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In Vitro and in Vivo characterization of alginate-chitosan-alginate artificial microcapsules for therapeutic oral delivery of live bacterial cells
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 105, No. 6, 660–665. 2008 DOI: 10.1263/jbb.105.660
© 2008, The Society for Biotechnology, Japan
In Vitro and in Vivo Characterization of Alginate-Chitosan-Alginate Artificial Microcapsules for Therapeutic Oral Delivery of Live Bacterial Cells Junzhang Lin,1,2 Weiting Yu,1 Xiudong Liu,3 Hongguo Xie,1,2 Wei Wang,1 and Xiaojun Ma1* Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P.R. China,1 Graduate School of the Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100039, P.R. China,2 and College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, P.R. China3 Received 9 January 2008/Accepted 25 March 2008
Oral administration of artificial cell microcapsules entrapping live bacterial cells is a promising approach in disease therapy. However, the current technology of microcapsules limits this approach. In this study, alginate-chitosan-alginate (ACA) microcapsules entrapping live bacterial cells were prepared with the purpose of oral delivery for therapy, and their in vitro and in vivo properties were investigated. Genetically engineered Escherichia coli DH5 were used as the model bacterial strain. ACA microcapsules remained intact and stable in simulated gastrointestinal fluid and the entrapped bacteria cells survived and grew normally. Moreover, ACA microcapsules were more stable than alginate-polylysine-alginate microcapsules in the rat gastrointestinal tract, which was attributed to the enhanced resistance of the ACA microcapsules to enzymatic digestion. Therefore, these results reinforce the potential of ACA microcapsules for the therapeutic oral delivery of live bacterial cells. [Key words: alginate/chitosan/alginate microcapsule, artificial cell, live bacterial cells, oral delivery]
able to smaller molecules, allowing the cells inside the microcapsules to metabolize small molecules found within the gut during passage through the intestine (18–20). If the microcapsule is protective in nature, the chances of survival of the bacterial cells are higher during transit through the GI tract thereby enhancing its function. Thus, the stability of the microcapsule membrane is critically important in the GI tract, where a myriad of membrane disruptive agents such as enzymatic action, chemical reactions, heat, low pH, diffusion, and mechanical pressure exists. Previous studies have demonstrated that oral administration of alginate-polylysine-alginate (APA) microcapsules containing the live bacterial cells have potential as an alternative therapy for several diseases such as kidney failure uremia and coronary heart disease (21). Unfortunately, however, APA microcapsules lacks stability in the GI tract (22–26). Alginate-chitosan (AC) microcapsules have been widely investigated for various reasons such as encapsulation of yeast cells in ethanol production (27), immobilization of hybridoma cells in the production of monoclonal antibodies (28), encapsulation of insulin producing islets for reversal of diabetes, and encapsulation of drugs for their sustained release (29, 30). However, the applicability of AC microcapsules to orally administrate live bacterial cells has not been studied previously. Thus, in this paper, Escherichia coli DH5, a genetically engineered strain harboring the gene encoding urease, was
The use of live bacterial cells for treating diseases such as inflammatory bowel disease, kidney failure uremia, cancer, diarrhea, and cholesteremia (1–8) has generated considerable attention and excitement among clinicians and health professionals. However, oral administration of live bacterial cells is limited by factors such as low survival of bacterial cells in the gastrointestinal (GI) tract, stimulation of the host immune response, replacement of normal intestinal flora, risk of systemic infection, deleterious metabolic activities, adjuvant side effects, immunomodulation, and risk of gene transfer (9). Thus, limiting the application of this therapy in regular clinical practice (10–16). To overcome these limitations, artificial cell microcapsules entrapping live bacterial cells have been developed for the disease treatment via oral delivery. Artificial cell microencapsulation is a technique used to encapsulate biologically active materials including viable bacteria in a specialized ultrathin and semipermeable polymer membrane (16, 17). The membrane not only protects the encapsulated bacteria from the harsh external environment but also facilitates metabolism of selected solutes capable of passing in and out of the microcapsule. In this way, the live bacteria is retained inside the microcapsule and isolated from the external environment. The membranes of the microcapsules are perme* Corresponding author. e-mail: [email protected] phone/fax: +86-411-84379096 660
