Impact of Orally Administered Microcapsules on Gastrointestinal Microbial Flora: In-Vitro Investigation Using Computer Controlled Dynamic Human Gastrointestinal Model

Abstract

Oral administration of artificial cell microcapsules has been proposed for various therapy procedures using biologically active materials. Recently we have designed novel APPPA microcapsules using alginate, poly-L-lysine, pectin, poly-L-lysine and alginate that have shown superior oral delivery features. This article investigates, in-vitro using a computer controlled dynamic gastrointestinal (GI) model, effects of APPPA microcapsules on health of gastrointestinal (GI) microbial flora. The impact of APPPA microcapsules on GI bacterial population, total anaerobes, total aerobes, Escherichia coli, Lactobacillus sp. and Staphylococcus sp. has been analyzed. In addition, the effects of microcapsules on GI microbial extracellular enzymatic activities have been investigated. Result shows the altered activities of microbial flora and enzymes due to the use of APPPA microcapsule. The most disparity is observed in the colon ascendans microbial activities. This study would have significant impact on future microcapsule design. However, further in-vivo studies are required.

Keywords:

• APPPA
• Alginate
• Artificial cell microcapsule
• Enzymes
• GI enzymes
• GI model
• Oral delivery
• Pectin

INTRODUCTION

Oral delivery provides practical advantages over other administration routes. It is the easiest route of administration, provides the most compliance for patients and eliminates the need for trained staff and equipment [[1]]. However the bioavailability of active materials in gastrointestinal tract is normally low because of several barriers in GI tract including proteolytic enzymes and high acidic condition of stomach [[2]]. Artificial cell [[3], [4]] microencapsulation technology is a potential solution to make the oral delivery of therapeutic agents possible [[5]]. In this system biologically active agents are entrapped in a protective barrier, which isolates them from the surrounding environment, including immune system of host and harsh condition of GI tract. Commonly this barrier is a polymeric ultra-thin membrane with selective permeability, which provides exchange of essential nutrients and waste and therapeutic products [[6-8]].

The idea of using artificial cell microencapsulation technology evolved many years ago [[9]]; since then research has been conducted to optimize the materials for membrane formation using different or modified polymers to improve mechanical and chemical stability and permeability and a few addressed the oral delivery of encapsulated proteins and enzymes [[10-14]]. And there are several diseases that can be potentially treated using artificial cell microcapsules. Thus, understanding the impact of orally administered microcapsules on native gastrointestinal (GI) bacterial flora and their activities is crucial. Currently there is a clear lack of data on impact of orally administered microcapsules on gastrointestinal (GI) microbial flora and their enzyme activities, which is one of the main goals of this study.

In this study, we use a novel microcapsule that we recently designed that has shown superior oral delivery features as model microcapsule [[15]]. Specifically, in the present we analyze the effects of oral delivery of the novel APPPA microcapsule on enzymatic activity and microbial contents of gastrointestinal (GI) tract in vitro. For this purpose, a computer controlled dynamic human gastro intestinal (GI) tract model has been applied (Figure 1). This in-vitro GI tract model is a simulator of the human (GI) tract, and consists of five vessels. Each vessel represents a part of the GI tract; stomach, small intestine and colon can be simulated in this model and valuable information can be potentially obtained in the early steps of characterizing the novel microcapsule [[16-18]]. In this article, the content of total anaerobic, total aerobic, Escherichia coli, Lactobacillus sp. and Staphylococcus sp. exit in GI model, which when exposed to microcapsules are analyzed and extracellular activity of β-galactosidase, β-glucosidase, β-glucuronidase, α-galactosidase and α-glucosidase has been investigated.

Figure 1 Schematic representation of the dynamic simulated human gastro-intestinal (GI) model. Vessel 1: Stomach; Vessel 2: Small intestine; Vessel 3: Ascending colon; Vessel 4: Transverse colon; and Vessel 5: Descending colon.

