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ArticleJanuary 9, 2013

CLSM Method for the Dynamic Observation of pH Change within Polymer Matrices for Oral Delivery
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Biomacromolecules

Cite this: Biomacromolecules 2013, 14, 2, 387–393

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https://doi.org/10.1021/bm301569r

Published January 9, 2013

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Abstract

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If acid-sensitive drugs or cells are administered orally, there is often a reduction in efficacy associated with gastric passage. Formulation into a polymer matrix is a potential method to improve their stability. The visualization of pH within these materials may help better understand the action of these polymer systems and allow comparison of different formulations. We herein describe the development of a novel confocal laser-scanning microscopy (CLSM) method for visualizing pH changes within polymer matrices and demonstrate its applicability to an enteric formulation based on chitosan-coated alginate gels. The system in question is first shown to protect an acid-sensitive bacterial strain to low pH, before being studied by our technique. Prior to this study, it has been claimed that protection by these materials is a result of buffering, but this has not been demonstrated. The visualization of pH within these matrices during exposure to a pH 2.0 simulated gastric solution showed an encroachment of acid from the periphery of the capsule, and a persistence of pHs above 2.0 within the matrix. This implies that the protective effect of the alginate-chitosan matrices is most likely due to a combination of buffering of acid as it enters the polymer matrix and the slowing of acid penetration.

Subjects

Introduction

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When delivering acid sensitive bioactives orally there are often problems associated with the low pH of the stomach adversely affecting the administration. Examples of such bioactives include acid-labile drugs, such as penicillin G, (1) antigens, (2) or microorganisms, such as live bacterial vaccines or probiotic bacteria. (3, 4) Although the entrapment of these species in enteric dosage forms has been shown to improve drug stability, (5) or reduce cell death, (4, 6) the mechanisms of protection are usually hypothesized and very rarely demonstrated or quantified dynamically. These mechanisms are likely to be either a result of the polymer’s insolubility in acid, halting penetration of acid into the polymer matrix, or the buffering capacity of the polymers in the formulation raising the pH inside the matrices to a level which is no longer harmful to the encapsulated material. (7, 8)

When an enterically formulated bioactive is taken orally there is only a short period of transition through the esophagus (around 10–14 s). (9) Upon entry to the stomach, a lower pH is encountered, due to hydrochloric acid secretion by parietal cells found in the gastric epithelium. (10) The pH of the gastric juices is highly variable; it can reach as low as pH 1 (11) in fasted patients and as high as pH 5 in a fed state. (12) These gastric secretions also include some enzymes which assist in the digestion of foods, the most abundant of which is the proteolytic enzyme, pepsin. Transit through the stomach varies due to a range of factors including age, gender, and meal volume (13) but usually occurs within 1–2 h after ingestion of the meal. (14) The enteric formulation will then pass from the stomach and into the lower digestive tract where pH rises to near neutral, at which point it will deliver the bioactive.

To this date, there are limited studies describing the visualization of the pH distribution in pharmaceutical formulations. (15) These focus on the measurement of pH within PLGA microspheres, (16-19) PLGA films, (20, 21) tablets, (22) and within pellets. (23) For example, Fu et al. (17) have entrapped SNARF (seminaphthorhodafluor)/NERF (carboxy-2,7-dimethyl-3-hydroxy-6-N-ethylaminospiro [isobenzofuran-1(3H),9-(9H)xanthen]-3-one)-dextran dyes into hollow PLGA microspheres to measure the pH change within the particles as the polymers biodegrade. While this method is fit for their purpose, it cannot be used for a highly porous polymer matrix system, when there is the possibility of diffusion out of the matrix or where there would be direct contact between the dye and polymers. Indeed, more recent studies by Liu et al. and Li et al. (16, 18) describe confocal visualization of pH change within PLGA microparticles containing acid-sensitive proteins using pH-responsive dyes. This study encounters a fluorescence quenching effect due to the interaction of the dye with entrapped proteins. To resolve this issue, an estimation of quenching was required and additional experimentation was needed to validate this. A study by Pygall et al. (22) measured the pH in HPMC tablets during exposure to 0.1 M HCl containing 0.5% (v/v) universal pH indicator. This allowed for the visualization of the hydration of the pellet and gave an indication of the pH in those regions. While providing useful information in that study, this method is limited in that the dye must diffuse in from the periphery of the formulation, meaning that pH visualization is not possible in regions were no indicator is present.

In this study we have developed a method that is, to our knowledge, the only currently available reliable technique for accurately visualizing the pH in hydrated polymer matrices dynamically and which can be applied to a variety of porous systems. This method has been developed in order to better understand the behavior of hydrogel systems containing bioactives during exposure to low pH. In brief, a Gram-positive Bifidobacterium strain was labeled with two pH-sensitive fluorophores, which could be independently excited. The pixel intensity ratio of images taken by confocal laser-scanning microscopy (CLSM) can be color coded, which allows the visualization of pH within formulations during exposure to acidic solutions. This method could be applied to any system in which the bacteria may be suspended within a matrix. The use of these microorganisms as a carrier for the dyes means that issues, such as fluorescence quenching, surrounding excipient–dye interactions are removed, thus, widening the application over other methods (16-18, 23) that require the entrapment, or uptake, of a dye into the formulation. Another advantage of using bacterial cells as a model bioactive is that bacteria are of a large enough size (>1 μm) that they will not diffuse or “reptate” out of all but the most porous formulations. This improves on previous methods that rely on small dye molecules being retained by the polymer matrix, which is not often the case in systems which are submerged in liquid. The method presented in this study serves as a means of investigating the protective effect of enteric formulations under these conditions. Additionally, both of the dyes used are nonselective, so can be used for various microbes.

The strain of bacteria used, B. breve, is a rod-shaped, nonmotile anaerobe that naturally inhabits the human intestine. Like all Bifidobacterium strains, it is gram-positive as a result of the thick layer of peptidoglycan constituting its cell wall. In addition to this, B. breve produces polysaccharides upon its surface that form a bacterial capsule. (24) The acid sensitivity of these cells arises mainly from the denaturing of proteins within the cell at lowered cytoplasmic pH. These proteins constitute part of the cell’s structure and give it some enzymatic activity. (25) Thus, damaging these proteins contributes to the death of the cell.

