http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2015.1022836
RESEARCH ARTICLE
Development of microbeads of chicken yolk antibodies against Clostridium difficile toxin A for colonic-specific delivery Shumin Zhang1*, Pingping Xing2*, Guiping Guo2, Hong Liu3, Donghai Lin2, Chuangchuang Dong3, Min Li3, and Dongxiao Feng1,3 School of Pharmceutical Sciences, Shandong Binzhou Medical College, Shandong Province, China, 2School of Pharmceutical Sciences, Yantai University, Shandong Province, China, and 3Center of Biotechnology, Shandong Bioasis Biotechnology Park, Shandong Province, China
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Abstract
Keywords
The incidence of Clostridium difficile infection has increased in Western world in the past 10 years, similar infection rates are also reported in developing countries such as China. Current antibiotics treatments have recurrence rates between 15% and 30%. IgY antibodies against toxin A of C. difficile could protect animal models from the challenge of lethal dose of C. difficile spores. However, IgY is sensitive to the low pH environment of the stomach and proteinases in the intestine. The objective of this study was to prepare colonic-specific delivery system of toxin A antigen-specific IgY to block the recognition of toxin A to the colon mucosa cells. Egg-laying hens were immunized with purified C. difficile toxin A C-terminal domain for 3 times, then egg IgY against the recombinant ToxA–C protein was purified from immunized egg yolk and frozen dried. IgY-loaded microbeads were prepared using mini fluid bed system; the loading efficiency was 21%. The pH and temperature stabilities of the microbeads were assayed. The IgY-loaded microbeads coated with 35% Eudragit S100 had colonic-specific IgY release specificity both in vitro and in vivo, the colonic-specific release of biological active IgY was 87.5% in the rat. Our study provides a new option for the biological treatment C. difficile infection.
Clostridium difficile, colonic-specific delivery, IgY, microbeads, toxin A
Introduction Clostridium difficile infection is one of the major causes of nosocomial infection. Reported cases of C. difficile infection have been more than doubled since 1996 in the Western world (Eaton & Mazuski, 2013). Between 0.5% and 1.5% of hospital admissions are infected by this bacterium. The incidence of community acquired C. difficile infection both in children and elderly has also increased dramatically in the past 10 years (Rao et al., 2013). Similar infection rate in developing countries such as China was also reported (Hawkey et al., 2013). The colonization of C. difficile occurs after the disruption of colonic flora because of long term of oral administration of antibiotic and/or anti-tumor drug. Clostridium difficile infection may cause mild to severe diarrhea (CDAD) and pseudomembranous colitis that may leads to death (Søes et al., 2013). Patients who acquire nosocomial infection of C. difficile need prolonged inhospital stay thus increases financial burden, it is estimated that the annual cost of this is over $6 billion in US and Europe (Voelker, 2012).
*These authors contributed equally to this article. Address for correspondence: Dongxiao Feng, Center of Biotechnology, Shandong Bioasis Biotechnology Park, 39, Keji Road, Yantai, Shandong Province 264670, PR China. Tel/Fax: +86 535 3800099. Email: [email protected]
History Received 7 January 2015 Revised 16 February 2015 Accepted 21 February 2015
Clostridium difficile is a Gram-positive, anaerobic, sporeforming bacterium which can be detected in the stool of 5% healthy adults. The spores of C. difficile are very stable and highly tolerant to regular hospital disinfection procedures (Barra-Carrasco et al., 2013). Seven different toxins can be produced by C. difficile; C. difficile disease is induced by toxin A and toxin B. Both toxin A and toxin B are mass molecular proteins that have cytotoxicity and enterotoxicity (O’Donoghue & Kyne, 2011). The repeat subunits at the C-terminal of toxin A and toxin B can recognize carbohydrate receptor on the colonic mucosa cells and mediate transportation of the toxins into the cells. The N-terminal domains of both toxins contain glucosyltransferase activity that can glycosylate Rho factor, which leads to cytoskeletal dysregulation in the toxified cells and the disruption of colonic epithelial tight junctions (Lima et al., 1988; Genisyuerek et al., 2011). Current therapies of CDAD with metronidazole, vancomycin and fidaxomicin has high recurrence rate between 15% and 30% (DuPont, 2011; Chaparro-Rojas & Mullane, 2013), which are inadequacy. Metronidazole and vancomycin resistance strains have also been isolated from clinical samples (Freeman et al., 2005). During the treatment of CDAD, oral administration of metronidazole and vancomycin may also promote persistent overgrowth of other vancomycin-resistant healthcare-associated pathogens such as Enterococci (Al-Nassir et al., 2008). Biological treatments such as vaccines and human monoclonal antibodies against
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toxin A and toxin B are under development at different stages of clinical trial (Lowy et al., 2010; Anosova et al., 2013; Koenigsknecht & Young, 2013). Several studies have revealed that serum anti C. difficile toxin A IgG concentrations were significantly correlated with protection from relapse of CDAD (Kelly & Kyne, 2011), which indicated the potential resolution of symptoms for patients with CDAD after vaccination or intravenous administration of IgG against toxin A and toxin B (Tian et al., 2012). Nevertheless, it is hard for antibodies in the blood to neutralize toxin A and toxin B in the colon for fully protection of C. difficile infection, high concentration of neutralizing antibodies in the colon should be able to provide better protection against CDAD than the antibodies in the blood. Intragastric administration of the toxin A- and toxin B-specific yolk antibodies (immunoglobulin Y [IgY]) can protect hamster model from morality induced by C. difficile (Roberts et al., 2011). However, the delivery of large protein molecules like IgY through digestion system to the colon in a non-degradable form is still a challenge for the fight against CDAD. Here, we describe the development of an oral colonspecific IgY drug delivery system using pH sensitive Eudragit S100 as the coating material, which can release IgY against C. difficile toxin A in the colon environment both in vitro and in vivo.