MATERIALS AND METHODS

Ca-alginate Beads Preparation

Microcapsules were prepared using an INOTECH Encapsulator. Alginate solution 1.65% (w/v) (Sigma-Aldrich, low viscosity) was loaded in a 60 ml syringe and extruded through 300 µm nozzle at frequency of 1052 HZ and voltage of 1.000 kv. Alginate droplets were collected in 0.1 M CaCl2 solution and stirred for 10 minutes for gel hardening.

APPPA Microcapsules Preparation

Ca-alginate beads were incubated respectively in these solutions: 0.1% (w/v) poly-l-lysine (Sigma, MW 27400), 0.1% (w/v) pectin (Sigma, degree of esterification 25%), 0.1% poly-l-lysine, and 0.1% alginate for 10 minutes in each. They were washed after each layer 2 times with saline (0.85% w/v). Microcapsules were washed with saline and stored in 4°C. All solutions were prepared in saline 0.85% w/v.

Computer Controlled Human Dynamic GI Model

This apparatus has been designed to simulate the human GI and gastrointestinal microbial ecosystem. It consists of 5 double layer vessels; each vessel represents a part of the gastrointestinal tract (Figure 1). The reactor was modified with the model described by Molly et al. [[16]]. Briefly, in each vessel the condition of temperature, pH, volume and retention time are simulated and computer controlled. The first vessel that works as stomach is fed by sterilized GI model medium [[16]]; food is passed to the next vessel, which represents the small intestine, while simulated pancreatic juice is added (Oxgall 6 g/l, Difco; Pancreatin 0.9 g/l Across; NaHCO₃ 12 g/l Fisher). All vessels are maintained in anaerobic conditions by flushing nitrogen every day for 20 minutes; temperature is constant at 37°C and pH controllers keep pH at desired condition by addition of 0.1 mol/l HCl or 0.1 mol/l NaOH.

Methods of Microbial Analysis in GI Model

The first 2 vessels of GI model are maintained in sterilized conditions, food is autoclaved and pancreatic juice is sterilized; therefore, microbial analysis was performed by taking samples of the last three vessels. While the GI model was setting up, the last three vessels were inoculated with human fecal suspension. In order to perform microbial analysis, a range of different agar media was prepared to enumerate formed colonies of various bacteria: brain heart infusion (BHI) agar (Difco) for total aerobes, BHI agar with cystein hydrochloride (Merck) for total anaerobes, MacConkey agar (Difco) for Escherichia coli, Mannitol salt agar (BBL) for Staphylococcus sp. and Rogasa agar (Difco) for Lactobacillus sp. Vessel 3 simulates colon ascendans with volume of 400 ml, pH of 5.5–6 and retention time of 9 hours. Considering the ratio of food materials and human body fluids, a suitable amount of APPPA microcapsules was weighed and exposed to liquids of this vessel and stored in anaerobic conditions; one container was considered for control that contains liquids without microcapsules. In certain time intervals, liquid samples were taken of each container and diluted serially in physiology solution (0.85%). Three plates were inoculated with 0.1 ml sample of suitable dilution. The plates were incubated in certain conditions. MacConkey agar plates for Escherichia coli were incubated at 43°C incubator in aerobic condition for 24 hours. BHI agar plates for total aerobes and Mannitol salt agar for Staphylococcus sp. were incubated at 37°C incubator for 24 and 48 hours, respectively. BHI agar and Rogosa agar plates were incubated in anaerobic condition at 37°C incubator with a gas atmosphere (80% N₂, 10% CO₂). The same method was applied for vessel 4 and vessel 5, which simulate colon transversum and colon descendans, respectively. Total volume of vessel 4 is 800 ml, pH is 6–6.4 and retention time is 18 hours and for vessel 5 total volume is 500 ml with the pH of 6.6–6.9 and retention time of 11 hours.