One formulation which has been shown to protect acid-sensitive bacteria, such as Bifidobacterium strains, is the ionic alginate microencapsulation system. (7, 8, 26-33) This method has been shown to greatly reduce cell death during exposure to gastric pHs, (7, 8) and that the subsequent coating of the alginate matrix with the cationic polysaccharide chitosan has improved the bacteria’s survival even further. (8, 34) Though a buffering effect has been attributed as the cause of protection, (7, 8) this claim has not been substantiated by experimentation. Herein a technique is developed in order to understand the change in pH within this alginate-chitosan system better.

Materials

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Bifidobacterium breve

NCIMB 8807 (B. breve) was purchased from the National Collections of Industrial Food and Marine Bacteria (Aberdeen, U.K.). Alginate (19–40 kDa), fluorescein isothiocyanate (FITC), and low molecular-weight chitosan (103 kDa, degree of deacetylation: 85.6%) were purchased from Sigma-Aldrich (Gillingham, U.K.). pHrodo succinimidyl ester was purchased from Invitrogen (California, U.S.A.). Wilkins-Chalgren (WC) anaerobe agar and phosphate-buffered saline (PBS) were purchased from Oxoid (U.K.). Alginate was purified by microfiltration (0.45 μm sartorius filter) before use; all other reagents were used without further purification. Materials other than alginate and chitosan were sterilized by autoclaving; alginate and chitosan were sterilized by microfiltration.

Methods

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Viability of B. breve at pH 2–7

B. breve was streaked onto WC agar plates from a previously prepared cell bank and allowed to grow anaerobically (48 h, 37 °C). After growth, an aliquot of the bacteria was used to inoculate tryptone–phytone–yeast (TPY) broth (10 mL) and the culture incubated (22 h, 37 °C) with shaking to late log phase; during this time, the cells grew to ∼9 log(CFU)/mL. The cell suspension was then divided into aliquots (1 mL) and centrifuged (11000 rpm, 5 min) before resuspending the cells into TPY (1 mL) that was adjusted to a variety of pHs (1.0, 2.0, 2.3, 2.6, 3.0, 4.0, 5.0, 6.0, and 7.0) using 1 M HCl. These samples were incubated for 1 h at 37 °C and enumerated by serial dilution in PBS and spreading onto WC agar plates before anareobic incubation (48 h, 37 °C). During incubation, each viable cell present on the plate produced one colony. From this, the total number of viable cells could then be calculated by the multiplication of colony numbers by the dilution factor used. Colonies produced were white and circular with a region of translucency around the edges. Those colonies that did not appear as such were not counted.

Survival of Alginate and Alginate-Chitosan Encapsulated B. breve During Simulated Gastrointestinal Passage

B. breve was grown as before and centrifuged (3200 rpm, 10 min, 4 °C). The supernatant was removed and the cells resuspended in 2% (w/v) alginate solution. This polymer/cell solution (1 mL) was extruded through a 21G needle into 0.05 M CaCl₂ (50 mL) and allowed to harden (30 min). After this period, the capsules were removed by filtration. For chitosan-coated capsules, the alginate encapsulated cells were then placed in a 0.4% (w/v) chitosan (in 0.1 M acetic acid adjusted to pH 6 with 1 M NaOH) for 10 min. From a previous study, this should result in a chitosan coat of approximately 7 μm thickness. (8) After coating, the capsules were removed by filtration. The produced capsules were then incubated at 37 °C in simulated gastric solution (pH 2 with 1 M HCl, 0.2% (w/v) NaCl, 10 mL) with shaking for 1 h. This was followed by exposure to simulated intestinal solution (6.8% (w/v) KH₂PO₄, pH 7.2 with 1 M NaOH, 50 mL) with shaking at 37 °C for 3 h. Samples were taken at 0, 60, 120, 180, and 240 min and the cells in solution were enumerated by the previously described method. By the 240 min mark, the capsules had completely dissolved. To determine the initial cell concentration, for each experiment a separate 1 mL batch of capsules was taken directly after production and placed into PBS (100 mL) and incubated (1 h, 37 °C). After incubation, the capsules were placed into a stomacher (Seward stomacher 400 circulator, 250 rpm, 20 min) to ensure their complete dissolution. The initial cell concentration was determined by enumerating the cells in this suspension. Simulated gastric solutions were made to pH 2.0 to represent the low pH found in the nonfasted stomach. (35-37)

Preparation of pHrodo/FITC Labeled B. breve

B. breve was streaked onto WC agar plates from a previously prepared cell bank and was grown anaerobically (48 h, 37 °C). After growth, the bacteria were inoculated into TPY broth (10 mL) and incubated (37 °C, 22 h) with shaking, during which time the cells grew to ∼9 log(CFU)/mL. After incubation, the cells were centrifuged (3200 rpm, 10 min, 4 °C) and the supernatant was removed before resuspension in phosphate-buffered saline (PBS, pH 9, 10 mL). To this cell suspension, pHrodo succinimidyl ester (1 μL of a 10 mg/mL stock solution in DMSO) was added and conjugation was allowed to proceed in darkness (37 °C, 30 min) with shaking. The cells were then centrifuged (3200 rpm, 10 min, 4 °C) and the supernatant removed before washing with, and resuspending in, PBS (pH 8, 10 mL). This suspension was then centrifuged once more (3200 rpm, 10 min, 4 °C), the supernatant discarded, and the cells resuspended in PBS (pH 7.2, 10 mL), and FITC solution (1 μL of a 10 mg/mL solution in water) was added. The reaction was allowed to proceed in darkness (37 °C, 30 min) with shaking before centrifugation (3200 rpm, 10 min, 4 °C), washing with PBS (pH 7.2, 1 mL), and resuspending in PBS (pH 7.2, 10 mL) to ensure the removal of excess dye. The tube containing the cells was then placed into boiling water (5 min) before switching to ice–water (5 min) to kill the cells and remove any potential for cell division. This was performed in order to reduce the motility of the bacteria when being visualized by microscopy. As the bacteria used are gram-positive and have an extracellular capsule, it is unlikely this treatment led to a large lysis of cells. Additionally, microscopic observation of the post-treated cells gave no indication of this. The suspension was then divided into 1 mL aliquots before centrifugation (13000 rpm, 5 min) and subsequent removal of the supernatant.