Materials and methods Ethics statement This study was performed in accordance with the recommendations of the US Department of Health for the care and use of laboratory animals, all the animals were exposed to CO2 for anesthesia before sacrificed, the protocols were approved by the Animal Ethics Committee of Shandong Binzhou Medical College (Permit number: 2013-08). Generation and purification of chicken yolk antibodies against ToxA–C The intact C-terminal fragment of toxin A (ToxA–C) of C. difficile was expressed and purified (Guo et al., 2013). Briefly, DNA sequence coding the C-terminal fragment of ToxA–C were cloned in pET-32b vector from Novagen and expressed as fusion protein with 6XHis tag and Thioredoxin (Trx) protein, the fusion protein was purified by Ni affinity purification column (Genescript, Nanjing, China). The fusion protein was digested with thrombin and Trx-6XHis tag was depleted by Ni affinity resin absorption. The generation, purification and bioactivity assay of IgY against ToxA–C were performed as described before (Guo et al., 2013). In brief, five egg-laying White Leghorn hens were immunized with the purified recombinant proteins that had been mixed with Freund’s adjuvant (SIGMA). The hens were immunized 3 times before eggs were harvested. IgY titers against ToxA– C were estimated by enzyme-linked immunoassays (EIAs) using recombinant ToxA–C antigen. IgY were fractionated from the collected yolks by a cold ethanol precipitation procedure (Hansen et al., 1998). The purified IgY was freezing dried and stored at 80 C. The bioactivity of the
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IgY against ToxA–C was assayed using in vitro rabbit intestinal loop toxin neutralization model. Preparation of the microbeads loaded with IgY against ToxA–C Water and organic solvent-soluble Eudragit S100 were kindly provided by Evonik Chemical Inc. (Shanghai, China), microcrystalline cellulose (MCC) was bought from Gaocheng Biotech & Health Co., Ltd (Hangzhou, China), triethyl citrate was bought from Fengyuan Pharmaceutical Co. Ltd (Anhui, China), talcum powder (medicine level, bacteria counting 5500) was bought from Jinying Talcum Co. Ltd (Qingdao, China). Mini laboratory fluid bed system was from Glatt Inc., Germany. Wistar rats (4 months old, 300 g) were bought from Beijing Vital River Laboratories, New Zealand rabbits (3 months old, 2.5 kg) were bought from Qingdao Kangda Rabbit Inc., all the animal experiments were approved by the animal ethics committees of the universities and company. Fifty grams of IgY was dissolved in 425 ml of water, 10 g of talcum powder was added before the mixture was sprayed into the coating tank of mini Glatt fluid bed system coating instrument (Glatt Inc. Germany), the fluid bed was preloaded with 100 g of MCC as core of the microbeads and prewarmed to 30 C. The spraying speed of the IgY solution was 2–3 ml/ min with the fluidization pressure was 0.3–0.4 bar and the aerosolization pressure was 0.5–0.6 bar. After all the IgY solution was loaded, kept the fluid bed running for 20 more minutes to dry the beads. Then 10% water-soluble Eudragit S100 solution (Eudragit S100 99.4 g, 1 mol/l ammonia solution 68 ml, triethyl citrate 49.9 g, talcum powder 49.7 g, H2O to 1 l) was sprayed into the fluid bed at 1 mg/ml/min with the aerosolization pressure at 0.4–0.6 bar. The coated microbeads were dried in the fluid bed for 20 min and recovered from the bed. For the organic solvent-soluble Eudragit S100, the coating solution was prepared by mixing 6.25 g Eudragit S100, 1.25 g triethyl citrate, 0.625 g talcum powder and 92 g 95% ethanol, the coating parameters were the same as those of the watersoluble Eudragit S100. IgY loading efficiency measurement IgY content of the beads was determined after completely dissolving prepared microbeads in phosphate buffer (pH 7.4). The concentrations of IgY were determined by Bradford protein assay kit (BCA method) bought from Genstar Biosolutions (Beijing, China). The ratio of the amount of the IgY to the weight of MCC was considered as loading efficiency of the microbeads. The coating ratio of Eudragit S100 was determined by the weight increased of the coated microbeads compared with the weight of the IgY-loaded beads before coating. Stability of IgY in the microbeads Stabilities of the IgY-loaded microbeads at different pH values and temperatures were determined by dissolving the IgY-loaded microbeads in buffers of different pH values between 2 and 13 at room temperature different temperatures between 25 C and 70 C for 3–24 h. The concentration of
DOI: 10.3109/10717544.2015.1022836
Microbeads of chicken yolk antibodies against Clostridium difficile toxin A
total protein was assayed by BCA method, the concentration of biofunctional IgY in the buffer was assayed by EIA method at 3, 12 and 24 h. For long-term stability of the IgY-loaded microbeads, the microbeads were stored in an incubator at 25 C for over 6 months. The functional IgY content of the microbeads was assayed by EIA method every month after dissolved the microbeads in PBS (pH 7.4) at room temperature for 3 h.
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In vitro release of the coated IgY microbeads For in vitro release assays, microbeads with known amount of IgY in a basket was put into the dissolution beaker containing 900 ml of 0.1 mol/l HCl solution of a dissolution apparatus (RZQ-8c, Tianfa Technology Inc. Tianjin). The beaker was rotated at 100 rpm and the temperature was fixed at 37 C + 0.5 for 2 h to mimic stomach release of the IgY. Then the basket was transferred to a new beaker containing 900 ml of PBS solution (pH 6.8) as the artificial intestinal fluid for 6 h. The pH value of the artificial intestinal fluid was adjusted to 7.2 to make the artificial colonic fluid by adding 0.1 mol/l of NaOH. The microbeads were kept in the artificial colonic fluid for 4 h. Five hundred microliter of the solutions was sampled every 30 min for the assays of IgY concentration by BCA method, the biofunctional IgY concentration was assayed by EIA methods using recombinant antigen as described in Step 2. The in vitro release of IgY-loaded microbeads in the enterosoluble capsule (An Hui Huangshan Capsule Inc., Huangshan, China) was also assayed. In vivo release of the coated IgY microbeads Fifteen Wistar rats were divided into five groups for the in vivo release assay of IgY-loaded microbeads. A tube of 2-mm diameter with an iron wire inside was inserted into the
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stomach of the rat first, then the iron wire was pulled out, the tube was connected to a 5-ml pipet tip, 10 mg of IgY-loaded microbeads was administrated into the tube through the tip, then the tube was connected to a syringe filled with 1 ml PBS to push the miceobeads down to the stomach of the rat. Three rats in each group of the animals were sacrificed every 2 h. Stomach, intestine, caecum and colon were collected separately from each rat. Blood sample from each rat was also collected from the eyes before the rat was sacrificed. After visual examination for the distribution and integrity of the microbeads in the organs, 10 ml of PBS (pH 7.4) was added to each sample and homogenized. After supersonication and centrifugation at 10 000 rpm for 30 min at 4 C, the supernatant from each sample was recovered for the assay of IgY concentration in the organs. The in vivo release efficiency was determined by the amount of recovered IgY to the amount of IgY administrated. The concentrations of IgY from different organs were assayed by EIA methods using recombinant ToxA–C antigen.
