new method for preparing more stable microcapsules for the
TRANSCRIPT
New Method for Preparing More StableMicrocapsules for the Entrapment of Genetically
Engineered Cells
Man-Yan Wang and Yao-Ting YuThe Key Laboratory of Bioactive Materials, Ministry of Education,
Institute for Molecular Biology, Nankai University,Tianjin, China
T. M. S. ChangArtificial Cells & Organs Research Center, Faculty of Medicine,
McGill University, Montreal, Quebec, Canada
Abstract: In this paper, we studied a new preparation method of microcapsulesfor entrapment of genetically engineered cells. Polyvinyl alcohol microcapsuleshaving well defined shape, high mechanical strength, good biochemical andpermeability properties were prepared by using low temperature physical cross-linking method. Comparing with currently used alginate-polylysine-alginatemicrocapsules, polyvinyl alcohol microcapsules have much higher mechanicalstrength. The low temperature physical crosslinking procedure of polyvinylalcohol is nontoxic to the genetically engineered E. coli DH5a cell, which attainedhigh activity in decomposing and metabolizing urea in vitro studies.
Keywords: Polyvinyl alcohol (PVA); Microcapsules; Genetically engineered E. coliDH5a cells; Low temperature physical crosslinking method
INTRODUCTION
Uremia is a rather widespread disease. To date, two therapeutic methodsare most commonly used for the treatment of the disease, i.e. transplan-tation and hemodialysis. Transplantation is limited by the availabilityof donors and hemodialysis is effective but expensive, especially for
The support from the seed project of 3� 3 collaboration is acknowledged.Address correspondence to Yao-Ting Yu, The Key Laboratory of Bioactive
Materials, Ministry of Education, Institute for Molecular Biology, Weijin Road#94,NankaiUniversity, Tianjin 300071,China. E-mail: [email protected]
Artificial Cells, Blood Substitutes, and Biotechnology, 33: 257–269, 2005
Copyright Q Taylor & Francis, Inc.
ISSN: 1073-1199 print/1532-4184 online
DOI: 10.1081/BIO-200066606
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developing countries, therefore scientists are searching for bettertherapeutic methods.
In the last two decades there has been an explosive increase inmolecular biology research [1,2]. As a result, a number of geneticallyengineered microorganisms with many special features have becomeavailable. They are being used widely in biotechnological and biomedicalprocesses. However, there are safety concerns about introducing geneticallyengineered microorganisms into the body for therapeutic applications. Toovercome this problem, genetically engineered cells were microencapsulatedin semipermeable microcapsules for oral administration.
This application has enormous potential for the removal of urea inkidney failure patients. Prakash and Chang have reported that geneticallyengineered E. coli DH5 cells microencapsulated in alginate-polylysine-alginate (APA) microcapsules administered orally were effective inmaintaining normal plasma urea level in uremic rats [3]. The micro-organisms were entrapped inside the microcapsules and after oraladministration passed down the gastrointestinal tract, finally excretedwith the stool in about 24 hr. During the passage of the microcapsulesthrough the intestine, urea can easily permeate through the membraneand react with the engineered E. coli cells that use urea in their complexenzyme reactions as nitrogen source. This technique could decreasedrastically the plasma urea level of uremic patients. However, there isa great demand for the strength of the membrane, which should notdeteriorate during its passage through the intestine. After oral adminis-tration, at least a small number of the APA microcapsules and othermicrocapsules would break down in the intestine. Therefore there isan urgent need to develop a new type of microcapsule with high mech-anical strength, which would overcome the peristaltic and other effectsof the intestine, and at the same time allow small molecules like urea topass freely across the membrane.
The use of polyvinyl alcohol (PVA) as an immobilization matrixoffers various advantages over the conventional alginate hydrogelsbecause of its good permeability, high durability, as well as chemical stab-ility and non-toxicity to viable cells. Several methods of immobilizationusing PVA have been reported, which include PVA-boric acid method[4,5], crosslinking by ultraviolet radition [6], freezing and incubating[7], as well as iterative freezing and thawing method [8,9].
We studied new microcapsules having much higher mechanicalstrength than APA microcapsules using PVA as a matrix to entrapgenetically engineered cells. Several crosslinking methods were studiedin preparing PVA microcapsules, and low temperature physical cross-linking method showed promising results. This process is mild comparedwith other methods and nontoxic to the genetically engineered E. coilDH5a cells, which attained high activity in decomposing and metabo-lizing urea in vitro studies.
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MATERIALS AND METHODS
Chemicals
Polyvinly alcohol (d.p. 2400–2500, degree of hydrolysis 98–99%) waspurchased from Shanghai Chemical Reagent Company (Shanghai, China).L-polylysine, Vitamin B12, urea, uric acid and creatinine were purchasedfrom Sigma Co. (St. Louis, MO). Tryptone, yeast extract powder, ampicillinwere purchased from Bebco Co. (Beijing, China). Sodium alginate was ofchemical grade and purified by microfiltration before use. Unless specified,other chemicals of analytical reagent grade were obtained from localchemical reagent companies and not further purified before use.