Preparation of Alginate-Chitosan Microcapsules Containing pHrodo/FITC Labeled B. breve

Alginate solution (2% w/v in water, 1 mL, filtered using Minisart Sterile-R 0.45 μm microfilters) was added to pHrodo/FITC-labeled B. breve pellets produced as above and vortexed to ensure complete mixing. This solution was then extruded using a syringe and a pump (2.0 mL/min) into 0.05 M calcium chloride solution (50 mL) and was left to harden for 30 min before filtration. In the case of chitosan-coated alginate microcapsules these were then placed into chitosan solution (0.4% w/v in 0.1 M acetic acid adjusted to pH 6, 10 mL) and left to stand (10 min). These chitosan-coated alginate microcapsules were then removed from the mixture by filtration.

Calibration of Microscope

To extract the pH values from the CLSM images a calibration curve was first constructed. pHrodo/FITC-labeled B. breve produced previously was resuspended into PBS (1 mL) that was adjusted accurately to pH 2, 2.5, 3, 4, 5, and 7.2. These new suspensions were placed onto a coverslip and imaged using a Leica SP2 CLSM. Samples were excited with 488 and 546 nm lasers sequentially, corresponding to the excitation wavelengths of FITC and pHrodo, respectively. From these 8-bit images, the pixel intensity of the cells was determined using the onboard software (Leica Confocal Software).

Measurement of pH within Alginate-Chitosan Microcapsules

A single alginate or alginate-chitosan microcapsule containing pHrodo/FITC labeled B. breve was placed onto a purpose designed coverslip (consisting of a 50 mm Petri dish with a central section removed which was then replaced with a thin glass coverslip), submerged in simulated gastric solution (pH 2 adjusted with 1 M HCl, 0.2% (w/v) NaCl, 100 μL, 37 °C), and imaged using a CLSM at 488 and 543 nm excitation wavelengths sequentially at 0, 1, 5, 10, 15, 30, 45, and 60 min intervals during incubation at 37 °C. The images were then manipulated using MATLAB (7.11.0) so that the individual pixel intensity could be related to a pH, which was then color coded according to its value to produce “pH maps” of the microcapsules. To do this, first the two images taken at the different excitation wavelengths of the dyes were divided by one another using the Boolean Logic function on the onboard software. This gave each pixel an intensity equivalent to the fluorescence of pHrodo/FITC; these images were then converted to an equivalent matrix of pixel intensities (Figure 1). These intensities were then put into “bins” defined by the range of intensity values equivalent to a known pH, which were then color coded. For example, values in the matrix between 3.3 and 5.8 (equivalent to fluorescence of pHrodo/FITC for pH 6 and 5, respectively) were binned together and assigned the value 20, which was then colored light blue to code for pH 5–6. The values that defined the bins were taken from a previously established calibration curve (Supporting Information).

Figure 1. Conversion of pixel intensity to pH. Image on left is produced by the division of the pixel intensities of a picture showing pHrodo fluorescence by the corresponding FITC image. Image on right is the result of coloration of the left image based on the intensity of each pixel.

Results and Discussion

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B. breve was exposed to a range of pHs between 2 and 7 and the viability of the cells measured after 1 h incubation at 37 °C (Figure 2). Gastric emptying time is highly variable, but 1 h was chosen as reasonable residence time of a particulate formulation. (38) There was a steady decrease from 9.3 ± 0.4 log(CFU)/mL to 8.7 ± 0.2 log(CFU)/mL between pH 7 and pH 3, after which the number of culturable cells dropped to ∼4.7 log(CFU)/mL as the pH reached 2.3. At pHs less than or equal to 2, there was no detection of any viable cells (detection limit is 3 log(CFU)/mL). Based on previous research, it is likely that cell death has a kinetic dependency on time in acid. (39) This data shows that this particular probiotic strain is acid-sensitive, showing very low numbers of viable cells at pHs less than 3. The human stomach pH is often lower than this threshold, so these bacteria are unlikely to survive gastric transit.

Figure 2. Viability of *B. breve* against TPY medium adjusted to various pH after 1 h incubation at 37 °C; N = 3 ± standard deviation; limit of detection, 3 log(CFU)/mL; line intended as guide to eye.

Alginate microcapsules have been shown previously to offer a good level of protection to acid sensitive bacteria when exposed to simulated gastric solutions and coating these matrices with chitosan improves survival further. (7, 8, 28, 30, 34, 40, 41) Our data, shown as release of viable cells to estimate the number of cells that may be deposited in the intestine in vivo, is consistent with these findings (Figure 3). During the first 60 min, no viable cells were detected in the simulated gastric solution of either pH 2.0 or 3.0. The enumeration of B. breve in simulated intestinal solution after exposure to pH 3.0 gastric solutions gave a release of viable cells amounting to 8.7 and 8.9 log(CFU)/mL for alginate and chitosan-coated alginate capsules, respectively (Figure 3). At pH 2, the viable cells released after 240 min amounted to 5.1 and 6.9 log(CFU)/mL for alginate and chitosan-coated alginate capsules, respectively. These values are high, relative to <3 log(CFU)/mL predicted in the control experiment (at pH 2 in Figure 2). The increased survival of cells in the chitosan coated system is believed to be due to an increase in buffering as the acid penetrates the capsule (7) and is also possibly due to reduced porosity at the capsule surface. (42) This provides a demonstration of the efficacy of this encapsulation system in protecting acid-sensitive bacteria. It should also be noted that the release of cells appears slower after exposure to pH 2 solution; this is most likely caused by the death of cells at the periphery first, which leads to a lag associated with the disintegration of the capsule from the extremities first. This would result in the release of the viable cells only once the region containing killed cells had disintegrated.

Figure 3. Release of viable cells from alginate and chitosan-coated alginate matrices during exposure to simulated gastric solution (pH 2.0/3.0, 60 min) followed by simulated intestinal solution (180 min) at 37 °C. By the 240 min mark, matrix dissolution was complete. Starting cell concentration included indicating maximum possible survival; N = 3 ± standard deviation; limit of detection, 3 log(CFU)/mL.