Results ToxA–C antigen-specific antibodies could be detected in the chicken yolk after the second immunization, the titer of the IgY in the egg yolk reached 1:50 000 after the third immunization and could last until 190 days. The titer of the IgY dropped dramatically after 196 days (Figure 1), 100 mg of IgY could be purified from each egg (Figure 1A). The purified IgY could neutralize the biological activity of ToxA–C in the rabbit intestine loop test by significantly reducing the ratio of weight/length (g/cm) of the ligated ileum loops as shown in Figure 1(C) (p50.05). The IgY was loaded on the MCC microbead cores in the fluid bed, the weight of the microbeads increased 40% after
Figure 1. Purification of IgY antibodies and the function assay. (A) SDS–PAGE analysis of purified IgY against toxin A. (B) The titer of chicken yolk antibodies against ToxA–C in eggs. (C) The injection ToxA and IgY antibodies of intestinal loops: (1) 100 mg/ml ToxA, (2) 50 mg/ml ToxA, (3) normal saline, (4) 100 mg/ml ToxA and IgY antibodies and (5) 50 mg/ml ToxA and IgY antibodies.
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Figure 2. Stability of IgY in the loaded microbeads. (A) pH stability of IgY in the loaded microbeads. The IgY-loaded microbeads were suspended in PBS buffer with different pH (2–11) value for 24 h, the buffer was aliquoted for ELISA assay to determine the binding activities of the IgY antibodies at 1, 12 and 24 h. IgY antibodies were stable between pH 4–10. (B) Temperature stability of IgY in the loaded microbeads. IgY-loaded microbeads were put in the incubators of different temperatures for 1, 12 and 24 h, then the IgY antibodies were dissolved in PBS (pH 7.4) and the antigenbinding activities of the IgY antibodies were assayed by ELISA. IgY antibodies were stable560 C. (C) Long-term storage stability of IgY at room temperature. IgY-loaded microbeads were put in the incubator at 25 C without light for 1–6 months, then the IgY antibodies were dissolved in PBS (pH 7.4), antigen-binding activities of IgY antibodies were assayed by EIA. IgY antibodies were stable for at least 6 months if stored at room temperature.
loading for 2 h, which equaled to 21% of IgY. No more IgY could be loaded to the microbeads even the loading time of the fluid bed was extended. The IgY-loaded microbeads were stable between pH 4 and pH 10. No significant decrease of the antigen-binding activities of IgY was observed after the microbeads was dissolved in the PBS buffers of different pH values for 24 h. The IgY was stable for 3 h at pH 3, after 12 h, the binding activity of IgY decreased dramatically. For pH 11 and above, the IgY lost its binding activity rapidly (Figure 2A). However, no significant difference was observed for the total protein concentrations at different pH values assayed by standard Bradford protein assay (BCA method, data not shown). The IgY-loaded microbeads showed same temperature stability as the IgY molecules. No significant decrease of the antigen-binding activity of the IgY in the microbeads after kept at as high as 60 C for 24 h compared with that of the microbeads at 25 C after dissolved in PBS (pH 7.4). The IgY
was stable for at least 1 h at 70 C, after 12 h, a significant decrease of the biological activity of the IgY was observed (Figure 2B). At 25 C, the IgY-loaded microbeads were stable for at least 6 months as shown in Figure 2(C), no significant difference of antigen-binding activity was observed. No difference was observed for the IgY concentrations dissolved in the PBS buffer at different temperatures as assayed by BCA method (data not shown). The IgY-loaded microbeads were coated with either water dispersible or organic solvent dispersible Eudragit S100 by the fluid bed (Figure 3). As shown in Figure 3(D), the antigen-binding activity of the IgY in the coated microbeads was not affected either in the water dispersable or organic solvent dispersable Eudragit S100 compared with that of the non-coated IgY. Five lots of IgY-loaded microbeads were coated with the water dispersable Eudragit S100, the weight of the microbeads increased by 15%, 20%, 25%, 30% and 35%, respectively. The average size of the coated IgY microbeads was 1.3 mm.
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DOI: 10.3109/10717544.2015.1022836
Microbeads of chicken yolk antibodies against Clostridium difficile toxin A
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Figure 3. Eudragit S100 coated IgY microbeads. (A) The unloaded beads. (B) IgY-loaded beads. The loading efficiency was 21%. (C) Coated microbeads. The coating rate was 35%. (D) The effect of coating process on the IgY biological activity in the microbeads. (1) Drug-loaded microbeads, (2) coated pellets of Eudragit S100 (aqueous dispersion solvent) and (3) coated pellets of Eudragit S100 (organic solvent).
As assayed by standard BCA method, no IgY was released from the 35% lot of Eudragit S100 coated IgY microbeads in the artificial stomach fluid in 2 h, in the artificial intestinal fluid, 14% IgY was released from the microbeads after 6 h. Over 90% of IgY was released in the artificial colonic fluid within 2 h. For the 15%, 20%, 25% and 30% lot of coated IgY microbeads, the total release of IgY in the artificial stomach fluid were 74%, 53%, 38% and 11%, respectively. In the artificial intestinal fluid, the total release of IgY for the four lots were 93%, 68%, 62% and 36%, respectively, which were not acceptable. Only the 35% lot of Eudragit S100 coated microbeads showed colonic-specific release of IgY (Figure 4A). For the in vivo release of IgY, microbeads could be found mostly in the stomach at 2 h, a few microbeads could also be observed in the intestine of the rats. At 4 h, the microbeads were mostly distributed in the intestine, some in the stomach and colon. At 6 h, most of the microbeads were found in the colon. At 8 h, the microbeads were distributed in the colon and caecum of the rat. At 10 h, most of the microbeads were excreted but quite a few of the microbeads could still be found in the colon and caecum. The release of IgY at different parts of the digestion system was showed in Figure 5. At 4 h, 3.8% of the IgY could be released in the intestine. At 6 h, 24.4% of
the IgY was detected in the intestine and 61.5% of the IgY was found in the colon. At 8 h, 87.5% of the IgY was released in the colon. After 10 h, only 4% and 5% of IgY could be detected in the intestine and colon, respectively. No IgY could be detected in the rat plasma at any time (Figure 5).
