiji , 1999 ). the unique approach according to the invention for preparing microcapsules for the immobilization of enzymes , cells , and microorganisms permits the biological agent to be protected in the inner , biocompatible core while the outer shell is fabricated to provide a selectively permeable layer . some potential applications of proteins and cells immobilized according to the invention are given in table 1 . the following examples are presented to illustrate the advantages of the present invention and to assist one of ordinary skill in making and using the same . these examples are not intended in any way otherwise to limit the scope of the disclosure . chitosan microcapsules without the alginate core were formed to optimize the conditions for the final alginate - chitosan hybrid microcapsules . the chitosan solution was prepared by dissolving 0 . 75 gram of chitosan in 100 ml of 0 . 1 - m acetic acid . the solution was mixed for 8 hours , then filtered through glass wool and degassed overnight . the suitable concentration of chitosan was found to be 0 . 75 % ( w / v ), and the optimum cross - linking time was found to be 1 . 5 hours . tests to optimize the conditions were done for alginate bead preparation . the type of alginate ( protanal ®, pronova , wash .) used was determined by cross - linking alginate with different g : m ratios , protanal ® lf 20 / 200 ( 55 : 45 g : m ratio ) was found to have the strongest walls at 0 . 34 m cacl 2 and 45 minutes cross - linking time . the optimum cacl 2 concentration to cross - link the alginate was determined by cross - linking alginate in different cacl 2 solutions ( 1 - 10 % w / v ). the 0 . 34 - m solution provided strong beads . no additional strength was seen at higher cacl 2 concentrations . to determine the cross - linking time , beads were cross - linked in a 0 . 34 - m aqueous cacl 2 solution for different times from 5 minutes up to 60 minutes . all beads showed good strength , and beads cross - linked for 45 minutes showed maximum strength . further cross - linking time provided no significant improvements in strength . however , later results suggested that a cross - linking time of 5 minutes simultaneously provides sufficient strength and an improved enzyme loading percentage . a 2 . 0 % w / v solution of na - alginate was prepared by dissolving 2 grams of na - alginate ( protanal lf 20 / 200 , pronova , wash .) in 100 ml of distilled water . the solution was mixed for 8 hours until the powder was completely dissolved . the solution was dropped into a 0 . 34 - m aqueous cacl 2 solution using a syringe with a 27½ - gauge needle . the formed microspheres were allowed to sit in the cacl 2 solution for 5 minutes . the final beads were collected , washed once with deionized distilled water , and were stored at 4 ° c . alginate beads cross - linked with bacl 2 were prepared using a 0 . 34 m bacl 2 as a cross - linking solution ; the cross - linking time was kept 5 minutes . the alginate microspheres , prepared as mentioned above , were dispersed in a 0 . 75 % ( w / v ) chitosan and allowed to sit for several seconds . using a plastic dropper with the end cut to provide the appropriately sized opening , the suspended alginate beads were sucked into the dropper and then dropped into a 3 % ( w / v ) sodium tripolyphosphate ( na - tpp ) aqueous solution . the microcapsules formed were allowed to sit in the na - tpp solution for 1 . 5 hours to ensure complete cross - linking . after the ca 2 + - alginate hybrid microcapsules were formed , the core was found to have a liquid consistency due to the extraction of the ca + 2 ions by the phosphate ions in the cross - linking step . the hybrid microcapsules containing ba 2 + - alginate beads did not liquefy , as ba 2 + ions are not leached out by the phosphate ions . thus , ba 2 + - alginate hybrid microcapsules have a solid core . scanning electron microscopy ( sem ) was performed on freeze - dried control chitosan microcapsules and freeze - dried alginate - chitosan hybrid microcapsules . the surface as well as cross - sections of the capsules were analyzed ( see fig3 , 4 a and 4 b ). the sem analysis was done using magnification of 40 × and 75 × for the chitosan microcapsules and the alginate - chitosan hybrid microcapsules , respectively . the strength of the microcapsules was studied by measuring the equilibrium water uptake by the hydrogels . equilibrium water uptake of the capsules is an indicator of the mechanical strength of the capsule . when the capsule takes up water , the wall swells and the matrix becomes less compacted . as the uptake of water increases , the strength of the capsules is usually decreased . five freeze - dried microcapsules ( alginate encapsulated in chitosan ) were weighed and then suspended in distilled water . after one hour , the capsules were taken out and the surface water was removed by placing the capsules on a dry kim - wipe ® tissue paper . after the excess surface water had been removed , the capsules were weighed again . five freeze - dried capsules made up of chitosan only were tested the same way , as controls . the procedure was carried out in triplicate for both the ca 2 + - and ba 2 + - alginate - encapsulated chitosan and the empty chitosan microcapsules . the equilibrium water uptake ( ewu ) was calculated using the following equation ( anderson et al ., 2001 ): where w s is the weight of the swollen capsules and w d is the weight of the dry capsules . the mechanical strength of the calcium and barium hybrid microcapsules as well as plain chitosan microcapsules was tested , and the force required to rupture the microcapsules was measured . in the case of dry microcapsules , chitosan ( control ) microcapsules had the lowest mechanical strength , having an average burst force of 16 . 1 g . liquid core microcapsules ( ca 2 + - alginate core ) required a force of 132 g to burst . solid core microcapsules had 2 burst points : the first representing the breaking of the outer chitosan shell happened at 14 g , and the second , corresponding to the ba 2 + - alginate bead breaking , was at 707 g . the wet chitosan microcapsules had a burst point of 9 . 6 g . the wet ca 2 + - alginate hybrid microcapsules required as force of 3 g . the wet ba 2 + - alginate hybrid microcapsules required a force of 21 . 1 g to break . not being bound by any theory , it is believed that the reason the wet ca 2 + - alginate hybrid microcapsules were weaker than the chitosan control microcapsules is that the control microcapsules have a smaller size ( represented by a smaller diameter − 2 . 133 mm for chitosan and 2 . 453 mm for the ca 2 + - alginate hybrid microcapsules ). in addition , the ca 2 + - alginate hybrid microcapsules have a higher inner osmotic pressure due to the alginate present in the core , which is absent in the control microcapsules . ba 2 + - alginate hybrid microcapsules had a higher mechanical strength in both the dry and the wet state . the permeability of chitosan is the rate - limiting step in the microencapsulated system . in order to estimate the molecular weight cut - off point of the cross - linked chitosan , a model representing the chitosan layer was developed in which a thin membrane acts as a layer surrounding the alginate core . the permeability coefficients for vitamin b2 ( molecular weight 376 daltons ) and vitamin b12 ( molecular weight 1355 daltons ) representing low - molecular weight substrates , and myoglobin ( molecular weight 14 , 000 daltons ) representing a high molecular weight substrate , were studied . chitosan solution was prepared by dissolving the polymer ( 750 kda , 87 . 6 % deacetylation ) obtained from pronova biopolymers ( raymond , wash .) in 0 . 1 - m acetic acid to make a 0 . 75 % ( w / v ) solution . films were made by pouring 10 ml of the solution into a petri dish ( 100 × 15 mm ) and air - drying for up to 48 hours . the resulting films were dipped into a 3 . 0 % ( w / v ) aqueous sodium tri - polyphosphate ( na - tpp , sigma chemical company , st . louis , mo .) solution and kept for 1 . 5 hours . the cross - linked membranes were washed with distilled water once and stored in pbs at 4 ° c . the thickness of the membranes was determined using a caliper after cross - linking the membranes and washing them with distilled water . the mean wet - thickness of the membranes was found to be 50 ± 4 . 