Enzyme Analysis of GI Model

Enzyme analysis specifically of the activity of β-galactosidase, β-glucosidase, β-glucuronidase, α-galactosidase and α-glucosidase was determined using methods described previously by others [[19]]. The substrates for these enzymes are ρ-nitrophenyl-β-D-galactopyranoside, ρ-nitrophenyl-β-D-glucopyranoside, ρ-nitrophenyl-β-D-glucuronide, ρ-nitrophenyl-α-D-galactopyranoside, ρ-nitrophenyl-α-D-glucopyranoside respectively (Sigma). Solution of substrate was prepared in phosphate buffer (0.1 mol/l pH 6.5) and stored in − 20°C.

As described for microbial analysis, considering the ratio of food materials and human body fluids, a suitable amount of APPPA microcapsules was weighed and exposed to liquids of each vessel and stored in anaerobic conditions; one container was considered for control, which contained liquids without microcapsules. In certain time intervals, samples of liquids were centrifuged at 10000 g for 10 min. 100 µl supernatant was transferred to a well of 96-well plate with 100 µl of 5 mmol/l solution of substrate. The absorbance was read and recorded at 405 nm immediately and 30 min after incubation at 37°C. Extracellular activity of these enzymes was obtained using absorbance values (405 nm) against a calibration curve of ρ-nitrophenol (Sigma) concentrations versus absorbance.

RESULTS

The Effects of Microcapsules on Colon Ascendans Microbial Flora

We investigated the effect of APPPA microcapsules on colon ascendans microbial flora and results are shown in Table 1. Result shows no significant effects in number of total aerobes after 6 and 12 hours APPPA microcapsules exposures. In addition, no significant difference in population of Escherichia coli after 6 and 12 hours APPPA microcapsule exposures compared to control group was observed.

Table 1. Effect of APPPA microcapsule on colon ascendance microbial population. The values have been shown in log colony-forming unit/ml (log CFU/ml)

	APPPA			Control
	0 h	6 h	12 h	0 h	6 h	12 h
Aerobes	3.16 ± 0.1	2.93 ± 0.01	2.94 ± 0.03	3.16 ± 0.1	2.86 ± 0.04	2.91 ± 0.11
Ecoli	3.28 ± 0.09	3.17 ± 0.09	3.4 ± 0.03	3.28 ± 0.09	2.83 ± 0.2	3.29 ± 0.18
Anaerobes	3.11 ± 0.05	2.99 ± 0.04	3.03 ± 0.01	3.11 ± 0.05	3.0 ± 0.03	2.97 ± 0.03
Lactobacilli	2.63 ± 0.04	2.56 ± 0.06	2.58 ± 0.6	2.63 ± 0.04	2.67 ± 0.1	2.55 ± 0.02

A reduction in total anaerobe number after 6 and 12 hours, however, was observed in APPPA microcapsules compared to control. At 6 hours the enumeration of Lactobacillus shows more bacteria in control compared to those exposed to APPPA microcapsules. However, after 12 hours the difference was not significant between control and APPPA groups.

The Effects of Microcapsules on Colon Transversum Microbial Flora

Results in Table 2 indicate the microbial analysis of colon transversum after APPPA microcapsule administration. Result shows that the amount of total aerobes has been almost reduced by the time. Clear reductions in the number of Escherichia coli after 24 hours of exposures were observed. The enumeration of Staphylococcus sp. indicates that the fluctuation in different times between microcapsules and control is not obviously high. Furthermore, the total anaerobes numbers were also clearly lowered. Lactobacillus sp. exposed to APPPA microcapsules were found reduced at 12 and 24 hours compared to control.