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used as a model for in vitro and in vivo assessment of the potential of alginate-chitosan-alginate (ACA) microcapsules in oral therapy of uremia. MATERIALS AND METHODS Materials Sodium alginate purchased from Qingdao China Oil Co. was dissolved to a concentration of 2% w/v, which was equivalent to 100 centipoise. Chitosan was modified from the raw material purchased from Ocean Biochemical (Zhejiang, China). The final product had a molecular weight (MW) of 7.5 kDa and was 96%–98% deacetylated. Poly-L-lysine (PLL) (MW =20700) and trypsinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and solvents were of reagent grade and were used without further purification. Microorganisms and cell culture E. coli DH5, harboring the urease gene obtained from Klebsiella aerogenes (31, 32), was cultivated in Luria–Bertani (LB) growth medium (10.0 g/l bactotryptone, 5.0 g/l bacto yeast extract, and 10.0 g/l sodium chloride; final pH adjusted to 7.5 with 1.0 N NaOH). The medium was sterilized in an autoclave for 30 min at 120°C. The inoculated medium 20 ml was incubated in 50 ml Erlenmeyer Flasks at 37°C in an orbital shaker operating at 120 rpm for 10 h. Preparation of calcium-alginate beads Sodium alginate was dissolved in 0.9% NaCl at a concentration of 1.5% (w/v), and then was passed through a 0.22 µm membrane filter. Calcium (Ca) alginate beads were formed in a high voltage electrostatic generator (DICP; Dalian, China) following the establishment of an electrostatic potential of 2–10 kV between the 0.4 mm outer diameter needle feeding the alginate solution and the gelling bath containing 0.1 M CaCl2. When the alginate solution was pumped at a rate of 30 ml/h through the needle with a voltage of 3 kV and a frequency of 120 Hz, beads with a diameter of about 500 µm were produced. The Ca-alginate beads were allowed to harden in the gelling bath for at least 30 min. Preparation of ACA microcapsules ACA microcapsules were prepared using a previously established two-stage procedure (33). In brief, Ca-alginate beads were transferred to a chitosan solution (0.5% w/w) containing 0.02 M sodium acetate/acetic acid buffer (pH 4.5), and shaken for 30 min. After being washed with ion-free water, the beads were immersed in 0.05% w/w alginate solution for 10 min. Finally, the microcapsules were washed again with ion-free water and stored at 4°C. Preparation of APA microcapsules APA microcapsules were prepared as per the procedure described for ACA microcapsules except that in the case of APA microcapsules PLL (0.1% w/w) was used instead of chitosan. Preparation of microcapsules containing E. coli DH5 E. coli DH5-encapsulated beads were prepared as previously described (6) with slight modification. In brief, E. coli DH5 was cultured in LB as described earlier in text and was centrifuged for 10 min at 10,000×g, the bacterial pellets were washed, suspended in 5 ml of physiological solution and mixed with 45 ml of sterile alginate solution, resulting in a final alginate concentration of 1.5% (w/v) and cell density of 3.22 ×108 colony forming units/ml. The bacteria were entrapped in ACA and APA microcapsules formed as described earlier in the text. The microencapsulated E. coli DH5 was stored in 1.0 l minimal solution (10% LB and 90% physiologic solution) at 4°C. In vitro study of urea removal For in vitro urea removal studies using microencapsulated E. coli DH5, a simulated culture medium containing urea was used. The medium consisted of 1.0 g/l glucose, 1.4 g/l sodium hydrogen phosphate, 0.3 g/l potassium dihydrogen phosphate, 1 ml/l of a trace mineral element, 0.07 g/l thiamin, and 1 mg/ml urea. In vitro studies were carried out using
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20 ml of the medium contained in 50 ml flasks at 37°C and were shaken using an orbital shaker at 150 rpm. Samples were collected at 0, 10, 30, 60, 120 and 240 min respectively and stored at 4°C for analysis. The concentrations of urea were quantitatively determined at 520 nm using a commercial urea diagnostics kit (Nanjing Jiancheng Bioengineering Institute of China). In the assay uricase catalyzes the oxidation of uric acid to allantoin, carbon dioxide, and hydrogen peroxide. Moreover, in the presence of peroxidase, hydrogen peroxide reacts with 4-aminoantipyrine dye (4-APP) and 3,5-dichloro-2-hydroxybenene sulfonate (DHBS) to form a quinoeimine dye exhibiting maximum absorbance at 540 nm. The intensity of the color produced is directly proportional to the concentration of the urea in the sample. Mechanical stability For mechanical stability evaluations, 200± 30 blank ACA or APA microcapsules were exposed to 30 ml of simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) in