0 μm . sem analysis of freeze - dried chitosan membranes cross - linked with na - tpp was performed . sem analysis was performed with an amr - 1000 scanning electron microscope ( amray instruments , bedford , mass .) at a voltage of 10 kv . the membrane surface and cross - section images were scanned at magnifications of 13 , 000 ×. using a side - by - side diffusion apparatus , the permeability coefficients of vitamin b2 , vitamin b12 , and myoglobin were determined . briefly , each apparatus was composed of a donor compartment and a receptor compartment , with a 15 - ml capacity . the donor and receptor compartments were separated by the membrane . the donor compartment was filled with 15 ml of vitamin b2 , vitamin b12 , or myoglobin solution , and the receptor compartment was filled with 15 ml of phosphate buffered - saline ( pbs ) ph 7 . 4 . the concentrations of vitamin b2 , vitamin b12 and myoglobin were 0 . 1 , 1 . 0 , and 0 . 1 mg / ml respectively . the receptor compartment was stirred and temperature was controlled in both compartments at 37 ° c . by a circulating water bath . samples were taken from the receptor compartment periodically for up to 24 hours . the absorbance values of vitamin b2 at 440 nm , vitamin b12 at 361 nm , and myoglobin at 410 nm were measured using a shimadzu 160u uv / iv spectrophotometer ( columbia , md .). the concentration of the diffused compound into the receptor compartment was calculated from calibration curves constructed earlier . the permeability coefficient p was calculated from the following equation : in ( c o / c t )=( pst )/( hv ) where c o is the initial concentration of each compound in the donor compartment . c t is the concentration at a given time . s is the surface area of the membrane ( 1 . 77 cm 2 ). v is the volume in the donor compartment and h is the thickness of the membrane . the plot of in ( c o / c t ) against t / h was used to calculate p . the permeability coefficients of vitamin b2 , vitamin b12 and myoglobin through cross - linked chitosan at 37 ° c . are presented in table 2 . as can be seen the membrane had a higher permeability coefficient for vitamin b2 than vitamin b12 due to the relative low molecular weight of vitamin b2 . myoglobin permeated at only a low level . using the log ( permeability coefficient ) versus log ( molecular weight ) profile , we calculated the molecular cut - off of the membrane to be 20 , 000 daltons and the preferred pore size to be & lt ; 100 nm . based on this study , it was determined that a chitosan layer would provide a selectively permeable screen for substrate and product diffusion in the enzyme - immobilized system of the invention . immobilization of horseradish peroxidase to determine loading efficiency and retained bioactivity horseradish peroxidase ( hrp ) is an enzyme that catalyzes the conversion of hydrogen peroxide to water . o - phenylenediamine opd ( h 2 )— a chromogen for hrp catalyzed reactions — is converted from a colorless compound to a yellow product opd (— h 2 ) when hrp catalyzes the conversion of hydrogen peroxide to water . the yellow product can be detected using visible spectroscopy at 495 nm . briefly , an opd ( h 2 ) solution ( 0 . 25 mg / ml ) was prepared in a 0 . 1 m citrate buffer at ph 6 . then 30 % ( v / v ) hydrogen peroxide was added to provide a final concentration of 0 . 6 % ( v / v ). one hundred microliters of hydrogen peroxide solution was placed in each well of a 96 - wells microtiter plate . one hundred microliters of hrp ( molecular weight 40 , 000 , icn ) solution was then added to the wells , and after 10 minutes , the reaction was stopped by the addition of 50 μl of 1 . 0 - n sodium hydroxide . hrp was tested for the efficiency of enzyme loading into the alginate beads . hrp was used in a concentration of 50 u / ml . the loading of the enzyme was measured by detecting enzymatic activity and comparing it to a standard curve of hrp activity . the hrp enzyme was suspended in ca 2 + - alginate solution and the beads were made as described earlier . the beads were cross - linked for 45 minutes . hrp was added to the 1 ml of alginate solution to give a final concentration of 5 , 10 and 20 μg / ml . fig5 shows the results of loading with various concentrations . a loading concentration of 10 μg / ml appeared to give the best results . a group of beads was loaded with 10 μg / ml hrp , or 100 ng of enzyme per bead , as described above , and the effect of cross - linking time on loading efficiency was studied . the beads cross - linked for 5 minutes were able to entrap approximately 100 % of the enzyme ( compared to a standard solution ), while the beads cross - linked for 45 minutes entrapped only 42 . 