Microorganism
Genetically engineered Escherichia coli DH5a cells was cloned in ourlab from the plasmid pKAU17 containing the urease gene, which was agenerous gift from Dr. S.B. Mulrooney at Michigan University.
Microorganism Culture Conditions
Luria-Bertani (LB) growth medium containing nickelous sulfate was usedfor primary cell cultivation. The culture medium was composed of10.00 g=L tryptone, 5.00 g=L yeast extract powder, 10.00 g=L sodiumchloride and 1mM nickelous sulfate. The pH was adjusted to 7.4 byadding about 10ml of 1.00 N sodium hydroxide. It was then sterilizedin Castle Labclaves for 30min at 121�C and added with 0.1 g=L ampi-cillin. Incubation was carried out in 200ml LB in 500ml Erlenmeyer flaskat 37�C in an orbital shaker at 100 r=min. Log phase bacteria cells wereharvested by centrifuging at 10,000 g for 20min at 4�C. The cell masswas then washed five times with cold 0.95% NaCl solution to removemedia components.
Preparation of PVA Microcapsules
PVA microcapsules were prepared by a modified low temperature physi-cal crosslinking method. A mixture of bacteria biomass and aqueoussolution of PVA was suspended in 100ml lubricating oil with 1.5ml span80 as dispersant. The whole system was frozen in cryohydrate-bath andthen placed into a cryostat at �20�C. After 18 hr, the temperature inthe cryostat was raised to �2�C and maintained constant for the sub-sequent 24 hr, then thawed to room temperature. The microcapsuleswas collected after washing and stored at 4�C.
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Mechanical Stability
In a 50ml flask, 100 microcapsules and 50 granules of glass beads (allglass beads referred to have a diameter of 5mm) were stirred in 5mlH2O at 180 r=min for 8 hr at 37�C. Mechanical stability of microcapsuleswas measured as the percentage of breakage of microcapsules calculatedby the following equation:
Breakage (%) ¼ Nb=Ns� 100%
where Nb stands for the number of broken microcapsules and Ns standsfor the total number of microcapsules in the sample.
Urea Removal
Urea removal was performed in 50ml Erlenmeyer flasks at 37�C 80r=minin an orbital shaker. For the microcapsules, urea removal experimentswere carried out the day after preparation. Reaction media in all experi-ments consisted of 1.00 g=L glucose, 20.00mg=L magnesium sulphate,30.00mg=L dipotassium hydrogen phosphate and 0.07mg=L Vitamin B12.Urea was added to the reaction media at 10mg=ml and samples weredrawn at designated periods. Supernatants were analyzed after samples werecentrifuged at 15,000 r=min. Urea concentrations were determined basedon the quantitative measurement using diacetylmonoxime reaction [10].
Permeability
Eight ml of a solution containing 500mg=L of Vitamin B12, urea, uricacid and creatinine, respectively, were stirred with 4ml of wet blankPVA beads at 100 r=min in a 50ml flask at 37�C. The permeability wasevaluated by measuring the change of concentration at designated times.
Concentration of Vitamin B12 was determined by spectrophotometerat 550 nm. Urea was evaluated by the colorimetric method of diacetylmo-noxime reaction. Uric acid was determined by oxidation-reductionmethod based on the reaction of sodium carbonate and phosphotungsticacid. Creatinine determination was based on Jaffe reaction betweensodium picrate and creatinine [10].
Chemical Stability
PVA microcapsules was stirred in buffer solutions at different acidity(a series of 0.2M Na2HPO4–0.2M NaH2PO4–0.1N HCl buffer solution,pH ¼ 1.0 � 11.0) and a variety of inorganic salt solutions of highconcentration (1N NaCl, 1N KCl, 1N NaHCO3) (these solution are
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searched into because they are the major components existing in enteron)for 12 hr at 100 r=min in a 50ml flask at 37�C.
Morphology Observation of PVA Microcapsules
The morphology of the microcapsules was examined by light opticalmicroscopy. For better visualization of the structure, the microcapsules werestained by immersing for 24hr in a 1% aqueous solution of Congo red,which is well known to form stable dyed conjugates with PVA [11,12].The excess of the dye was removed by washing with distilled water.
RESULTS AND DISCUSSION
The low temperature physical crosslinking methods has a much lower toxiceffect on the viability of genetically engineered E. coli DH5 cells, whichattained high activity in vitro studies when compared with chemicalcrosslinking methods that used organic solvents (data not shown).