Discussion As the major cytotoxin and enterotoxin of C. Difficile, toxin A is responsible for the binding of colonic mucosa cells and mediates transportation of the toxins into the cells, thus causes the symposiums of CDAD. Molecules which can block the recognition of toxin A to the carbohydrate receptors of the colonic mucosa cells should be able to protect the patients from CDAD (Kink & Williams, 1998). Using recombinant expressed receptor-binding domain of toxin A, we have generated egg IgY which can neutralize the toxicity of ToxA– C in vitro. The ToxA–C antigen-specific IgY can protect the rabbit intestine from liquid accumulation caused by the ToxA–C injection. Although the stability of IgY is much higher than other type of antibodies, it is still sensitive to the low pH condition of the stomach and proteinases in the intestine. Carbonate buffer is required for orogastric administration of IgY in
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Figure 4. In vitro release of ToxA–C antigen binding-specific IgY from the microbeads. (A) In vitro release The IgY-loaded microbeads. (B) In vitro IgY release from the microbeads in exterosoluble capsules. The IgY-loaded microbeads or the capsules were suspended in artificial gastric fluid for 2 h, artificial intestinal fluid for 6 h and artificial colonic fluid for 4 h, the fluids were sampled every 30 min to detected IgY concentrations using EIA and BCA methods.
animal models to protect the IgY from degradation in the stomach (Roberts et al., 2011). Colonic-specific delivery of IgY can protect ToxA–C antigen-specific IgY from degradation in the stomach and intestine, therefore protect the animal from the colonization of C. difficile in the colon. IgY-loaded microbeads were prepared using fluid bed system. IgY could be loaded on the MCC core at maximal 21% of the microbead weight. The surface of the IgY-loaded microbeads was uneven compared to the smooth shape of the MCC core. As large protein molecule, the IgY solution has high viscosity, it cannot form tight and regular structure on the MCC core like small molecular drugs, powder of IgY broken down from the microbeads could be found from the bottom of the fluid bed. Although 50% (W/W) of talcum powder was added as anti-tackiness agent, the maximal loading efficiency of IgY on the MCC core was 21%, which
was lower than that of the small molecule drugs. To increase the loading efficiency of IgY to the carrier of colonic-specific delivery formulation, other agents or technologies may be necessary to be applied, e.g. pectinate beads (Atyabi et al., 2005). The IgY in the microbeads is stable in broad ranges of pH and temperature. The antigen-binding activity of the IgY did not change between pH 4–10 C and 25–60 C in 24 h. No significant decrease of antigen-binding activity of the IgY microbeads was observed after stored for over 6 months at 25 C. In addition, the 95% ethanol solvent used in the coating process did not affect the antigen-binding activity of the IgY molecules, either, indicating that IgY is suitable for the development of oral administrative formulation. Because of the rough surface of the IgY-loaded microbeads, big amount of Eudragit S100 was needed to coat the
DOI: 10.3109/10717544.2015.1022836
Microbeads of chicken yolk antibodies against Clostridium difficile toxin A
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IgY is a large protein molecule with high immunogenicity. If absorbed in the blood through the digestion system, severe allergic reaction might be evoked in the patients. No detectable IgY activity was found in the animal plasma during the in vivo release assay, indicating that IgY cannot be absorbed by the colon mucosa cells, extra IgY molecules will be excreted with no harm to the patients and the environment.
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Conclusion
Figure 5. In vivo IgY release in the rat digestion system. The rats were intragastrically administrated with same amount of microbeads containing 10 mg IgY against the ToxA of C. difficile. Stomach, intestine, caecum and colon samples were collected every 2 h. Blood sample from each rat was also collected. After visual examination, 10 ml of PBS (pH 7.4) was added to each sample and homogenated. After supersonication and centrifugation at 10 000 rpm for 30 min at 4 C, the supernatant from each sample was recovered for the assay of IgY concentration in the organs using ELISA method.
microbeads completely. Only the 35% lot of coated IgY microbeads showed colonic-specific release in the in vitro release assay. Eudragit S-100 is a pH sensitive material which can be dissolved at pH46.8 (Ramasamy et al., 2013; Tsai et al., 2013), so active compounds in the microbeads coated with Eudragit S100 should not be released in the stomach (pH54) and intestine (pH & 6.8). However, a large percentage of IgY was released earlier in the artificial stomach and intestinal fluid due to the incomplete coating of IgY microbeads in the other four lots of IgY-loaded microbeads. No significant improvement was observed when the coated IgY microbeads were filled into enterosoluble capsule. The capsule could protect the IgY from releasing in the artificial stomach fluid, but not in the artificial intestinal fluid (Figure 4B). Although mouse and hamster were used as the infection model of C. difficile, however, these animals are too small for the intragastric administration of microbeads, so rat was selected for the in vivo release of IgY microbeads. The results of in vivo release of coated IgY microbeads was consistence with the results of in vitro assay. The microbeads stayed in the stomach of the rat for 2 h and then migrated to intestine, colon and caecum subsequently. IgY activity could be detected in the intestine between 4 and 6 h at low percentage (3.8– 24.4%), but 87.5% in the colon at 8 h, Eudragit S100 could protect the IgY from releasing and degradation in the stomach and the intestine, almost 90% active IgY was released in the colon from the microbeads coated with the pH sensitive Eudragit S100, which demonstrated colonic-specific delivery of active ToxA–C antigen-specific IgY in rat. Future in vivo assays are needed to test the anti C. difficile colonization activity of the IgY microbeads in the colonic mucosa of animal model challenged with lethal dose of C. difficile spores.
In this study, we have generated chicken IgY antibodies against the receptor-binding domain of C. difficile toxin A, which could neutralize the toxicity of the toxin. A colonicspecific delivery formulation of IgY microbeads coated with pH sensitive coating material Eudragit S100 was developed, the loading efficiency of IgY on the MCC core was 21%. The IgY-loaded microbeads are stable in broad ranges of pH and temperature. IgY microbeads coated with 35% of Eudragit S100 showed colonic-specific release of active IgY both in vitro and in vivo.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Rao K, Micic D, Chenoweth E, et al. (2013). Poor functional status as a risk factor for severe Clostridium difficile infection in hospitalized older adults. J Am Geriatr Soc 61:1738–42. Roberts A, McGlashan J, Al-Abdulla I, et al. (2011). Development and evaluation of an ovine antibody-based platform for treatment of Clostridium difficile infection. Infect Immun 80:875–82. Søes LM, Holt HM, Bo¨ttiger B, et al. (2013). Risk factors for Clostridium difficile infection in the community: a case–control study in patients in general practice, Denmark, 2009–2011. Epidemiol Infect 27:1–12. Tian JH, Fuhrmann SR, Kluepfel-Stahl S, et al. (2012). A novel fusion protein containing the receptor binding domains of C. difficile toxin A and toxin B elicits protective immunity against lethal toxin and spore challenge in preclinical efficacy models. Vaccine 30:4249–58. Tsai SW, Yu DS, Tsao SW, et al. (2013). Hyaluronan-cisplatin conjugate nanoparticles embedded in Eudragit S100-coated pectin/alginate microbeads for colon drug delivery. Int J Nanomed 8:2399–407. Voelker R. (2012). Study: vast majority of C. difficile infections occur in medical settings. JAMA 307:1356.