8 % of hrp . the loading efficiency appears to have increased with a shorter cross - linking time because of a decreased contact time of the beads with the cross - linking solution . thus , leaching of the enzyme out of the beads was decreased . therefore , in subsequent studies , the cross - linking time was decreased from 45 to 5 minutes to increase the loading efficiency , although the strength of the alginate bead was slightly reduced . a fluorescent method was used to measure hrp kinetics . amplex reds ( molecular probes , eugene , oreg . ), a fluorophore for hrp , is converted to the highly fluorescent resorufin giving a red color with an excitation / emission wavelengths of 570 / 582 nm . a 200 μm amplex reds solution was prepared using 50 μm phosphate buffer ph 7 . 4 . hydrogen peroxide was used at a concentration of 20 mm . hrp was used at a concentration of 10 μg / 100 μl , the amount of immobilized hrp that is equivalent to 100 % loading . the final volume was brought to 3 . 0 ml with phosphate buffer . due to the instability of amplex red ® in the presence of light or when in solution for a short time , the kinetics of the enzyme were not measured . an enzyme having a more stable substrate and product was chosen for further studies . however , beads loaded with hrp were incubated with the amplex red for 30 minutes in a phosphate buffer ph 7 . 4 to qualitatively detect enzymatic activity . fig6 shows a qualitative image of ca 2 + - alginate hybrid microcapsules loaded with hrp and incubated with the substrate amplex red loading of β - galactosidase was studied by incorporating the enzyme into alginate beads and then measuring the enzymatic activity of β - galactosidase after dissolving the beads in 3 % ( w / v ) na - tpp aqueous solution . β - galactosidase converts o - nitrophenolgalactopyranoside ( onpg ) to o - nitrophenol ( onp ) and galactose . onp has a yellow color and can be detected using visible spectroscopy at 405 nm . β - galactosidase ( 333 u / ml ) was mixed in 3 ml of 2 % ( w / v ) alginate solution at room temperature for 3 minutes . following the mixing process , 1 ml ( 100 beads ) of the alginate solution was dropped using a 3 . 0 ml syringe with a 27½ gauge needle into either a 0 . 34 m cacl 2 aqueous solution or a 0 . 34 m bacl 2 aqueous solution . the beads were cross - linked for 5 minutes , removed from the cross - linking solution and then washed with distilled water . the ca 2 + - alginate loaded beads were placed in 20 ml of 3 % na - tpp solution and stirred until the beads completely dissolved . as a control , alginate beads without the enzyme were formed and dissolved in 20 ml of 3 % w / v na - tpp . after complete dissolution , the control solution was spiked with 100 μl of 333 u / ml β - galactosidase ( theoretical amount of enzyme present in the 100 beads of alginate ). one hundred microliters of the dissolved bead solution was added to 2 . 9 ml of 1 mm onpg and allowed to react for 5 minutes . in addition , 100 μl of the spiked control solution was added to 2 . 9 ml of 1 mm onpg . the reaction was stopped with 100 μl of 1 . 0m sodium carbonate . the concentration of enzyme in the control solution was considered 100 %, and the concentration of the enzyme in the sample solution was compared to the control solution . loading of the enzyme in the ca 2 + - alginate beads resulted in a 60 % yield , while retention of the enzyme in the ba 2 + - alginate beads was 100 %. the binding of the alginate polymer chains in the presence of the ba 2 + ion are stronger , leading to the high enzyme retention within the matrix of the polymer . the binding in the presence of the ca 2 + ions is possibly slower and the binding is not as strong , which allows the enzyme to leach out into the cross - linking solution . the enzyme concentration in the bacl 2 beads was measured indirectly by measuring the enzyme concentration in the cross - linking solution . two methods were used ; uv absorbance at 280 nm to determine protein concentration and enzymatic