The pHrodo succinimidyl ester and FITC were conjugated to bacteria via amine residues that were present within the cell (Figure 4A). These dyes were shown to be present by UV–vis spectrofluorometry (λ_exc: 488 and 546, for FITC and pHrodo, respectively) and the images showed a change in pixel intensity of the cells when exposed to pH between pH 2 and 7 (Supporting Information). Conjugated pHrodo increased in intensity when the pH was decreased from 7 to 2 (Figure 4B), whereas the fluorescence intensity of FITC decreased (Figure 4C). These cells were then encapsulated within alginate and alginate-chitosan matrices before exposure to simulated gastric solution and visualization by confocal microscopy.

Figure 4. Schematic diagram of dye conjugation to amine moieties within bacteria, producing thiourea and amide moieties when reacting with pHrodo succinimidyl ester and FITC, respectively (A). Fluorescence of pHrodo (B) and FITC (C) when conjugated to *B. breve* showing variation of intensity with pH. For this experiment, a solution of fluorescently labeled *B. breve* conjugate (100 μL) at ∼9 log(cells)/mL was diluted into PBS (900 μL) adjusted to pH 2.0, 5.0, and 7.0. The fluorescence of conjugated pHrodo and FITC was then quantified by UV spectrofluorometry (λ_ex: 546 and 488, respectively) over the ranges shown. Fluorescence changes with pH are most commonly the result of protonation or deprotonation around a fluorophore’s pK_a. (43).

This method allows for the production of “pH maps” of matrices (Figure 5). These maps allow the visualization of the pH as the acid penetrates into the polymer network. This lets the viewer not only see the rate of diffusion of the acid, but also the color-coding, which allows for the identification of specific pH environments within the matrix. It should be noted that cells were killed after labeling to reduce movement during imaging. In the case of alginate capsules, a thick band of red, associated with pHs between 2.5 and 2 appears after only 1 min, accompanied by a clear bright blue region of pH 7–6 in the center of the matrix. As time passes, this central circle can be seen to darken, and the encroachment of the external red band thickens, showing the penetration of acid into the capsule. In the case of chitosan-coated alginate capsules, the appearance of the thick band of red on the perimeter of the capsule is considerably slower and the persistence of higher pH in the center is observably longer than in the uncoated matrices. These images imply a combination of buffering effects, signified by the presence of pH above that of the simulated gastric solution (pH 2), even at the periphery, and a slowing of acid penetration into the polymer network, most likely a result of the formation of an acid-gel. This example demonstrated the ability of our method to produce pH maps and elucidate pH changes occurring within matrices.

Figure 5. pH maps of an alginate and a chitosan-coated alginate microcapsule during exposure to simulated gastric solution at pH 2.0. Scale: 1 mm.

To obtain a better view of the pH distribution within the microcapsules, a close comparison of the pH within the matrix after 60 min (from a repeat of the experiment) is shown in Figure 6. In the case of alginate only matrices, there is a thick dark red color, associated with pHs nearing 2.0 around the perimeter of the capsules, in which it would be expected all cells to be dead. In the most part, there is an orange-red color, pertaining to pHs between 2.5 and 3 throughout the rest of the matrix. pHs between 2.3 and 3 correlate to an approximately 4.5 log(CFU)/mL survival of bacteria (based in Figure 2). In the case of alginate encapsulated cells, 60 min exposure to gastric solution resulted in a 5 log(CFU)/mL survival of cells, showing some consistency with our findings (Figure 3), though direct comparison is complicated due to the kinetic dependency of the viability studies. In the alginate-chitosan matrix, a thinner, lighter colored ring of red is seen around the perimeter, most likely due to increased buffering or reduced porosity. Toward the center of the capsules, there is the emergence of yellow and some blue regions associated with pHs up to 5, at which very high cell survival would be seen. This increased pH results in an increase in cell survival, which is seen in the simulated gastric viability assay (Figure 3).

Figure 6. Comparison of pH within alginate and alginate-chitosan matrices after 60 min exposure to simulated gastric juice at pH 2.0 (scale bar: 1 mm). Images taken from a separate experiment to those shown in Figure 5.

From the images captured on the confocal microscope it is possible to measure the thickness of the aforementioned ring of red, associated with pHs in the region 2–3, using image analysis software (ImageJ). These pHs should be associated with a relatively rapid cell death according to the viability testing already conducted (Figure 2). The thickness of the red band could then be used to give a representation of encroachment of low pH into the matrix (Figure 7). In both the alginate and chitosan-coated alginate there is a sudden penetration of acid into the periphery of the matrix, which slows over time. This nonlinear behavior is most likely a result of acid-gel formation at the periphery of the capsules during penetration, reducing the rate of diffusion into the matrix. The chitosan-coated alginate matrices seem to slow encroachment of the low pH into the gel, relative to the uncoated samples. The depth of penetration of low pH into the samples measures 367.6 ± 13.4 and 290.4 ± 17.0 μm for the uncoated and chitosan-coated alginate matrices, respectively. This reaffirms the qualitative findings discussed as there is a reduced area of pHs associated with a particularly rapid cell death in the chitosan-coated samples.

Figure 7. Encroachment of pH < 3.0 into alginate (open squares) and chitosan-coated alginate (closed circles) matrices during incubation in simulated gastric solution at 37 °C; N = 3 ± standard deviation, band thickness is the average of five measurements at equidistant points on the matrix.

Experimentally, this method is very quick and straightforward. The dyes used may be tailored to provide information at different pH ranges assuming that cell staining dyes with pH dependent fluorescence in this region may be found. There are currently numerous amine-active dyes available at a range of pHs which will allow for higher accuracy at particular pH ranges. Co-staining and pH determination by ratiometric means should make this method resistant to changes in bacterial concentration if it were to be applied with other systems or microbes.

Concluding Remarks

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A method for the reliable visualization of pH within polymer matrices has been developed. This method allows for the production of “pH maps” showing the distribution of pH within a cross-section of a hydrated polymer matrix. This method was then applied to alginate and alginate-chitosan matrices, which are commonly used for the protection of acid-sensitive bacteria. After demonstration of this protective effect in vitro, the pH environment within the matrices was visualized using pH maps. These maps revealed what we suggest to be a combination of buffering, which was seen to increase after coating with the basic chitosan and an encroaching of low pH from the periphery into the matrix rather than a bulk pH change, which slowed with chitosan coating. The use of microbes as carriers of fluorophores for this purpose offers an alternative to free dyes that may otherwise not be retained by the polymer network or the conjugation of dyes to the polymers, which may affect their fluorescence. It is also possible that microparticles could be used as an alternative, such as the polystyrene microparticles with surface-bound dyes used by Sánchez-Martin et al. (44) to measure intracellular pH. There are various studies that use pH-sensitive fluorophores conjugated to polymer particles, (45-47) which could be adapted and evaluated for the observation of pH within porous polymer matrices.