Table 2. Effect of APPPA microcapsules on colon transversum bacterial populations. The values have been shown in log colony-forming unit/ml (log CFU/ml)

	APPPA				Control
	0 h	6 h	12 h	24 h	0 h	6 h	12 h	24 h
Aerobes	3.4 ± 0.04	3.27 ± 0.02	3.3 ± 0.002	3.21 ± 0.02	3.4 ± 0.04	3.36 ± 0.01	3.37 ± 0.03	3.16 ± 0.1
Ecoli	3.28 ± 0.04	3.27 ± 0.06	3.24 ± 0.02	3.15 ± 0.04	3.28 ± 0.04	3.17 ± 0.06	3.31 ± 0.02	3.07 ± 0.01
Staph.	2.97 ± 0.05	2.49 ± 0.007	2.74 ± 0.02	2.6 ± 0.08	2.97 ± 0.05	2.79 ± 0.08	2.77 ± 0.08	2.54 ± 0.07
Anaerobes	3.41 ± 0.04	3.36 ± 0.01	3.29 ± 0.03	3.2 ± 0.05	3.41 ± 0.04	3.4 ± 0.05	3.4 ± 0.03	2.99 ± 0.01
Lactobacilli	3.29 ± 0.03	3.29 ± 0.02	3.22 ± 0.04	3.21 ± 0.06	3.29 ± 0.03	3.28 ± 0.08	3.38 ± 0.04	3.35 ± 0.03

The Effects of Microcapsules on Colon Descendans Microbial Flora

The influences of microcapsules on the microbial population of colon descendans were investigated and results are shown in Table 3. Result shows a reduction in total aerobes at 6 and 12 hours of exposures; however, the difference between APPPA microcapsules and control was not significant. The number of Escherichia coli colonies was found reduced after 12 hours significantly for APPPA microcapsules compared to control. At 6 hours the Staphylococcus sp. in control was found higher than that exposed to microcapsules. However, the population of total anaerobes did not vary significantly. The results for Lactobacillus sp. analysis show bacteria population exposed to APPPA microcapsules after 12 hours were found lower than control.

Table 3. Impact of APPPA microcapsule on colon descendans bacterial populations. The values have been shown in log colony-forming unit/ml (log CFU/ml)

	APPPA			Control
	0 h	6 h	12 h	0 h	6 h	12 h
Aerobes	3.16 ± 0.09	2.97 ± 0.02	3.0 ± 0.08	3.16 ± 0.09	3.02 ± 0.05	3.06 ± 0.05
Ecoli	3.0 ± 0.13	3.03 ± 0.04	2.84 ± 0.02	3.0 ± 0.13	3.19 ± 0.001	2.86 ± 0.05
Staph.	3.11 ± 0.009	2.86 ± 0.1	2.75 ± 0.03	3.11 ± 0.009	3.09 ± 0.01	2.79 ± 0.01
Anaerobes	3.22 ± 0.12	3.26 ± 0.15	2.9 ± 0.02	3.22 ± 0.12	3.02 ± 0.19	2.88 ± 0.12
Lactobacilli	2.91 ± 0.01	2.89 ± 0.02	2.71 ± 0.05	2.91 ± 0.01	2.87 ± 0.005	2.88 ± 0.09

The Effects of Microcapsules on Colon Ascendan Enzymatic Activities

Figure 2 shows the results of the influence of APPPA microcapsules on colon ascendan enzymatic activity. At time 0 experiments start assuming microcapsules have not induced any effects on enzymatic activity. After 6 hours, the activity of enzymes did not change significantly compared to 0 hours. The most difference is observed in α-galactosidase from 62 at 0 hours to 118 (U/l) at 6 hours for APPPA microcapsules compared to control were observed. The values for APPPA samples and control were similar. However, after 12 hours of exposure, the difference between microcapsules and control was found more obvious for α-galactosidase. β-glucosidase activity after 12 hours of exposures activities were zero. Activity of α-glucosidase exposed to APPPA microcapsules for 12 hours was 17 (U/l) compared to control activities of 205 (U/l). Thus, microcapsule significantly diminishes α-glucosidase expression (Figure 2).

Figure 2 Impact of APPPA microcapsules on colon ascendance extracellular microbial enzymes.