a 50 ml conical flask from 2 h to 24 h while being shaken at 150 rpm at 37.2°C. Samples were withdrawn and observed with a physical light microscope. It was seen that SGF was composed of NaCl, HCl and pepsin in distilled water, final pH 1.2, and SIF was composed of monobasic potassium hydrogen phosphate, sodium hydroxide in distilled water, final pH 7.5. The ingredient concentrations of both fluids were as specified by the United States Pharmacopoeia (USP 24, 2000). In order to study the effect of enzymatic degradation on capsules, 1% (w/v) trypsinase was incorporated into the SIF. Survival of free and encapsulated bacteria in simulated gastric conditions Ten ml aliquots of encapsulated and free bacteria were added to SGF and the pH was adjusted to 2.0 with 1 M NaOH. Samples of individual treatments were then incubated anaerobically at 37°C and sampled every 2 h. Survival of free and encapsulated bacteria was determined by spread plate count on LB agar after incubation at 37°C for 48 h. In vivo study The in vivo studies conformed to the Principles of Laboratory Animal Care of China. Twelve male Wistar rats were fasted overnight and were randomly divided into two groups. The two groups were orally administered with ACA microencapsulated E. coli DH5 and APA microencapsulated E. coli DH5, respectively. Each rat was administered with 2.5 ml of physiological solution containing 2000± 200 of microcapsules into its stomach by rat douche. Dosed rats were fasted throughout the experiment before being sacrificed in 2 h intervals following microcapsule installation for up to 12 h. The gastric pouch and intestine were immediately dissected upon sacrifice and rinsed to separate the contents, and the rinsed mixture was collected. The microcapsules were retrieved by filtration and observed by light microscopy.
RESULTS AND DISCUSSION ACA microcapsules containing E. coli DH5 cells The materials used for developing microcapsules are critically important in addressing the complex problems associated with oral delivery of live bacterial cells. They should provide mild encapsulation conditions, be non-toxic and biocompatible to the bacteria and host, have appropriate membrane permeability (permeable to nutrients and substrate, but impermeable to antibody-sized molecules), and have the ability to reduce the acidic and enzymatic damage during transit through the GI tract. Chitosan is highly biocompatible (34) and is widely used in biomedical research. Therefore, it is potentially a good candidate in preparing microcapsules for oral delivery of live bacterial cells. Moreover, it is essential that microcapsules remain intact, with no leakage of the entrapped bacteria (Fig. 1a). After being incubated in LB medium for 15 h, E. coli DH5 aggre-
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FIG. 1. Optical micrographs of ACA microcapsules with entrapped E. coli DH5 at 0 h (a) and after 15 h incubation in YPD (b). Bars: 40 µm.
FIG. 2. In vitro urea degradation by ACA and APA artificial cell microcapsules. The closed squares represent ACA microencapsulated E. coli DH5, the upward oriented triangles represent APA microencapsulated E. coli DH5, downward oriented triangles represent free cells, and closed circles represent ACA microcapsules without cells.
gates distributed in the microcapsules were evident, implying marked proliferation of the bacteria (Fig. 1b). Thus, these observations indicated that ACA microcapsules are stable and intact during culture, and provide an environment suitable for the growth and proliferation of E. coli DH5. Urea removal capacity of microencapsulated E. coli DH 5 In vitro experiments were designed to evaluate the urea removal capacity of ACA or APA microencapsulated E. coli DH5 harboring urease. However, no significant differences for ACA and APA microencapsulated cells in urea removal capacity were noted (P > 0.05) (Fig. 2). In the presence of E. coli-containing ACA microcapsules, the urea concentration in the simulated culture medium was significantly reduced from 429.20 mg/l to 37.06 mg/l in 120 min, and was undetectable at 240 min. Urea removal was accomplished better by free cells than by bacteria encapsulated with ACA or APA, which may be attributed to the easy diffusion of the urea molecule through the cell than compared to immobilized cells. However, the shape of the degradation curve for free cells was similar to those of ACA and APA encapsulated bacteria (Fig. 2). Degradation was not delayed in the encapsulated samples, indicating that the ACA and APA membranes exhibit good permeability for small molecules such as urea. Furthermore, the observations of degradative activity in the microcapsules attests to the continued viabil-
FIG. 3. Survival rate of ACA and APA microencapsulated E. coli DH5 in SGF. Microencapsulated cells were inoculated in SGF for 2 h at 37°C (n= 3).