Supporting Information

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The calibration curve determined for the fluorescence ratio of pHrodo/FITC with pH. This material is available free of charge via the Internet at http://pubs.acs.org.

bm301569r_si_001.pdf (107.88 kb)

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Author Information

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Corresponding Authors
- Dimitris Charalampopoulos - †Department of Food and Nutritional Sciences and ‡Reading School of Pharmacy, University of Reading, Reading, RG6 6AD, United Kingdom; Department of Medical Physics and Bioengineering, University College London, Gower Street, London, WC1E 6BT, United Kingdom; Clasado Research Services Ltd., Science and Technology Centre, University of Reading, Earley Gate, Whiteknights Road, Reading, RG6 6BZ, United Kingdom; Email: [email protected] [email protected]
- Vitaliy V. Khutoryanskiy - †Department of Food and Nutritional Sciences and ‡Reading School of Pharmacy, University of Reading, Reading, RG6 6AD, United Kingdom; Department of Medical Physics and Bioengineering, University College London, Gower Street, London, WC1E 6BT, United Kingdom; Clasado Research Services Ltd., Science and Technology Centre, University of Reading, Earley Gate, Whiteknights Road, Reading, RG6 6BZ, United Kingdom; Email: [email protected] [email protected]
Authors
- Michael T. Cook - †Department of Food and Nutritional Sciences and ‡Reading School of Pharmacy, University of Reading, Reading, RG6 6AD, United Kingdom; Department of Medical Physics and Bioengineering, University College London, Gower Street, London, WC1E 6BT, United Kingdom; Clasado Research Services Ltd., Science and Technology Centre, University of Reading, Earley Gate, Whiteknights Road, Reading, RG6 6BZ, United Kingdom
- Teedah Saratoon - Department of Medical Physics and Bioengineering, University College London, Gower Street, London, WC1E 6BT, United Kingdom
- George Tzortzis - Clasado Research Services Ltd., Science and Technology Centre, University of Reading, Earley Gate, Whiteknights Road, Reading, RG6 6BZ, United Kingdom
- Alexander Edwards - †Department of Food and Nutritional Sciences and ‡Reading School of Pharmacy, University of Reading, Reading, RG6 6AD, United Kingdom; Department of Medical Physics and Bioengineering, University College London, Gower Street, London, WC1E 6BT, United Kingdom; Clasado Research Services Ltd., Science and Technology Centre, University of Reading, Earley Gate, Whiteknights Road, Reading, RG6 6BZ, United Kingdom
Notes
The authors declare no competing financial interest.

Acknowledgment

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This research was supported by Clasado Ltd. and the University of Reading.