RESEARCH ARTICLE
Development of microbeads of chicken yolk antibodies against Clostridium difficile toxin A for colonic-specific delivery Shumin Zhang1*, Pingping Xing2*, Guiping Guo2, Hong Liu3, Donghai Lin2, Chuangchuang Dong3, Min Li3, and Dongxiao Feng1,3 School of Pharmceutical Sciences, Shandong Binzhou Medical College, Shandong Province, China, 2School of Pharmceutical Sciences, Yantai University, Shandong Province, China, and 3Center of Biotechnology, Shandong Bioasis Biotechnology Park, Shandong Province, China
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Abstract
Keywords
The incidence of Clostridium difficile infection has increased in Western world in the past 10 years, similar infection rates are also reported in developing countries such as China. Current antibiotics treatments have recurrence rates between 15% and 30%. IgY antibodies against toxin A of C. difficile could protect animal models from the challenge of lethal dose of C. difficile spores. However, IgY is sensitive to the low pH environment of the stomach and proteinases in the intestine. The objective of this study was to prepare colonic-specific delivery system of toxin A antigen-specific IgY to block the recognition of toxin A to the colon mucosa cells. Egg-laying hens were immunized with purified C. difficile toxin A C-terminal domain for 3 times, then egg IgY against the recombinant ToxA–C protein was purified from immunized egg yolk and frozen dried. IgY-loaded microbeads were prepared using mini fluid bed system; the loading efficiency was 21%. The pH and temperature stabilities of the microbeads were assayed. The IgY-loaded microbeads coated with 35% Eudragit S100 had colonic-specific IgY release specificity both in vitro and in vivo, the colonic-specific release of biological active IgY was 87.5% in the rat. Our study provides a new option for the biological treatment C. difficile infection.
Clostridium difficile, colonic-specific delivery, IgY, microbeads, toxin A
Introduction Clostridium difficile infection is one of the major causes of nosocomial infection. Reported cases of C. difficile infection have been more than doubled since 1996 in the Western world (Eaton & Mazuski, 2013). Between 0.5% and 1.5% of hospital admissions are infected by this bacterium. The incidence of community acquired C. difficile infection both in children and elderly has also increased dramatically in the past 10 years (Rao et al., 2013). Similar infection rate in developing countries such as China was also reported (Hawkey et al., 2013). The colonization of C. difficile occurs after the disruption of colonic flora because of long term of oral administration of antibiotic and/or anti-tumor drug. Clostridium difficile infection may cause mild to severe diarrhea (CDAD) and pseudomembranous colitis that may leads to death (Søes et al., 2013). Patients who acquire nosocomial infection of C. difficile need prolonged inhospital stay thus increases financial burden, it is estimated that the annual cost of this is over $6 billion in US and Europe (Voelker, 2012).
*These authors contributed equally to this article. Address for correspondence: Dongxiao Feng, Center of Biotechnology, Shandong Bioasis Biotechnology Park, 39, Keji Road, Yantai, Shandong Province 264670, PR China. Tel/Fax: +86 535 3800099. Email: [email protected]
History Received 7 January 2015 Revised 16 February 2015 Accepted 21 February 2015
Clostridium difficile is a Gram-positive, anaerobic, sporeforming bacterium which can be detected in the stool of 5% healthy adults. The spores of C. difficile are very stable and highly tolerant to regular hospital disinfection procedures (Barra-Carrasco et al., 2013). Seven different toxins can be produced by C. difficile; C. difficile disease is induced by toxin A and toxin B. Both toxin A and toxin B are mass molecular proteins that have cytotoxicity and enterotoxicity (O’Donoghue & Kyne, 2011). The repeat subunits at the C-terminal of toxin A and toxin B can recognize carbohydrate receptor on the colonic mucosa cells and mediate transportation of the toxins into the cells. The N-terminal domains of both toxins contain glucosyltransferase activity that can glycosylate Rho factor, which leads to cytoskeletal dysregulation in the toxified cells and the disruption of colonic epithelial tight junctions (Lima et al., 1988; Genisyuerek et al., 2011). Current therapies of CDAD with metronidazole, vancomycin and fidaxomicin has high recurrence rate between 15% and 30% (DuPont, 2011; Chaparro-Rojas & Mullane, 2013), which are inadequacy. Metronidazole and vancomycin resistance strains have also been isolated from clinical samples (Freeman et al., 2005). During the treatment of CDAD, oral administration of metronidazole and vancomycin may also promote persistent overgrowth of other vancomycin-resistant healthcare-associated pathogens such as Enterococci (Al-Nassir et al., 2008). Biological treatments such as vaccines and human monoclonal antibodies against
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toxin A and toxin B are under development at different stages of clinical trial (Lowy et al., 2010; Anosova et al., 2013; Koenigsknecht & Young, 2013). Several studies have revealed that serum anti C. difficile toxin A IgG concentrations were significantly correlated with protection from relapse of CDAD (Kelly & Kyne, 2011), which indicated the potential resolution of symptoms for patients with CDAD after vaccination or intravenous administration of IgG against toxin A and toxin B (Tian et al., 2012). Nevertheless, it is hard for antibodies in the blood to neutralize toxin A and toxin B in the colon for fully protection of C. difficile infection, high concentration of neutralizing antibodies in the colon should be able to provide better protection against CDAD than the antibodies in the blood. Intragastric administration of the toxin A- and toxin B-specific yolk antibodies (immunoglobulin Y [IgY]) can protect hamster model from morality induced by C. difficile (Roberts et al., 2011). However, the delivery of large protein molecules like IgY through digestion system to the colon in a non-degradable form is still a challenge for the fight against CDAD. Here, we describe the development of an oral colonspecific IgY drug delivery system using pH sensitive Eudragit S100 as the coating material, which can release IgY against C. difficile toxin A in the colon environment both in vitro and in vivo.