ity of the entrapped bacterial populations. Survival of free and encapsulated bacteria in simulated gastric conditions The protective effects of ACA and APA microcapsules to E. coli in simulated gastric conditions are seen in Fig. 3. The survival rates of E. coli DH5 in alginate gel beads, ACA and APA microcapsules exceeded 55%, which was markedly higher than 8.4% survival rate of free cells. ACA and APA microcapsules impart almost the same protection to cells in SGF (P > 0.05); hence, implicating that alginate attributed to the protective factor of encapsulated cells rather than using chitosan or PLL. GI tract stability of ACA microcapsules When the microcapsules pass though the GI tract, they are subjected to various environmental physiochemical stresses that include reaction with GI fluid, mechanical strength, enzyme activity, and temperature. Therefore, microcapsules suited for oral delivery of bacterial cells should be capable to resist these stresses. Light microscopic analysis revealed that the ACA microcapsules remained in a uniform spherical shape with a smooth surface in SGF, whereas APA microcapsules appeared wrinkled (Fig. 4). One possible reason for the morphological difference may due to the fact that ACA microcapsules are more resistance to low pH-mediated deformation by virtue of their membrane thickness (approximately
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FIG. 4. Photomicrograph of ACA (a) and APA (b) microcapsules in SGF after shaking at 150 rpm for 24 h. Bars: 40 µm.
FIG. 5. Mechanical stability of ACA and APA microcapsules in SGF after shaking at 150 rpm (n = 3). No significant differences were obtained between the two groups (P >0.05). FIG. 7. Photographs of ACA (a) and APA (b) microcapsules containing E. coli DH5 prior to installation in rat GI tract, and of retrieved ACA microcapsules (c) and APA microcapsules (d) 6 h later. Bars: 100 µm.
FIG. 6. Mechanical stability of ACA and APA microcapsules in SIF after shaking at 150 rpm (n = 3). No significant differences were obtained between the two groups (P >0.05).
40 µm) as compared with APA microcapsules, whose membranes averaged about 10 µm in thickness. Figure 5 and 6 depict the stability of ACA and APA microcapsules in SGF and SIF. No significant difference was observed between the two groups (P >0.05). In vivo stability of ACA microcapsules To evaluate the microcapsule stability in vivo, an oral dose of ACA or APA microcapsule suspension was administered to rats via
oral gavage. Figure 7 shows the morphological changes of ACA and APA microcapsules before and after passing though the intestine. The original ACA and APA microcapsules were spherical and uniform in shape with a smooth surface. When residing in the intestine for 6 h, ACA microcapsules remained intact and retained the spherical shape, while APA microcapsules became wrinkled or broke open. Moreover, many intact ACA microcapsules could be retrieved from the cecum of rats, while very few APA microcapsules were retrieved (data not shown). These results validated that in vivo stability of ACA microcapsules was higher than APA microcapsules. This stability likely reflects the difference between chitosan and PLL in tolerance to enzymatic degradation in the rat digestive tract. ACA and APA microcapsules are hydrolyzed by lysozyme and trypsinase, respectively (35). Presently, when trypsinase was mixed with SIF, it markedly disrupted the stability of APA microcapsule membranes, but had little effect on ACA microcapsule membrane (Fig. 8). This effect is because trypsinase can hydrolyze the peptide bond of lysine residues, indicating that microcapsules composed of ACA are more resistant to GI enzymatic degradation than APA microcapsules. Conclusions This study demonstrates that ACA microcapsules can be used for the entrapment of live bacterial
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FIG. 8. Effect of trypsinase on mechanical stability of ACA and APA microcapsules (n = 3). Significant differences were obtained between the two (P <0.05).
cells and are capable of supporting the cell growth of bacteria. Moreover, the ACA microcapsule membrane also has superior mechanical and chemical stability in simulated gastrointestinal conditions. In vivo experiments demonstrated that the ACA microcapsule is more stable than the APA microcapsule, as a consequence of increased resistance to GI enzymatic degradation. Therefore, it is anticipated that ACA microcapsules could allow safe and effective oral delivery of live bacterial cell for various clinical applications. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant 20736006) and the Youth Talent Science Foundation of Dalian City (grant 2007J23JH036).