References

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Cell survival of Lactobacillus rhamnosus GG entrapped in gelled whey protein isolate (WPI) micro-beads was elucidated relative to cells suspended in native WPI and free-cell controls during ex vivo porcine gastro-intestinal incubation. Probiotic gastric tolerance was investigated as a function of pH (2.0-3.4) and time with subsequent intestinal incubation (pH 7.2). Free cells showed no survival after 30 min ex vivo stomach incubation (≤pH 3.4), while native WPI enhanced survival by 5.7 ± 0.1, 5.1 ± 0.2 and 2.2 ± 0.2 log10 cfu mL-1 following 180 min incubation at pH 3.4, 2.4 and 2.0, resp. Protein micro-beads augmented ex vivo probiotic acid resistance (8.9 ± 0.1 log10 cfu mL-1) and demonstrated significant micro-bead adsorption capacity coupled with micro-bead digestion and controlled release of viable, functional probiotics within 30 min intestinal incubation. This technol. potentially envisions whey protein micro-beads as efficacious entrapment matrixes and binding vehicles for delivery of bioactive ingredients.
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Artificial cell microcapsule for oral delivery of live bacterial cells for therapy: design, preparation, and in-vitro characterization
Ouyang, Wei; Chen, Hongmei; Jones, Mitchell L.; Metz, Terrence; Haque, Tasima; Martoni, Christopher; Prakash, Satya
Journal of Pharmacy & Pharmaceutical Sciences (2004), 7 (3), 315-324CODEN: JPPSFY; ISSN:1482-1826. (Canadian Society for Pharmaceutical Sciences)
Bacterial cells can be engineered to synthesize a wide array of disease modifying substrates such as cytokines, vaccines and antibodies; however, their use as an orally delivered therapeutic is limited by poor gastrointestinal (GI) survival and instigation of immunogenic response. Artificial cell microcapsules have been well studied as a means to overcome such problems, however, presently obtainable microcapsules have limitations. This study summarizes a novel microcapsule design specifying its prepn. and GI stability in-vitro. Multilayer APPPA microcapsules were designed, prepd. and characterized in-vitro for bacterial cell oral delivery using Lactobacillus reuteri cells as a model. Microcapsule structural integrity, mech. stability, and GI survival studies were performed in simulated gastric (SGF) and intestinal (SIF) fluids in various pH conditions at 37.2° and compared with presently available alginate/poly-L-lysine/alginate (APA) microcapsules. HPLC was used for the microcapsule membrane permeability study. Results show that APPPA microcapsules can be prepd. for bacterial cell encapsulation and are stable in simulated GI conditions. No microcapsule damage was reported when exposed to SGF and SIF for 12 h at 250 rpm mech. shaking at 37.2°. In addn., 93.2±2.3% and 98.9±0.6% of microcapsules were undamaged after 24 h in SGF and SIF resp. Microcapsule pH stability results show that 92.8±3.1% of microcapsules remained intact at pH 1, 3, 5, and 7 and no damage was obsd. at pH 9.0 when challenged for 24 h. When exposed for 3 h with 250 rpm shaking at 37.2°, no damage of the microcapsules in SGF and SIF at pH, 1, 3, 5, 7, and 9 was obsd. Compared to APA microcapsules, APPPA membranes showed superior GI stability and permeability for cell encapsulation. Novel APPPA microcapsules have superior features for oral delivery of live bacterial cells and they can be used for various clin. applications. However, further study such as membrane permeability, cytotoxicity, immune protection capacity, and suitability for live bacterial cell oral delivery in-vivo is required.
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Gastrointestinal (GI) pH has been measured in 66 normal subjects using a pH sensitive radiotelemetry capsule passing freely through the gastrointestinal tract. Signals were recorded with a portable solid state receiver and recording system, enabling unconstrained measurements with normal ambulatory activities for up to 48 h during normal GI transit. Capsule position in the gut was monitored by surface location using a directional detector. Gastric pH was highly acidic (range 1.0-2.5) in all subjects. The mean pH in the proximal small intestine was 6.6 (0.5) for the first hour of intestinal recording. By comparison the mean pH in the terminal ileum was 7.5 (0.4) (p less than 0.001). In all subjects there was a sharp fall in pH to a mean of 6.4 (0.4) (p less than 0.001) as the capsule passed into the caecum. Values are means (SD). pH then rose progressively from the right to the left colon with a final mean value of 7.0 (0.7) (p less than 0.001).
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Pygall, Samuel R.; Kujawinski, Sarah; Timmins, Peter; Melia, Colin D.
International Journal of Pharmaceutics (2009), 370 (1-2), 110-120CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
Few studies report the effects of alkalizing buffers in HPMC matrixes. These agents are incorporated to provide micro-environmental buffering, protection of acid-labile ingredients, or pH-independent release of weak acid drugs. In this study, the influence of sodium citrate on the release kinetics, gel layer formation, internal gel pH and drug release mechanism was investigated in HPMC 2910 and 2208 (Methocel E4M and K4M) matrixes contg. 10% felbinac 39% HPMC, dextrose and sodium citrate. Matrix dissoln. at pH 1.2 and pH 7.5 resulted in complex release profiles. HPMC 2910 matrixes exhibited biphasic release, with citrate increasing the immediate release phase (<60 min) and reducing the extended release. HPMC 2208 matrixes were accelerated, but without the loss of extended release characteristics. Studies of early gel layer formation suggested gel barrier disruption and enhanced liq. penetration. PH modification of the gel layer was transitory (<2 h) and corresponded temporally with the immediate release phase. Results suggest that in HPMC 2910 matrixes, high initial citrate concns. within the gel layer suppress particle swelling, interfere with diffusion barrier integrity, but are lost rapidly whereupon drug soly. reduces and the diffusion barrier recovers. These Hofmeister or osmotic-mediated effects are better resisted by the less methoxylated HPMC 2208.
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Development of microparticulate systems for intestinal delivery of Lactobacillus acidophilus and Bifidobacterium lactis
Albertini, Beatrice; Vitali, Beatrice; Passerini, Nadia; Cruciani, Federica; Di Sabatino, Marcello; Rodriguez, Lorenzo; Brigidi, Patrizia
European Journal of Pharmaceutical Sciences (2010), 40 (4), 359-366CODEN: EPSCED; ISSN:0928-0987. (Elsevier B.V.)
In the present study intestinal delivery systems resistant to gastric juice, loaded with the probiotic bacteria Lactobacillus acidophilus LA14 and Bifidobacterium lactis BI07, were produced by the polyelectrolyte complexation. First, beads were prepd. by the traditional extrusion method and nine formulations were developed using alginate as main carrier and the biopolymer, xanthan gum (XG), as hydrophilic retardant polymer or the cellulose deriv., cellulose acetate phthalate (CAP), as gastro-resistant polymer. The results showed that the incorporation of the 0.5% (w/v) of XG or the 1% (w/v) of CAP within the 3% (w/v) of alginate soln. increased the survival of the probiotic bacteria in acid conditions from 63% of the freeze-dried bacteria up to 76%. Subsequently, these formula was used to prep. smaller microcapsules by means of an atomization device. Despite of the high viscosity of the biomass suspension, the spraying system produced spherical and non-aggregated microcapsules able to survive in harsh condition better than beads: the survival of the probiotic bacteria after acid incubation was 91%. The performance of the microcapsules in simulated gastric fluid (SGF) contg. pepsin and in gut medium (GM) contg. bile salts was excellent (viability > 95%). Furthermore, the viability of probiotic bacteria was maintained after an incubation of 24 h in GM. Finally, stability tests performed at 5 °C highlighted a bacterial viability of about 82% and 70% after 6 and 9 mo, resp.