Materials and methods Ethics statement This study was performed in accordance with the recommendations of the US Department of Health for the care and use of laboratory animals, all the animals were exposed to CO2 for anesthesia before sacrificed, the protocols were approved by the Animal Ethics Committee of Shandong Binzhou Medical College (Permit number: 2013-08). Generation and purification of chicken yolk antibodies against ToxA–C The intact C-terminal fragment of toxin A (ToxA–C) of C. difficile was expressed and purified (Guo et al., 2013). Briefly, DNA sequence coding the C-terminal fragment of ToxA–C were cloned in pET-32b vector from Novagen and expressed as fusion protein with 6XHis tag and Thioredoxin (Trx) protein, the fusion protein was purified by Ni affinity purification column (Genescript, Nanjing, China). The fusion protein was digested with thrombin and Trx-6XHis tag was depleted by Ni affinity resin absorption. The generation, purification and bioactivity assay of IgY against ToxA–C were performed as described before (Guo et al., 2013). In brief, five egg-laying White Leghorn hens were immunized with the purified recombinant proteins that had been mixed with Freund’s adjuvant (SIGMA). The hens were immunized 3 times before eggs were harvested. IgY titers against ToxA– C were estimated by enzyme-linked immunoassays (EIAs) using recombinant ToxA–C antigen. IgY were fractionated from the collected yolks by a cold ethanol precipitation procedure (Hansen et al., 1998). The purified IgY was freezing dried and stored at 80 C. The bioactivity of the
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IgY against ToxA–C was assayed using in vitro rabbit intestinal loop toxin neutralization model. Preparation of the microbeads loaded with IgY against ToxA–C Water and organic solvent-soluble Eudragit S100 were kindly provided by Evonik Chemical Inc. (Shanghai, China), microcrystalline cellulose (MCC) was bought from Gaocheng Biotech & Health Co., Ltd (Hangzhou, China), triethyl citrate was bought from Fengyuan Pharmaceutical Co. Ltd (Anhui, China), talcum powder (medicine level, bacteria counting 5500) was bought from Jinying Talcum Co. Ltd (Qingdao, China). Mini laboratory fluid bed system was from Glatt Inc., Germany. Wistar rats (4 months old, 300 g) were bought from Beijing Vital River Laboratories, New Zealand rabbits (3 months old, 2.5 kg) were bought from Qingdao Kangda Rabbit Inc., all the animal experiments were approved by the animal ethics committees of the universities and company. Fifty grams of IgY was dissolved in 425 ml of water, 10 g of talcum powder was added before the mixture was sprayed into the coating tank of mini Glatt fluid bed system coating instrument (Glatt Inc. Germany), the fluid bed was preloaded with 100 g of MCC as core of the microbeads and prewarmed to 30 C. The spraying speed of the IgY solution was 2–3 ml/ min with the fluidization pressure was 0.3–0.4 bar and the aerosolization pressure was 0.5–0.6 bar. After all the IgY solution was loaded, kept the fluid bed running for 20 more minutes to dry the beads. Then 10% water-soluble Eudragit S100 solution (Eudragit S100 99.4 g, 1 mol/l ammonia solution 68 ml, triethyl citrate 49.9 g, talcum powder 49.7 g, H2O to 1 l) was sprayed into the fluid bed at 1 mg/ml/min with the aerosolization pressure at 0.4–0.6 bar. The coated microbeads were dried in the fluid bed for 20 min and recovered from the bed. For the organic solvent-soluble Eudragit S100, the coating solution was prepared by mixing 6.25 g Eudragit S100, 1.25 g triethyl citrate, 0.625 g talcum powder and 92 g 95% ethanol, the coating parameters were the same as those of the watersoluble Eudragit S100. IgY loading efficiency measurement IgY content of the beads was determined after completely dissolving prepared microbeads in phosphate buffer (pH 7.4). The concentrations of IgY were determined by Bradford protein assay kit (BCA method) bought from Genstar Biosolutions (Beijing, China). The ratio of the amount of the IgY to the weight of MCC was considered as loading efficiency of the microbeads. The coating ratio of Eudragit S100 was determined by the weight increased of the coated microbeads compared with the weight of the IgY-loaded beads before coating. Stability of IgY in the microbeads Stabilities of the IgY-loaded microbeads at different pH values and temperatures were determined by dissolving the IgY-loaded microbeads in buffers of different pH values between 2 and 13 at room temperature different temperatures between 25 C and 70 C for 3–24 h. The concentration of
DOI: 10.3109/10717544.2015.1022836
Microbeads of chicken yolk antibodies against Clostridium difficile toxin A
total protein was assayed by BCA method, the concentration of biofunctional IgY in the buffer was assayed by EIA method at 3, 12 and 24 h. For long-term stability of the IgY-loaded microbeads, the microbeads were stored in an incubator at 25 C for over 6 months. The functional IgY content of the microbeads was assayed by EIA method every month after dissolved the microbeads in PBS (pH 7.4) at room temperature for 3 h.
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In vitro release of the coated IgY microbeads For in vitro release assays, microbeads with known amount of IgY in a basket was put into the dissolution beaker containing 900 ml of 0.1 mol/l HCl solution of a dissolution apparatus (RZQ-8c, Tianfa Technology Inc. Tianjin). The beaker was rotated at 100 rpm and the temperature was fixed at 37 C + 0.5 for 2 h to mimic stomach release of the IgY. Then the basket was transferred to a new beaker containing 900 ml of PBS solution (pH 6.8) as the artificial intestinal fluid for 6 h. The pH value of the artificial intestinal fluid was adjusted to 7.2 to make the artificial colonic fluid by adding 0.1 mol/l of NaOH. The microbeads were kept in the artificial colonic fluid for 4 h. Five hundred microliter of the solutions was sampled every 30 min for the assays of IgY concentration by BCA method, the biofunctional IgY concentration was assayed by EIA methods using recombinant antigen as described in Step 2. The in vitro release of IgY-loaded microbeads in the enterosoluble capsule (An Hui Huangshan Capsule Inc., Huangshan, China) was also assayed. In vivo release of the coated IgY microbeads Fifteen Wistar rats were divided into five groups for the in vivo release assay of IgY-loaded microbeads. A tube of 2-mm diameter with an iron wire inside was inserted into the
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stomach of the rat first, then the iron wire was pulled out, the tube was connected to a 5-ml pipet tip, 10 mg of IgY-loaded microbeads was administrated into the tube through the tip, then the tube was connected to a syringe filled with 1 ml PBS to push the miceobeads down to the stomach of the rat. Three rats in each group of the animals were sacrificed every 2 h. Stomach, intestine, caecum and colon were collected separately from each rat. Blood sample from each rat was also collected from the eyes before the rat was sacrificed. After visual examination for the distribution and integrity of the microbeads in the organs, 10 ml of PBS (pH 7.4) was added to each sample and homogenized. After supersonication and centrifugation at 10 000 rpm for 30 min at 4 C, the supernatant from each sample was recovered for the assay of IgY concentration in the organs. The in vivo release efficiency was determined by the amount of recovered IgY to the amount of IgY administrated. The concentrations of IgY from different organs were assayed by EIA methods using recombinant ToxA–C antigen.