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© 2008, The Society for Biotechnology, Japan
In Vitro and in Vivo Characterization of Alginate-Chitosan-Alginate Artificial Microcapsules for Therapeutic Oral Delivery of Live Bacterial Cells Junzhang Lin,1,2 Weiting Yu,1 Xiudong Liu,3 Hongguo Xie,1,2 Wei Wang,1 and Xiaojun Ma1* Laboratory of Biomedical Material Engineering, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, P.R. China,1 Graduate School of the Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100039, P.R. China,2 and College of Environment and Chemical Engineering, Dalian University, Dalian Economic Technological Development Zone, Dalian 116622, P.R. China3 Received 9 January 2008/Accepted 25 March 2008
Oral administration of artificial cell microcapsules entrapping live bacterial cells is a promising approach in disease therapy. However, the current technology of microcapsules limits this approach. In this study, alginate-chitosan-alginate (ACA) microcapsules entrapping live bacterial cells were prepared with the purpose of oral delivery for therapy, and their in vitro and in vivo properties were investigated. Genetically engineered Escherichia coli DH5 were used as the model bacterial strain. ACA microcapsules remained intact and stable in simulated gastrointestinal fluid and the entrapped bacteria cells survived and grew normally. Moreover, ACA microcapsules were more stable than alginate-polylysine-alginate microcapsules in the rat gastrointestinal tract, which was attributed to the enhanced resistance of the ACA microcapsules to enzymatic digestion. Therefore, these results reinforce the potential of ACA microcapsules for the therapeutic oral delivery of live bacterial cells. [Key words: alginate/chitosan/alginate microcapsule, artificial cell, live bacterial cells, oral delivery]
able to smaller molecules, allowing the cells inside the microcapsules to metabolize small molecules found within the gut during passage through the intestine (18–20). If the microcapsule is protective in nature, the chances of survival of the bacterial cells are higher during transit through the GI tract thereby enhancing its function. Thus, the stability of the microcapsule membrane is critically important in the GI tract, where a myriad of membrane disruptive agents such as enzymatic action, chemical reactions, heat, low pH, diffusion, and mechanical pressure exists. Previous studies have demonstrated that oral administration of alginate-polylysine-alginate (APA) microcapsules containing the live bacterial cells have potential as an alternative therapy for several diseases such as kidney failure uremia and coronary heart disease (21). Unfortunately, however, APA microcapsules lacks stability in the GI tract (22–26). Alginate-chitosan (AC) microcapsules have been widely investigated for various reasons such as encapsulation of yeast cells in ethanol production (27), immobilization of hybridoma cells in the production of monoclonal antibodies (28), encapsulation of insulin producing islets for reversal of diabetes, and encapsulation of drugs for their sustained release (29, 30). However, the applicability of AC microcapsules to orally administrate live bacterial cells has not been studied previously. Thus, in this paper, Escherichia coli DH5, a genetically engineered strain harboring the gene encoding urease, was
The use of live bacterial cells for treating diseases such as inflammatory bowel disease, kidney failure uremia, cancer, diarrhea, and cholesteremia (1–8) has generated considerable attention and excitement among clinicians and health professionals. However, oral administration of live bacterial cells is limited by factors such as low survival of bacterial cells in the gastrointestinal (GI) tract, stimulation of the host immune response, replacement of normal intestinal flora, risk of systemic infection, deleterious metabolic activities, adjuvant side effects, immunomodulation, and risk of gene transfer (9). Thus, limiting the application of this therapy in regular clinical practice (10–16). To overcome these limitations, artificial cell microcapsules entrapping live bacterial cells have been developed for the disease treatment via oral delivery. Artificial cell microencapsulation is a technique used to encapsulate biologically active materials including viable bacteria in a specialized ultrathin and semipermeable polymer membrane (16, 17). The membrane not only protects the encapsulated bacteria from the harsh external environment but also facilitates metabolism of selected solutes capable of passing in and out of the microcapsule. In this way, the live bacteria is retained inside the microcapsule and isolated from the external environment. The membranes of the microcapsules are perme* Corresponding author. e-mail: [email protected] phone/fax: +86-411-84379096 660