The PVA beads prepared by low temperature physical crosslinkingmethod were highly elastic. The effect of polymer concentration on mech-anical stability was examined. It was found that polymer concentrationsplayed a significant role in the formation of insoluble and thermo-stablePVA-cryogel beads. The stability of PVA-cryogel beads increases as thepolymer concentration increases and reaches an optimal condition at10–14% by weight of polymer solution (Fig. 1A, B).
Figure 1. Effect of PVA concentration on mechanical strength of PVA-cryogelbeads. (A) weight loss of PVA beads after 1 g PVA beads was stirred in 10mlof distilled water in a 50ml Erlenmeyer flask at 37�C on an orbital shaker at180 r=min for 8 hr, (B) breakage percentage of PVA beads after 100 granules ofPVA beads was stirred with 50 granules of glass beads in 5ml distilled water ina 50ml Erlenmeyer flask at 37�C on an orbital shaker at 180 r=min for 8 hr.
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The mechanical strength of PVA-cryogel beads was compared withAPA beads, which is currently used for entrapment of genetically engi-neered E. coli DH5a cell in oral administration [3,13]. When breakageof APA microcapsules reached 100%, under the same condition thebreakage of PVA microcapsules was only 3% (Fig. 2). The result showeda promising application for PVA-cryogel to be used for entrapment ofgenetically engineered E. coli DH5 cells in oral administration.
PVAmicrocapsules were prepared from 1g wet free bacteria (a numberof 1� 1011 counted using microscope) and 20ml of 12% PVA aqueoussolution by low temperature physical crosslinking method. The free bacteriain the residue was collected and compared with 0.2 g wet free bacteria for itsability to remove urea, and the yield of encapsulated cells was determined.Result showed that 0.2 g wet free bacteria can remove 991.5mg urea in150ml of 10mg=ml urea solution in an hour, and the free bacteria in theresidue can remove 932.4mg. Thus we calculated the yield of encapsulatedcells to be 81.2% by the following equation.
Yield of encapsulated cells ¼ f1� ½ð932:4=991:5Þ � 0:2�=1:0g� 100 ¼ 81:2
Urea removal by free bacteria and encapsulted bacteria (0.1 g) weredetermined in 50ml of 10mg=ml urea solution (Fig. 3). High viabilityof encapsulated genetically engineered E. coli DH5 cells was observed.
The studies also revealed that the highest viability of the bacteriaPVA-cryogel microcapsules was achieved when 12% solution ofPVA was employed during the low temperature physical crosslinking
Figure 2. Mechanical strength of PVA-cryogel beads compared with APA beads.(PVA-cryogel beads was prepared from 12% PVA solution by low temperaturephysical crosslinking method, APA beads was prepared according to reference[14]; breakage percentage was determined by using 100 granules of PVA-cryogelor APA beads stirred with 150 granules of glass beads in 5ml of distilled water ina 50ml Erlenmeyer flask at 37�C on an orbital shaker at 180 r=min for 24 hr.)
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procedure (Fig. 4). The lowest viability of cells was observed in 14%polymer solution. The result is in accord with the PVA gel preparedfrom iterative freezing-thawing methods. It was reported that thechemical structure of PVA could provide stability to cells and proteins[15,16]. The high viability of bacteria may be attributed to the presenceof the water-soluble PVA polymer that attains water and displays theprotective effect on the cells under rather adverse condition. Thishypothesis is also conformed by the fact that the viability of cellsdecreased in 14% polymer solution, resulting from a lower content ofwater subsequently formed the hard gel.
It was observed that the addition of a small amount of poly-saccharides (0 � 0.5% aginate) could assist the encapsulated cells to
Figure 3. Comparative study of urea removal of 0.1 g free and PVA-encapsulatedbacteria in 50ml of 10mg=ml urea solution (^) free bacteria, (�) encapsulatedbacteria.
Figure 4. Effect of polymer concentration on ability of 0.1 g encapsulatedbacteria for urea removal in 10ml of 10mg=ml urea solution (�) 6%, (~) 8%,(^) 10%, (&) 12%, (4) 14%.
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maintain a high ability of decomposing urea of encapsulated geneticallyengineered E. coli DH5 cells after long-term storage (Fig. 5A, B), whilehigher concentration of sodium alginate apparently decreased the abilityto remove urea. This may be attributed to the water adsorption propertyof sodium alginate that affords to keep a water-rich microenvironmentfor the bacteria to move freely and promotes its multiplication. Thedecrease of the ability to remove urea in the case high concentrationsof sodium alginate may be induced by the increase of viscosity and adecrease of water content.