Results ToxA–C antigen-specific antibodies could be detected in the chicken yolk after the second immunization, the titer of the IgY in the egg yolk reached 1:50 000 after the third immunization and could last until 190 days. The titer of the IgY dropped dramatically after 196 days (Figure 1), 100 mg of IgY could be purified from each egg (Figure 1A). The purified IgY could neutralize the biological activity of ToxA–C in the rabbit intestine loop test by significantly reducing the ratio of weight/length (g/cm) of the ligated ileum loops as shown in Figure 1(C) (p50.05). The IgY was loaded on the MCC microbead cores in the fluid bed, the weight of the microbeads increased 40% after
Figure 1. Purification of IgY antibodies and the function assay. (A) SDS–PAGE analysis of purified IgY against toxin A. (B) The titer of chicken yolk antibodies against ToxA–C in eggs. (C) The injection ToxA and IgY antibodies of intestinal loops: (1) 100 mg/ml ToxA, (2) 50 mg/ml ToxA, (3) normal saline, (4) 100 mg/ml ToxA and IgY antibodies and (5) 50 mg/ml ToxA and IgY antibodies.
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Figure 2. Stability of IgY in the loaded microbeads. (A) pH stability of IgY in the loaded microbeads. The IgY-loaded microbeads were suspended in PBS buffer with different pH (2–11) value for 24 h, the buffer was aliquoted for ELISA assay to determine the binding activities of the IgY antibodies at 1, 12 and 24 h. IgY antibodies were stable between pH 4–10. (B) Temperature stability of IgY in the loaded microbeads. IgY-loaded microbeads were put in the incubators of different temperatures for 1, 12 and 24 h, then the IgY antibodies were dissolved in PBS (pH 7.4) and the antigenbinding activities of the IgY antibodies were assayed by ELISA. IgY antibodies were stable560 C. (C) Long-term storage stability of IgY at room temperature. IgY-loaded microbeads were put in the incubator at 25 C without light for 1–6 months, then the IgY antibodies were dissolved in PBS (pH 7.4), antigen-binding activities of IgY antibodies were assayed by EIA. IgY antibodies were stable for at least 6 months if stored at room temperature.
loading for 2 h, which equaled to 21% of IgY. No more IgY could be loaded to the microbeads even the loading time of the fluid bed was extended. The IgY-loaded microbeads were stable between pH 4 and pH 10. No significant decrease of the antigen-binding activities of IgY was observed after the microbeads was dissolved in the PBS buffers of different pH values for 24 h. The IgY was stable for 3 h at pH 3, after 12 h, the binding activity of IgY decreased dramatically. For pH 11 and above, the IgY lost its binding activity rapidly (Figure 2A). However, no significant difference was observed for the total protein concentrations at different pH values assayed by standard Bradford protein assay (BCA method, data not shown). The IgY-loaded microbeads showed same temperature stability as the IgY molecules. No significant decrease of the antigen-binding activity of the IgY in the microbeads after kept at as high as 60 C for 24 h compared with that of the microbeads at 25 C after dissolved in PBS (pH 7.4). The IgY
was stable for at least 1 h at 70 C, after 12 h, a significant decrease of the biological activity of the IgY was observed (Figure 2B). At 25 C, the IgY-loaded microbeads were stable for at least 6 months as shown in Figure 2(C), no significant difference of antigen-binding activity was observed. No difference was observed for the IgY concentrations dissolved in the PBS buffer at different temperatures as assayed by BCA method (data not shown). The IgY-loaded microbeads were coated with either water dispersible or organic solvent dispersible Eudragit S100 by the fluid bed (Figure 3). As shown in Figure 3(D), the antigen-binding activity of the IgY in the coated microbeads was not affected either in the water dispersable or organic solvent dispersable Eudragit S100 compared with that of the non-coated IgY. Five lots of IgY-loaded microbeads were coated with the water dispersable Eudragit S100, the weight of the microbeads increased by 15%, 20%, 25%, 30% and 35%, respectively. The average size of the coated IgY microbeads was 1.3 mm.
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Microbeads of chicken yolk antibodies against Clostridium difficile toxin A
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Figure 3. Eudragit S100 coated IgY microbeads. (A) The unloaded beads. (B) IgY-loaded beads. The loading efficiency was 21%. (C) Coated microbeads. The coating rate was 35%. (D) The effect of coating process on the IgY biological activity in the microbeads. (1) Drug-loaded microbeads, (2) coated pellets of Eudragit S100 (aqueous dispersion solvent) and (3) coated pellets of Eudragit S100 (organic solvent).
As assayed by standard BCA method, no IgY was released from the 35% lot of Eudragit S100 coated IgY microbeads in the artificial stomach fluid in 2 h, in the artificial intestinal fluid, 14% IgY was released from the microbeads after 6 h. Over 90% of IgY was released in the artificial colonic fluid within 2 h. For the 15%, 20%, 25% and 30% lot of coated IgY microbeads, the total release of IgY in the artificial stomach fluid were 74%, 53%, 38% and 11%, respectively. In the artificial intestinal fluid, the total release of IgY for the four lots were 93%, 68%, 62% and 36%, respectively, which were not acceptable. Only the 35% lot of Eudragit S100 coated microbeads showed colonic-specific release of IgY (Figure 4A). For the in vivo release of IgY, microbeads could be found mostly in the stomach at 2 h, a few microbeads could also be observed in the intestine of the rats. At 4 h, the microbeads were mostly distributed in the intestine, some in the stomach and colon. At 6 h, most of the microbeads were found in the colon. At 8 h, the microbeads were distributed in the colon and caecum of the rat. At 10 h, most of the microbeads were excreted but quite a few of the microbeads could still be found in the colon and caecum. The release of IgY at different parts of the digestion system was showed in Figure 5. At 4 h, 3.8% of the IgY could be released in the intestine. At 6 h, 24.4% of
the IgY was detected in the intestine and 61.5% of the IgY was found in the colon. At 8 h, 87.5% of the IgY was released in the colon. After 10 h, only 4% and 5% of IgY could be detected in the intestine and colon, respectively. No IgY could be detected in the rat plasma at any time (Figure 5).