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used as a model for in vitro and in vivo assessment of the potential of alginate-chitosan-alginate (ACA) microcapsules in oral therapy of uremia. MATERIALS AND METHODS Materials Sodium alginate purchased from Qingdao China Oil Co. was dissolved to a concentration of 2% w/v, which was equivalent to 100 centipoise. Chitosan was modified from the raw material purchased from Ocean Biochemical (Zhejiang, China). The final product had a molecular weight (MW) of 7.5 kDa and was 96%–98% deacetylated. Poly-L-lysine (PLL) (MW =20700) and trypsinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and solvents were of reagent grade and were used without further purification. Microorganisms and cell culture E. coli DH5, harboring the urease gene obtained from Klebsiella aerogenes (31, 32), was cultivated in Luria–Bertani (LB) growth medium (10.0 g/l bactotryptone, 5.0 g/l bacto yeast extract, and 10.0 g/l sodium chloride; final pH adjusted to 7.5 with 1.0 N NaOH). The medium was sterilized in an autoclave for 30 min at 120°C. The inoculated medium 20 ml was incubated in 50 ml Erlenmeyer Flasks at 37°C in an orbital shaker operating at 120 rpm for 10 h. Preparation of calcium-alginate beads Sodium alginate was dissolved in 0.9% NaCl at a concentration of 1.5% (w/v), and then was passed through a 0.22 µm membrane filter. Calcium (Ca) alginate beads were formed in a high voltage electrostatic generator (DICP; Dalian, China) following the establishment of an electrostatic potential of 2–10 kV between the 0.4 mm outer diameter needle feeding the alginate solution and the gelling bath containing 0.1 M CaCl2. When the alginate solution was pumped at a rate of 30 ml/h through the needle with a voltage of 3 kV and a frequency of 120 Hz, beads with a diameter of about 500 µm were produced. The Ca-alginate beads were allowed to harden in the gelling bath for at least 30 min. Preparation of ACA microcapsules ACA microcapsules were prepared using a previously established two-stage procedure (33). In brief, Ca-alginate beads were transferred to a chitosan solution (0.5% w/w) containing 0.02 M sodium acetate/acetic acid buffer (pH 4.5), and shaken for 30 min. After being washed with ion-free water, the beads were immersed in 0.05% w/w alginate solution for 10 min. Finally, the microcapsules were washed again with ion-free water and stored at 4°C. Preparation of APA microcapsules APA microcapsules were prepared as per the procedure described for ACA microcapsules except that in the case of APA microcapsules PLL (0.1% w/w) was used instead of chitosan. Preparation of microcapsules containing E. coli DH5 E. coli DH5-encapsulated beads were prepared as previously described (6) with slight modification. In brief, E. coli DH5 was cultured in LB as described earlier in text and was centrifuged for 10 min at 10,000×g, the bacterial pellets were washed, suspended in 5 ml of physiological solution and mixed with 45 ml of sterile alginate solution, resulting in a final alginate concentration of 1.5% (w/v) and cell density of 3.22 ×108 colony forming units/ml. The bacteria were entrapped in ACA and APA microcapsules formed as described earlier in the text. The microencapsulated E. coli DH5 was stored in 1.0 l minimal solution (10% LB and 90% physiologic solution) at 4°C. In vitro study of urea removal For in vitro urea removal studies using microencapsulated E. coli DH5, a simulated culture medium containing urea was used. The medium consisted of 1.0 g/l glucose, 1.4 g/l sodium hydrogen phosphate, 0.3 g/l potassium dihydrogen phosphate, 1 ml/l of a trace mineral element, 0.07 g/l thiamin, and 1 mg/ml urea. In vitro studies were carried out using
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20 ml of the medium contained in 50 ml flasks at 37°C and were shaken using an orbital shaker at 150 rpm. Samples were collected at 0, 10, 30, 60, 120 and 240 min respectively and stored at 4°C for analysis. The concentrations of urea were quantitatively determined at 520 nm using a commercial urea diagnostics kit (Nanjing Jiancheng Bioengineering Institute of China). In the assay uricase catalyzes the oxidation of uric acid to allantoin, carbon dioxide, and hydrogen peroxide. Moreover, in the presence of peroxidase, hydrogen peroxide reacts with 4-aminoantipyrine dye (4-APP) and 3,5-dichloro-2-hydroxybenene sulfonate (DHBS) to form a quinoeimine dye exhibiting maximum absorbance at 540 nm. The intensity of the color produced is directly proportional to the concentration of the urea in the sample. Mechanical stability For mechanical stability evaluations, 200± 30 blank ACA or APA microcapsules were exposed to 30 ml of simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) in a 50 ml conical flask from 2 h to 