Preparation Of Macroporous & Controllable Porous NaCS-PDMDAAC Microcapsules And Their Applications

Sodium cellulose sulfate (NaCS)/ poly(dimethyldiallylammonium chrolide) (PDMDAAC) is a very interesting microcapsule system formed by dropping a solution of polyanion NaCS into a solution of polycation PDMDAAC. The microcapsules with high mechanical strength, stable physicochemical property and good bio-compatibility have been widely applied in the cell culture and organ transplantation.It is well known that an efficient diffusion and a minimal delay in the transport of the substances through the membrane are crucial for cell growth in a favorable environment in the capsules. However, the membrane formed by the reaction of NaCS and PDMDAAC is relatively compact and has low molecular weight cut-off, which would limit its further application to the high density cell culture or the continuous production of biomacromolecules.In this paper, the macroporous NaCS-PDMDAAC microcapsules were prepared using starch as pore forming agent in the encapsulation solution. The starch was firstly immobilized in the membrane of the capsules and then degraded by amylase. The effects of some key parameters in the process, such as the kind of pore forming agent, pretreatment of starch, the location starch added and the reaction time were studied. A fundamental mathematics model on the amylase diffusion into the microcapsules was established, by which the effective diffusion coefficients of the amylase in the membrane of capsules from T_d could be easily calculated. T_d was defined as the time period from the capsules dropping into the amylase solution to the end of starch degradation in the capsules by the amylase. An optimized process of macroporous capsules preparation was gotten. The cassava starch gelatinized less than 3min was used as pore forming agent. 4% NaCS aqueous solution with 1.2% gelatinized cassava starch was added dropwise into stirred 6% PDMDAAC aqueous solution. After 40min reaction, the capsules were separated from the solution and then immerged into 1% amylase aqueous solution at 30°C until nearly no starch was detected in the capsules.There was no obvious difference of membrane thickness and diameter between the macroporous and the standard capsules. The mechanical strength of the former is a little lower than the latter. It implied the pore forming agent was an independent parameter in the adjustment of the membrane permeability. The membrane of the capsules was observed by SEM. The layers of the standard capsules were dense while those of macroporous were relatively incompact. Using the cell lysate diffusion, the MWCO of the capsules was gotten. The MWCO of the macroporous capsules was 70kDa, which is over 4 times than that of the standard ones. The diffusion of some solutions with low molecular weight including glucose, VB12, three kinds of amino acids and macromolecules including four kinds of proteins in the capsules were investigated. According to these data, the diffusion coefficients in the membrane and the combined diffusion coefficients in the capsules were determined. It indicated the permeability of the macroporous capsules was improved significantly especially for the substance of higher molecular weight. Meanwhile adding the different concentration of starch could control the permeability of the capsules.As model cells, Candida kruse and E.coli immobilized in the capsules were cultured in the shaking flasks and bubble column respectively. The cell density-immobilized in the capsules were 5-60 times than that in free culture. It was shown that the cell density immobilized in the microcapsules cultured in the bubble column was higher than that cultured in the shaking flasks. It implied that the limiting factor of the cell growth in the capsule lied in the diffusion of the oxygen. Since the rate of the oxygen transport across the membrane was greatly enhanced due to the enlarged pore size, the maximum cell density in the macroporous capsules is 20%~110% over than that in the standard capsules in the bubble column. However, the increase extent of E.coli cell density is higher than that of the yeast, which may be due to the difference of the oxygen requirement between the two microbes.Macroporous capsules system was used to immobilize Candida valida in a semi-continuous culture for producing lipase. Since the lipase diffusing to the media outside of the capsules, the product could be separated from the cell, which would relieve the product inhibition to some extent. Encapsuled Candida valida was cultured sequentially 15 batches. The cell released lipase 249 U/mL,1.56 U/mL ? h, which increased 3.1 times than that in the free culture.Polyelectrolyte complex (PEC) formation is strongly influenced by the presence of salts due to the charge screen effect, which was used to prepare the controllable porous capsules. MWCO of the capsules could be controlled from 10 to 70kDa with different concentrations of low molecular weight salt. Meanwhile the membrane thickness and mechanical strength were changed. Adding different kinds of salt in the formation of the capsules, the ions order for improving the mechanical strength of capsules was Mg +>Ca +>Na+>K+ for cation and C1">SO42" for anion, which was inversed proportional to the ion radius. However, the order was of secondary importance. Low molecular weight substance diffusion showed the permeability of the membrane increased with the increase of the salt concentration. However, the permeability of the capsules reduced because of the increase of the membrane thickness. Protein diffusion showed the diffusion rate of the macromolecules increased with the increase of salt concentration. The permeability of the controllable porous capsules is inferior to that of the macroporous capsules. The effect of the addition of the low molecular weight salt on PEC capsules formation could be discussed by measurement of the viscosity and zeta potential of polyelectrolyte solution. The understanding on the controllable porous capsules preparation and their characteristics could provide us some fundamental data for the further study.