The Effects of Microcapsules on Colon Transversum Enzymatic Activities

Results in Figure 3 indicate enzyme activity analysis in colon transversum after exposure to APPPA microcapsules. After 6 hours of exposure the activity of all enzymes was found reduced compared to control at time 0 hours except for β-glucosidase. The β-glucosidase enzyme for samples with no microcapsules was found increased from 566 at 0 hours to 859 (U/l) after 6 hours. From hours 6 to 12 activities of all enzymes were found increased. We further investigated and found that the activities of all enzymes for samples containing no microcapsules from time 12 to 24 have been increased. At this point APPPA microcapsule groups, however, show a slight decrease in activity of β-glucosidase and α-galactosidase from 673 (U/l) after 12 hours to 664 (U/l) in 24 hours and from 151 (U/l) in 12 hours to 137 (U/l) after 24 hours, respectively, while at the same time in this sample activity of β-galactosidase was found increased and activity of α-glucosidase remained stable.

Figure 3 Impact of APPPA microcapsule on colon transversum microbial extracellular enzymes.

The Effects of Microcapsules on Colon Descendans Enzymatic Activities

The impact of microcapsules on enzymatic activity of colon descendans is shown in Figure 4. From time 0 to 6 hours there is a decrease in activity of all enzymes in both controlled and APPPA microcapsules samples observed. From 6 to 12 hours, activities of enzymes in sample with APPPA microcapsules were found increased slightly. However, activity of α-glucosidase decreased from 628 (U/l) to 516 (U/l) were observed in the same time periods. From 6 to 12 hours, activity of all enzymes were found increased significantly in control groups. And at this point activity of all enzymes in samples with no microcapsules were found clearly higher than sample with APPPA microcapsules.

Figure 4 Impact of APPPA microcapsule on colon descendans microbial extracellular enzyme activities.

DISCUSSION

Artificial cell microencapsulation with the combination of oral delivery offers advantages over the other drug delivery systems. This system takes advantage of oral delivery as a simple, easy and convenient route of administration and specific properties of microencapsulation such as immunoisolating and protecting the active agents.

The overall objective of microencapsulation is replacing deficient organs by encapsulating therapeutic cells, slow release of therapeutic materials from semi- permeable membrane and protecting the active agents from biodegradation. This technique can be applied in a very broad area in therapy [[20-30]]. For example, encapsulating secreting islets is a potential solution for diabetic patients to save them from painful daily injection of insulin. Another application of microencapsulation is in gene therapy. It can not be imagined that a patient can simply take a pill containing a gene of interest while conventional gene therapy has several difficulties including inefficient gene delivery to target site, unstable gene activity and host immune response. Development of this system can also save a large number of patients with organ deficiency. Conventional treatment for these diseases is usually painful and affects their normal life. Some efforts so far have been performed to make the oral delivery of therapeutic agents possible. Examples include oral delivery of recombinant peptide hormones and proteins, oral delivery of encapsulated enzymes for degrading urea, uric acid, and creatinine and Chitosan-Polyethylene-Glycol-Alginate microcapsules for oral delivery of hirudin [[31-33]]. Despite all these successive efforts, we have yet to find a microcapsule that can be applied to a broad range of applications.

As an answer to this necessity, a novel microcapsule has been designed consisting of multi-layer membrane using alginate, polylysine, and pectin. Alginate and polylysine are very commonly used biomaterials for encapsulation. Pectin is a natural polysaccharide present in the cell wall of most plants. Pectin increases the stability of microcapsules in acidic pH of GI tract [[34], [35]]. The combination of these materials may constitute a new chemical entity that needs performing in vitro analysis to prove biomaterials composing the membrane do not lower microbial population significantly, hence it does not affect the functionality of GI tract.