24 h while being shaken at 150 rpm at 37.2°C. Samples were withdrawn and observed with a physical light microscope. It was seen that SGF was composed of NaCl, HCl and pepsin in distilled water, final pH 1.2, and SIF was composed of monobasic potassium hydrogen phosphate, sodium hydroxide in distilled water, final pH 7.5. The ingredient concentrations of both fluids were as specified by the United States Pharmacopoeia (USP 24, 2000). In order to study the effect of enzymatic degradation on capsules, 1% (w/v) trypsinase was incorporated into the SIF. Survival of free and encapsulated bacteria in simulated gastric conditions Ten ml aliquots of encapsulated and free bacteria were added to SGF and the pH was adjusted to 2.0 with 1 M NaOH. Samples of individual treatments were then incubated anaerobically at 37°C and sampled every 2 h. Survival of free and encapsulated bacteria was determined by spread plate count on LB agar after incubation at 37°C for 48 h. In vivo study The in vivo studies conformed to the Principles of Laboratory Animal Care of China. Twelve male Wistar rats were fasted overnight and were randomly divided into two groups. The two groups were orally administered with ACA microencapsulated E. coli DH5 and APA microencapsulated E. coli DH5, respectively. Each rat was administered with 2.5 ml of physiological solution containing 2000± 200 of microcapsules into its stomach by rat douche. Dosed rats were fasted throughout the experiment before being sacrificed in 2 h intervals following microcapsule installation for up to 12 h. The gastric pouch and intestine were immediately dissected upon sacrifice and rinsed to separate the contents, and the rinsed mixture was collected. The microcapsules were retrieved by filtration and observed by light microscopy.
RESULTS AND DISCUSSION ACA microcapsules containing E. coli DH5 cells The materials used for developing microcapsules are critically important in addressing the complex problems associated with oral delivery of live bacterial cells. They should provide mild encapsulation conditions, be non-toxic and biocompatible to the bacteria and host, have appropriate membrane permeability (permeable to nutrients and substrate, but impermeable to antibody-sized molecules), and have the ability to reduce the acidic and enzymatic damage during transit through the GI tract. Chitosan is highly biocompatible (34) and is widely used in biomedical research. Therefore, it is potentially a good candidate in preparing microcapsules for oral delivery of live bacterial cells. Moreover, it is essential that microcapsules remain intact, with no leakage of the entrapped bacteria (Fig. 1a). After being incubated in LB medium for 15 h, E. coli DH5 aggre-
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FIG. 1. Optical micrographs of ACA microcapsules with entrapped E. coli DH5 at 0 h (a) and after 15 h incubation in YPD (b). Bars: 40 µm.
FIG. 2. In vitro urea degradation by ACA and APA artificial cell microcapsules. The closed squares represent ACA microencapsulated E. coli DH5, the upward oriented triangles represent APA microencapsulated E. coli DH5, downward oriented triangles represent free cells, and closed circles represent ACA microcapsules without cells.
gates distributed in the microcapsules were evident, implying marked proliferation of the bacteria (Fig. 1b). Thus, these observations indicated that ACA microcapsules are stable and intact during culture, and provide an environment suitable for the growth and proliferation of E. coli DH5. Urea removal capacity of microencapsulated E. coli DH 5 In vitro experiments were designed to evaluate the urea removal capacity of ACA or APA microencapsulated E. coli DH5 harboring urease. However, no significant differences for ACA and APA microencapsulated cells in urea removal capacity were noted (P > 0.05) (Fig. 2). In the presence of E. coli-containing ACA microcapsules, the urea concentration in the simulated culture medium was significantly reduced from 429.20 mg/l to 37.06 mg/l in 120 min, and was undetectable at 240 min. Urea removal was accomplished better by free cells than by bacteria encapsulated with ACA or APA, which may be attributed to the easy diffusion of the urea molecule through the cell than compared to immobilized cells. However, the shape of the degradation curve for free cells was similar to those of ACA and APA encapsulated bacteria (Fig. 2). Degradation was not delayed in the encapsulated samples, indicating that the ACA and APA membranes exhibit good permeability for small molecules such as urea. Furthermore, the observations of degradative activity in the microcapsules attests to the continued viabil-
FIG. 3. Survival rate of ACA and APA microencapsulated E. coli DH5 in SGF. Microencapsulated cells were inoculated in SGF for 2 h at 37°C (n= 3).