Discussion As the major cytotoxin and enterotoxin of C. Difficile, toxin A is responsible for the binding of colonic mucosa cells and mediates transportation of the toxins into the cells, thus causes the symposiums of CDAD. Molecules which can block the recognition of toxin A to the carbohydrate receptors of the colonic mucosa cells should be able to protect the patients from CDAD (Kink & Williams, 1998). Using recombinant expressed receptor-binding domain of toxin A, we have generated egg IgY which can neutralize the toxicity of ToxA– C in vitro. The ToxA–C antigen-specific IgY can protect the rabbit intestine from liquid accumulation caused by the ToxA–C injection. Although the stability of IgY is much higher than other type of antibodies, it is still sensitive to the low pH condition of the stomach and proteinases in the intestine. Carbonate buffer is required for orogastric administration of IgY in
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Figure 4. In vitro release of ToxA–C antigen binding-specific IgY from the microbeads. (A) In vitro release The IgY-loaded microbeads. (B) In vitro IgY release from the microbeads in exterosoluble capsules. The IgY-loaded microbeads or the capsules were suspended in artificial gastric fluid for 2 h, artificial intestinal fluid for 6 h and artificial colonic fluid for 4 h, the fluids were sampled every 30 min to detected IgY concentrations using EIA and BCA methods.
animal models to protect the IgY from degradation in the stomach (Roberts et al., 2011). Colonic-specific delivery of IgY can protect ToxA–C antigen-specific IgY from degradation in the stomach and intestine, therefore protect the animal from the colonization of C. difficile in the colon. IgY-loaded microbeads were prepared using fluid bed system. IgY could be loaded on the MCC core at maximal 21% of the microbead weight. The surface of the IgY-loaded microbeads was uneven compared to the smooth shape of the MCC core. As large protein molecule, the IgY solution has high viscosity, it cannot form tight and regular structure on the MCC core like small molecular drugs, powder of IgY broken down from the microbeads could be found from the bottom of the fluid bed. Although 50% (W/W) of talcum powder was added as anti-tackiness agent, the maximal loading efficiency of IgY on the MCC core was 21%, which
was lower than that of the small molecule drugs. To increase the loading efficiency of IgY to the carrier of colonic-specific delivery formulation, other agents or technologies may be necessary to be applied, e.g. pectinate beads (Atyabi et al., 2005). The IgY in the microbeads is stable in broad ranges of pH and temperature. The antigen-binding activity of the IgY did not change between pH 4–10 C and 25–60 C in 24 h. No significant decrease of antigen-binding activity of the IgY microbeads was observed after stored for over 6 months at 25 C. In addition, the 95% ethanol solvent used in the coating process did not affect the antigen-binding activity of the IgY molecules, either, indicating that IgY is suitable for the development of oral administrative formulation. Because of the rough surface of the IgY-loaded microbeads, big amount of Eudragit S100 was needed to coat the
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Microbeads of chicken yolk antibodies against Clostridium difficile toxin A
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IgY is a large protein molecule with high immunogenicity. If absorbed in the blood through the digestion system, severe allergic reaction might be evoked in the patients. No detectable IgY activity was found in the animal plasma during the in vivo release assay, indicating that IgY cannot be absorbed by the colon mucosa cells, extra IgY molecules will be excreted with no harm to the patients and the environment.
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Conclusion
Figure 5. In vivo IgY release in the rat digestion system. The rats were intragastrically administrated with same amount of microbeads containing 10 mg IgY against the ToxA of C. difficile. Stomach, intestine, caecum and colon samples were collected every 2 h. Blood sample from each rat was also collected. After visual examination, 10 ml of PBS (pH 7.4) was added to each sample and homogenated. After supersonication and centrifugation at 10 000 rpm for 30 min at 4 C, the supernatant from each sample was recovered for the assay of IgY concentration in the organs using ELISA method.
microbeads completely. Only the 35% lot of coated IgY microbeads showed colonic-specific release in the in vitro release assay. Eudragit S-100 is a pH sensitive material which can be dissolved at pH46.8 (Ramasamy et al., 2013; Tsai et al., 2013), so active compounds in the microbeads coated with Eudragit S100 should not be released in the stomach (pH54) and intestine (pH & 6.8). However, a large percentage of IgY was released earlier in the artificial stomach and intestinal fluid due to the incomplete coating of IgY microbeads in the other four lots of IgY-loaded microbeads. No significant improvement was observed when the coated IgY microbeads were filled into enterosoluble capsule. The capsule could protect the IgY from releasing in the artificial stomach fluid, but not in the artificial intestinal fluid (Figure 4B). Although mouse and hamster were used as the infection model of C. difficile, however, these animals are too small for the intragastric administration of microbeads, so rat was selected for the in vivo release of IgY microbeads. The results of in vivo release of coated IgY microbeads was consistence with the results of in vitro assay. The microbeads stayed in the stomach of the rat for 2 h and then migrated to intestine, colon and caecum subsequently. IgY activity could be detected in the intestine between 4 and 6 h at low percentage (3.8– 24.4%), but 87.5% in the colon at 8 h, Eudragit S100 could protect the IgY from releasing and degradation in the stomach and the intestine, almost 90% active IgY was released in the colon from the microbeads coated with the pH sensitive Eudragit S100, which demonstrated colonic-specific delivery of active ToxA–C antigen-specific IgY in rat. Future in vivo assays are needed to test the anti C. difficile colonization activity of the IgY microbeads in the colonic mucosa of animal model challenged with lethal dose of C. difficile spores.
In this study, we have generated chicken IgY antibodies against the receptor-binding domain of C. difficile toxin A, which could neutralize the toxicity of the toxin. A colonicspecific delivery formulation of IgY microbeads coated with pH sensitive coating material Eudragit S100 was developed, the loading efficiency of IgY on the MCC core was 21%. The IgY-loaded microbeads are stable in broad ranges of pH and temperature. IgY microbeads coated with 35% of Eudragit S100 showed colonic-specific release of active IgY both in vitro and in vivo.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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