For the aim of implantation of live cells, some criteria should be considered including the effect of microcapsules on the surrounded environment. Therefore the variation in microbial population and enzymatic condition of GI tract was investigated using GI model. This model provides valuable and reliable [[17]] information. Some research groups have already applied GI models to study food or drug products [[36], [37]]. We applied this model to characterize the novel microcapsules by investigating the impact of microcapsules on the GI microbial flora. Vessel 3 of GI model simulates colon ascendans. In this vessel, the population of total aerobes and total anaerobes and Lactobacillus exposed to microcapsules has lowered after 6 and 12 hours. There was an exception in control samples for Lactobacillus plates after 6 hours. Enumeration of formed colony of bacteria at this time showed that samples without microcapsules indicated the higher number of bacteria. The variation observed in the number of formed colony for Escherichia coli is lower than other bacteria. In colon ascendans enzymatic analysis at 12 hours all enzymes in samples with no microcapsules showed higher activities. However, information obtained from enzymatic analysis does not clarify the main reason of microcapsules influence on lowering enzymatic activity. It is required to apply a proper method to monitor enzymatic activity in samples containing microcapsules. Enumeration of total aerobic bacteria showed that in the colon transversum within 24 hours, APPPA samples caused bacterial counts to remain relatively stable and controls slightly decreased. Numbers of counting Escherichia coli for 24 hours contact with APPPA samples and controls showed a slight drop. The enumeration of Staphylococcus sp. in the colon transversum simulating vessel showed that over a 24-hour period, cell counts for bacteria in contact with APPPA microcapsules were not significantly different from the controls with approximately 2.549 log CFU/ml. For total anaerobe bacteria, numbers of counting colonies remained somewhat steady for APPPA samples and lessened for controls over 24 hours. Lactobacilli counts in the colon transversum simulator were not significantly altered in 24 hours for APPPA samples at 3.216 log CFU/ml or for the control at 3.357 log CFU/ml. Colon transversum enzymatic analysis of indicated that activity of enzymes in samples contact with microcapsules decreased over 24 hours; however, these numbers for samples with no microcapsules increased by the time.

The total aerobe count on incubation with APPPA capsules and in controls was again relatively stable in 12 hours within the colon descendans vessel. The counts of E. coli in the vessel simulating colon descendans over 12 hours showed a decrease on contact with APPPA membrane microcapsule as well as with controls. For Staphylococcus sp., in 12 hours the numbers decreased for APPPA microcapsule samples and for control samples. The total anaerobe count, over a 12-hour period, for samples exposed to APPPA microcapsules and also for controls was almost stable. In Lactobacillus, the numbers slightly went down for samples that contain APPPA microcapsules whereas the controls stayed at around 2.883 log CFU/ml. Enzymatic analysis of colon descendans simulator showed that over 12 hours activity of enzymes in controls with no microcapsules increased while activity of enzymes in samples contact with APPPA microcapsules decreased by the time.

Although using GI model provides valid information in variation of bacteria population, knowledge obtained from enzyme activity is not sufficient to describe the impact of microcapsules on enzymatic activity. Whether microcapsules affect the functionality of enzyme producing cells or they might reduce enzyme activity directly, applying an accurate method is required for more realistic information.

CONCLUSION

The present study indicates the influence of APPPA microcapsules on microbial content of simulator of GI tract and on enzyme activities. Microbial analysis of simulated GI fluids has demonstrated that the difference between samples exposed to microcapsules and control is not very clear, indicating biomaterials used in new membrane do not affect bacterial flora of GI tract significantly. In enzymatic analysis of simulated GI tract there are some differences between microcapsules and control, which demands an accurate method to clarify the impact of microcapsules on enzyme activity. In the present study, APPPA microcapsule has shown encouraging results for oral delivery; however, supplementary research is required to evaluate this membrane for therapeutic application. In vivo experiments in experimental animal models are particularly required to consider for further research.

This work was supported by research grants (to SP) from the Canadian Institute of Health Research (CIHR). FA acknowledges PhD. Scholarship form Iranian Ministry of Health and Education. HC and TL acknowledge graduate fellowships from National Science Research Council (NSERC) of Canada. WO acknowledges research associate support from CIHR, Canada.