ity of the entrapped bacterial populations. Survival of free and encapsulated bacteria in simulated gastric conditions The protective effects of ACA and APA microcapsules to E. coli in simulated gastric conditions are seen in Fig. 3. The survival rates of E. coli DH5 in alginate gel beads, ACA and APA microcapsules exceeded 55%, which was markedly higher than 8.4% survival rate of free cells. ACA and APA microcapsules impart almost the same protection to cells in SGF (P > 0.05); hence, implicating that alginate attributed to the protective factor of encapsulated cells rather than using chitosan or PLL. GI tract stability of ACA microcapsules When the microcapsules pass though the GI tract, they are subjected to various environmental physiochemical stresses that include reaction with GI fluid, mechanical strength, enzyme activity, and temperature. Therefore, microcapsules suited for oral delivery of bacterial cells should be capable to resist these stresses. Light microscopic analysis revealed that the ACA microcapsules remained in a uniform spherical shape with a smooth surface in SGF, whereas APA microcapsules appeared wrinkled (Fig. 4). One possible reason for the morphological difference may due to the fact that ACA microcapsules are more resistance to low pH-mediated deformation by virtue of their membrane thickness (approximately
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FIG. 4. Photomicrograph of ACA (a) and APA (b) microcapsules in SGF after shaking at 150 rpm for 24 h. Bars: 40 µm.
FIG. 5. Mechanical stability of ACA and APA microcapsules in SGF after shaking at 150 rpm (n = 3). No significant differences were obtained between the two groups (P >0.05). FIG. 7. Photographs of ACA (a) and APA (b) microcapsules containing E. coli DH5 prior to installation in rat GI tract, and of retrieved ACA microcapsules (c) and APA microcapsules (d) 6 h later. Bars: 100 µm.
FIG. 6. Mechanical stability of ACA and APA microcapsules in SIF after shaking at 150 rpm (n = 3). No significant differences were obtained between the two groups (P >0.05).
40 µm) as compared with APA microcapsules, whose membranes averaged about 10 µm in thickness. Figure 5 and 6 depict the stability of ACA and APA microcapsules in SGF and SIF. No significant difference was observed between the two groups (P >0.05). In vivo stability of ACA microcapsules To evaluate the microcapsule stability in vivo, an oral dose of ACA or APA microcapsule suspension was administered to rats via
oral gavage. Figure 7 shows the morphological changes of ACA and APA microcapsules before and after passing though the intestine. The original ACA and APA microcapsules were spherical and uniform in shape with a smooth surface. When residing in the intestine for 6 h, ACA microcapsules remained intact and retained the spherical shape, while APA microcapsules became wrinkled or broke open. Moreover, many intact ACA microcapsules could be retrieved from the cecum of rats, while very few APA microcapsules were retrieved (data not shown). These results validated that in vivo stability of ACA microcapsules was higher than APA microcapsules. This stability likely reflects the difference between chitosan and PLL in tolerance to enzymatic degradation in the rat digestive tract. ACA and APA microcapsules are hydrolyzed by lysozyme and trypsinase, respectively (35). Presently, when trypsinase was mixed with SIF, it markedly disrupted the stability of APA microcapsule membranes, but had little effect on ACA microcapsule membrane (Fig. 8). This effect is because trypsinase can hydrolyze the peptide bond of lysine residues, indicating that microcapsules composed of ACA are more resistant to GI enzymatic degradation than APA microcapsules. Conclusions This study demonstrates that ACA microcapsules can be used for the entrapment of live bacterial
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FIG. 8. Effect of trypsinase on mechanical stability of ACA and APA microcapsules (n = 3). Significant differences were obtained between the two (P <0.05).
cells and are capable of supporting the cell growth of bacteria. Moreover, the ACA microcapsule membrane also has superior mechanical and chemical stability in simulated gastrointestinal conditions. In vivo experiments demonstrated that the ACA microcapsule is more stable than the APA microcapsule, as a consequence of increased resistance to GI enzymatic degradation. Therefore, it is anticipated that ACA microcapsules could allow safe and effective oral delivery of live bacterial cell for various clinical applications. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant 20736006) and the Youth Talent Science Foundation of Dalian City (grant 2007J23JH036).
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