The ability to remove urea by microencapsulated cells increased asthe concentration of bacteria increased (Fig. 6). But when the initial
Figure 5. Effect of concentration of sodium alginate on ability of 0.1 g encapsu-lated bacteria for urea removal in 20ml of 10mg=ml urea solution. (A) Freshlyprepared PVA-cryogel microcapsules made from 20ml of 12% PVA solutionand 1 g wet bacteria. (B) The microcapsules were stored at 4�C for 45 days (&)0.0%, (�) 0.10%, (~) 0.5%, (^) 1.0%, (4) 1.5%.
Figure 6. Effect of initial bacteria concentration on the ability of microcapsulesfor urea removal in 20ml of 10mg=ml urea solution (&) 0.50%, (^) 2.50%,(~) 5.00%, (&) 10.00%, (�) 15.00%.
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concentration of bacteria increased up to 20.0% (w=w), the encapsulatedcells broke down. The optimal concentration of bacteria for microencap-sulating should be kept at 15.0% (w=w) when both good yield and highability of decomposing urea were obtained. Higher concentration ofbacteria could decrease the strength of microcapsules, therefore decreasethe availability of encapsulated bacteria cells.
Figure 7. The concentration variation of (A) urea, (B) uric acid, (C) creatinine,(D) vitamin B12 in the presence of PVA-cryogel microcapsules.
Table 1. Loss of weight and bulk density of PVA-cryogel micro-capsules after stirring in buffer solutions of different acidity for12 hr (P > 0:05Þ
Buffer pH Loss of weight (%) Loss of bulk density (%)
1 �0.65� 2.61 �0.94� 2.843 �2.60� 1.30 �1.89� 1.895 �1.30� 1.95 �0.94� 1.767 �0.65� 1.30 �1.89� 1.899 �1.30� 1.95 �0.92� 2.8411 �2.60� 1.30 �2.83� 1.88
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Table 2. Loss of weight and bulk density of PVA-cryogel microcapsules afterstirring in inorganic salt solutions of high concentration for 8 hr ðP > 0:05Þ
Inorganic salt solution 1 N NaCl 1 N KCl 1 N NaHCO3
Loss of weight (%) �1.30� 1.30 �0.65� 1.30 �1.95� 1.95Loss of bulk density (%) �0.94� 1.76 0� 2.86 �0.94� 2.84
Figure 8. Effect of acidity on ability of encapsulated bacteria for urea removalin 20ml of 10mg=ml urea solution (&) pH 5, (^) pH 6, (~) pH 7, (&) pH 8,(�) pH 9.
Figure 9. Effect of inorganic salt of high concentration on ability of encapsulatedbacteria for urea removal in 20ml of 10mg=ml urea solution (&) 0.5N NaCl,(^) 0.5N KCl, (~) 0.5N NaHCO3, (&) untreated.
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PVA-cryogel microcapsules have good permeability for small mole-cules. Urea can permeate through PVA-cryogel microcapsules within1min, uric acid within 10min, creatinine within 3min and Vitamin B12
min (Fig. 7A, B, C, D). The results clearly indicate that the permeabilityproperty is satisfactory. Herewith small molecules can easily infiltrate
Figure 10. Surface structure of PVA-cryogel microcapsules (A) unstainedmicrocapsules at �350 magnification, (B) stained by immersing for 24 hr in a1% aqueous solution of Congo red (at �4200 magnification), (C) inner structureof PVA microcapsules by SEM.
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into the PVA-cryogel membranes in quite a short time, which is aprerequisite to obtain a high clearance of uremic toxins when the micro-capsules goes down through the intestine. No leakage of genetically engi-neered E.coli DH5a cells from the microcapsules was detected. Thus thecells can reside in the microcapsules and decompose the toxins effectively.
Good biochemical stability was observed from PVA-cryogel micro-capsules. Table 1 and 2, Figs. 8 and 9 show that the PVA-cryogelmicrocapsules are quite stable in acidic medium (buffer solutions ofdifferent acidity, pH ¼ 1�11) and in high salt concentration solutions(1.0N NaCl, 1.0N KCl, 1.0N NaHCO3) in spite of some small change,and also shows high ability of decomposing urea in buffer solutions atdifferent acidity (pH ¼ 4�9) and salt solution of high concentration0.5N NaCl, 0.5N KCl, 0.5N NaHCO3). The experimental results helpto prognosticate that the microcapsules can pass through the intestinestably and remove toxins without rupture.
Light optical microscopy observation revealed that the surface of thespherical PVA-cryogel microcapsules was undulating with numerouspores (Fig. 10A, B). and SEM photo demonstrated the porous innerstructure (Fig. 10C). The pore size varied over a large range, whichexplained the good permeability of the microcapsules for uremic toxins.
As a conclusion, PVA-cryogel microcapsules prepared by lowtemperature physical crosslinking method have a high potential to beused for oral administration in the treatment of uremia patients. It alsoprovides a feasible nontoxic method for the immobilization of otherbioactive ingredients.
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