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Mazumder, M. A. J.; Burke, N. A. D.; Shen, F.; Potter, M. A.; Stover, H. D. H. Biomacromolecules 2009, 10, 1365– 1373
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Microencapsulation of Lactobacillus plantarum spp in an alginate matrix coated with whey proteins
Gbassi, Gildas Komenan; Vandamme, Thierry; Ennahar, Said; Marchioni, Eric
International Journal of Food Microbiology (2009), 129 (1), 103-105CODEN: IJFMDD; ISSN:0168-1605. (Elsevier B.V.)
Whey proteins were used as a coating material to improve encapsulation of Lactobacillus plantarum strains in calcium alginate beads. L. plantarum 299v, L. plantarum 800 and L. plantarum CIP A159 were used in this study. Inactivation expts. were carried out in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Cross-sections of freeze-dried beads revealed the random distribution of bacteria throughout the alginate network. From an initial count of 10.04 ± 0.01 log10 CFU g- 1 for L. plantarum 299v, 10.12 ± 0.04 for L. plantarum CIP A159 and 10.03 ± 0.01 for L. plantarum 800, bacteria in coated beads and incubated in SGF (37 °C, 60 min) showed a better survival for L. plantarum 299v, L. plantarum CIP A159 and L. plantarum 800 (resp. 7.76 ± 0.12, 6.67 ± 0.08 and 5.81 ± 0.25 log10 CFU g- 1) when compared to uncoated beads (2.19 ± 0.09, 1.89 ± 0.09 and 1.65 ± 0.10 log10 CFU g- 1) (p < 0.05). Only bacteria in the coated beads survived in the SIF medium (37 °C, 180 min) after SGF treatment. This preliminary work showed that whey proteins are a convenient, cheap and efficient material for coating alginate beads loaded with bacteria.
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Lin, J. Z.; Yu, W. T.; Liu, X. D.; Xie, H. G.; Wang, W.; Ma, X. J. J. Biosci. Bioeng. 2008, 105, 660– 665
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Microencapsulation of Bifidobacterium animalis subsp. lactis in modified alginate-chitosan beads and evaluation of survival in simulated gastrointestinal conditions
Liserre, Alcina Maria; Re, Maria Ines; Franco, Bernadette D. G. M.
Food Biotechnology (Philadelphia, PA, United States) (2007), 21 (1), 1-16CODEN: FBIOEE; ISSN:0890-5436. (Taylor & Francis, Inc.)
Bifidobacterium animalis subsp. lactis was entrapped in alginate, alginate-chitosan, alginate-chitosan-Sureteric and alginate-chitosan-Acryl-Eze. Survival and in vitro release of bifidobacteria from the microparticles were investigated under conditions simulating gastrointestinal fluids covering the pH range from 1.5 to 7.5, with and without pepsin (3gL-1), pancreatin (1gL-1), and bile (10gL-1). All types of microcapsules protected B. animalis, but the use of chitosan and enteric polymers in the formulation of the beads, esp. Acryl-Eze, enhanced the beneficial effects of the microencapsulation technique. Besides promoting the controlled release of bifidobacteria in simulated gastrointestinal juices, the microencapsulation with enteric polymers improved the survival rate of these microorganisms.
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Mandal, S.; Puniya, A. K.; Singh, K. Int. Dairy J. 2006, 16, 1190– 1195
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Effect of alginate concentrations on survival of microencapsulated Lactobacillus casei NCDC-298
Mandal, S.; Puniya, A. K.; Singh, K.
International Dairy Journal (2006), 16 (10), 1190-1195CODEN: IDAJE6; ISSN:0958-6946. (Elsevier B.V)
This study reports the tolerance of Lactobacillus casei NCDC-298 encapsulated in different alginate concns. (2%, 3% or 4%), to low pH (1.5), high bile salt concn. (1% or 2%) and heat processing (55, 60 or 65°C for 20 min). The release of encapsulated cells in simulated aq. soln. of colonic pH was also assessed. The survival of encapsulated L. casei was better at low pH, high bile salt concn. and during heat treatment as compared to free cells. The survival increased proportionately with increasing alginate concns. without affecting the release of entrapped cells in soln. of colonic pH.
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Krasaekoopt, W.; Bhandari, B.; Deeth, H. Int. Dairy J. 2004, 14, 737– 743
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The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria
Krasaekoopt, Wunwisa; Bhandari, Bhesh; Deeth, Hilton
International Dairy Journal (2004), 14 (8), 737-743CODEN: IDAJE6; ISSN:0958-6946. (Elsevier Science B.V)
The probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, were encapsulated into uncoated calcium alginate beads and the same beads were coated with three types of material, chitosan, sodium alginate, and poly-L-lysine in combination with alginate. The thickness of the alginate beads increased with the addn. of coating materials. No differences were detectable in the bead strength by texture anal. or in the thickness of the beads with different types of coating materials by TEM. The survivability of three probiotics in uncoated beads, coated beads, and as free cells (unencapsulated) was conducted in 0.6% bile salt soln. and simulated gastric juice (pH 1.55) followed by incubation in simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for L. acidophilus and L. casei in all treatments. However, B. bifidum did not survive the acidic conditions of gastric juice even when encapsulated in coated beads.
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Chandramouli, V.; Kailasapathy, K.; Peiris, P.; Jones, M. J. Microbiol. Methods 2004, 56, 27– 35
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An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions
Chandramouli, V.; Kailasapathy, K.; Peiris, P.; Jones, M.
Journal of Microbiological Methods (2004), 56 (1), 27-35CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier Science B.V.)
An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 μm), different sodium alginate concns. (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concns. of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concn. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concn. of calcium chloride was increased. Increase in cell load during encapsulation increased the no. of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride soln. for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concns. was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concn. (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 109 CFU/mL, 30 min hardening in 0.1 M CaCl2 and capsule size 450 μm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.
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Krasaekoopt, W.; Bhandari, B.; Deeth, H. C. Lebensm. Wiss. Technol. 2006, 39, 177– 183
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Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated milk during storage
Krasaekoopt, Wunwisa; Bhandari, Bhesh; Deeth, Hilton C.
LWT--Food Science and Technology (2005), 39 (2), 177-183CODEN: LSTWB3 ISSN:. (Elsevier B.V.)
Survival of the microencapsulated probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, in stirred yoghurt from UHT- and conventionally treated milk during low temp. storage was investigated. The probiotic cells both as free cells and microencapsulated cells (in alginate beads coated with chitosan) were added into 20 g/100 g total solids stirred yoghurt from UHT-treated milk and 16 g/100 g total solids yoghurt from conventionally treated milk after 3.5 h of fermn. The products were kept at 4° for 4 wk. The survival of encapsulated probiotic bacteria was higher than free cells by approx. 1 log cycle. The no. of probiotic bacteria was maintained above the recommended therapeutic min. (107 cfu g-1) throughout the storage except for B. bifidum. The viabilities of probiotic bacteria in yoghurts from both UHT- and conventionally treated milks were not significantly (P > 0.05) different.
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Metabolic, hormonal and gastric fluid and pH changes after different preoperative feeding regimens