activity . ba 2 + - alginate beads cannot be dissolved in na - tpp solution . it is important to note that when enzyme loading in ca 2 + - alginate beads was determined from the cross - linking solution , the results were similar to the method described above . the kinetics of β - galactosidase activity was studied in the free enzyme ( non - immobilized ) and the immobilized enzyme form . the rational was to see if the outer shell membrane ( chitosan ) had any effect on the kinetics of the enzyme . when the alginate core is liquefied ( ca 2 + - alginate beads ), the rate - limiting step for enzymatic catalysis is assumed to be diffusion across the outer chitosan membrane . in the case of the non - liquified core ( ba 2 + - alginate beads ), diffusion is presumed to be hampered by both the outer chitosan layer and the cross - linked alginate matrix . briefly , the kinetics of the free enzyme was studied by conventional enzyme kinetics assessments . the enzyme concentration was held constant while the substrate concentration was varied . the initial rate was calculated for each concentration . for the free enzyme kinetics studies , a final volume in each tube was 3 . 0 ml . an enzyme concentration of 3 . 33 u / 100 μl was used . the amounts of onpg used ranged from 0 . 05 to 0 . 25 μmole . the reactions were started and absorption readings at 405 nm were determined . readings were taken initially at 5 second intervals , and then after 20 seconds , the absorption readings were taken at 10 second intervals . the kinetics of the immobilized enzyme were studied in a similar fashion as the free enzyme with some modifications to accommodate for the diffusion of substrate and product . briefly , 8 enzyme - containing beads ( either ca 2 + -- alginate or ba 2 + - alginate hybrids ) were placed in each vial containing 2 , 2 . 5 , 2 . 75 and 3 ml of 1 mm onpg . phosphate buffer ( 0 . 1 m , ph 7 . 4 ) was added to any vial to complete the volume up to 3 ml . samples were taken at time intervals starting at 5 minutes and for up to 60 minutes and absorbance was measured at 405 nm to determine the appearance of product . the following effectiveness ratio was used as a comparison parameter for immobilized systems . the effectiveness factor ( ef ) can be calculated according to the following formula ( gemeiner , p ., 1992 , shuler et al ., 2002 , kennedy et al ., 1985 ): the ( ef ) value gives an indication of the barrier effect the immobilization has on the enzyme activity . if the value of ( ef ) is equal to or greater than one , then there is no effect on diffusion due to the immobilization process . if the ( ef ) is smaller than one , then immobilization has an effect on substrate and product diffusion . the ( ef ) value is usually less than one in the case of physical immobilization . as shown in fig9 and 10 , a lag time before product detection was observed . this lag time was due to the diffusion barrier for both the substrate onpg and the product onp by the capsule wall . in the case of ca 2 + - alginate hybrids ( fig9 ), a lag time of around 7 minutes was observed before the product could be detected , and at 10 min . up to 60 min . an increase in the absorption was observed , indicating that the conversion of onpg was occurring continuously . in the case of ba 2 + - alginate hybrids ( fig1 ), a longer lag time was observed ; this effect is attributed to the cross - linked ba 2 + - alginate matrix , which did not liquefy ( unlike ca 2 + - alginate ). in addition , the amount of product produced was lower over a given period of time . the stability of β - galactosidase was studied in both the free and the immobilized form at 4 , 25 , and 37 ° c . the free enzyme was diluted to a concentration of 20 u / ml and placed in substrate solutions at the three different temperature . initial uv absorbance of the reaction with 1 - mm substrate was measured and taken as reference . the hybrid microcapsules were loaded with an enzyme concentration of 0 . 5 u / bead . referring to fig9 - 12 , it can be seen that at 4 ° c ., there was not much of a difference in terms of enzyme stability between the free enzyme and the immobilized enzyme forms ( 93 % for the free enzyme , 89 % for the ca 2 + - 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