Naguib M; Samarkandimb A H; Al-Hattab Y; Turkistani A; Delvi M B; Riad W; Attia M
Canadian journal of anaesthesia = Journal canadien d'anesthesie (2001), 48 (4), 344-50 ISSN:0832-610X.
PURPOSE: To evaluate the metabolic, hormonal and gastric fluid and pH changes after administration of a small volume of different preoperative feeding regimens. METHODS: In a prospective, randomized, double-blind study 375 adult patients were allocated to one of five groups. Patients ingested 60 ml honey, glucose-fructose-sucrose-maltose mixture (GFSM), apple juice or water two hours before surgery or continued their overnight fast (controls). Blood samples were obtained from an indwelling venous catheter before the administration of feeding regimens and before induction of anesthesia for determination of glucose, triglycerides, insulin, epinephrine and norepinephrine concentrations. Before anesthesia induction, patients were asked to grade the degree of thirst and hunger. After tracheal intubation residual gastric volume (RGV) was suctioned through an orogastric tube. RESULTS: Administration of honey, GFSM, apple juice or water resulted in increases in RGV without changes in the gastric pH. The median RGV values were 15 ml in controls and 20-25 ml in other groups. Thirst was noted after administration of fluids containing sugars. Hunger was noted in the apple juice group. Plasma concentrations of glucose increased and triglycerides decreased after ingestion of fluids containing sugars. Plasma insulin concentrations decreased in GFSM and apple juice groups. Norepinephrine concentrations increased in the control, apple juice and water groups. CONCLUSIONS: Small volumes of fluid increased RGV (P < 0.05). Apple juice resulted in increased incidence of thirst and hunger and plasma glucose and norepinephrine concentrations. Compared with GFSM or apple juice, honey had a gentler effect on plasma glucose and insulin concentrations.
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Survival of Ca-alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions
Hansen, L. Truelstrup; Allan-Wojtas, P. M.; Jin, Y.-L.; Paulson, A. T.
Food Microbiology (2002), 19 (1), 35-45CODEN: FOMIE5; ISSN:0740-0020. (Elsevier Science)
Nine bifidobacteria were tested for their tolerance to simulated gastrointestinal tract conditions at 37°C. Resistance to simulated gastric juice (pH 2·0 and 3·0) and bile salts (5 and 10 g l-1) varied among test strains. Bifidobacteria lactis Bb-12 was significantly more resistant to low pH and bile than any other test strain. Bifidobacteria adolescentis 15703, Bifidobacteria breve 15700, Bifidobacteria lactis Bb-12 and Bifidobacteria longum Bb-46, with the best overall resistance, were encapsulated in alginate microspheres (mean diams. of 20 and 70 μ m). Cold-stage SEM revealed groups of encapsulated bacteria in the porous alginate microspheres. The alginate microspheres retained their integrity at pH 2·0. Microencapsulation of Bifidobacteria did not significantly improve survival over free cells when exposed to simulated gastric juice. Survival was improved for microencapsulated Bifidobacteria longum Bb-46 over that of free cells during refrigerated storage in milk with 2% fat. However, the presence of microencapsulated Bifidobacteria longum and Bifidobacteria lactis in milk resulted in off-flavors not found in samples with free cells, indicating the metab. of entrapped cells had been altered.
42
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Gombotz, Wayne R.; Wee, SiowFong
Advanced Drug Delivery Reviews (1998), 31 (3), 267-285CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)
A review with 129 refs. There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins. Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent. Alginate, a naturally occurring biopolymer extd. from brown algae, has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biol. agents. Alginate polymers are a family of linear unbranched polysaccharides which contain varying amts. of 1,4'-linked β-d-mannuronic acid and α-l-guluronic acid residues. The residues may vary widely in compn. and sequence and are arranged in a pattern of blocks along the chain. Alginate can be ionically crosslinked by the addn. of divalent cations in aq. soln. The relatively mild gelation process has enabled not only proteins, but cells and DNA to be incorporated into alginate matrixes with retention of full biol. activity. Furthermore, by selection of the type of alginate and coating agent, the pore size, degrdn. rate, and ultimately release kinetics can be controlled. Gels of different morphologies can be prepd. including large block matrixes, large beads (>1 mm in diam.) and microbeads (<0.2 mm in diam.). In situ gelling systems have also been made by the application of alginate to the cornea, or on the surfaces of wounds. Alginate is a bioadhesive polymer which can be advantageous for the site specific delivery to mucosal tissues. All of these properties, in addn. to the nonimmunogenicity of alginate, have led to an increased use of this polymer as a protein delivery system. This review will discuss the chem. of alginate, its gelation mechanisms, and the phys. properties of alginate gels. Emphasis will be placed on applications in which biomols. have been incorporated into and released from alginate systems.
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Abstract
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Figure 1
Figure 1. Conversion of pixel intensity to pH. Image on left is produced by the division of the pixel intensities of a picture showing pHrodo fluorescence by the corresponding FITC image. Image on right is the result of coloration of the left image based on the intensity of each pixel.
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Figure 2
Figure 2. Viability of B. breve against TPY medium adjusted to various pH after 1 h incubation at 37 °C; N = 3 ± standard deviation; limit of detection, 3 log(CFU)/mL; line intended as guide to eye.
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Figure 3
Figure 3. Release of viable cells from alginate and chitosan-coated alginate matrices during exposure to simulated gastric solution (pH 2.0/3.0, 60 min) followed by simulated intestinal solution (180 min) at 37 °C. By the 240 min mark, matrix dissolution was complete. Starting cell concentration included indicating maximum possible survival; N = 3 ± standard deviation; limit of detection, 3 log(CFU)/mL.
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Figure 4
Figure 4. Schematic diagram of dye conjugation to amine moieties within bacteria, producing thiourea and amide moieties when reacting with pHrodo succinimidyl ester and FITC, respectively (A). Fluorescence of pHrodo (B) and FITC (C) when conjugated to B. breve showing variation of intensity with pH. For this experiment, a solution of fluorescently labeled B. breve conjugate (100 μL) at ∼9 log(cells)/mL was diluted into PBS (900 μL) adjusted to pH 2.0, 5.0, and 7.0. The fluorescence of conjugated pHrodo and FITC was then quantified by UV spectrofluorometry (λ_ex: 546 and 488, respectively) over the ranges shown. Fluorescence changes with pH are most commonly the result of protonation or deprotonation around a fluorophore’s pK_a. (43).
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Figure 5
Figure 5. pH maps of an alginate and a chitosan-coated alginate microcapsule during exposure to simulated gastric solution at pH 2.0. Scale: 1 mm.
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Figure 6
Figure 6. Comparison of pH within alginate and alginate-chitosan matrices after 60 min exposure to simulated gastric juice at pH 2.0 (scale bar: 1 mm). Images taken from a separate experiment to those shown in Figure 5.
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Figure 7
Figure 7. Encroachment of pH < 3.0 into alginate (open squares) and chitosan-coated alginate (closed circles) matrices during incubation in simulated gastric solution at 37 °C; N = 3 ± standard deviation, band thickness is the average of five measurements at equidistant points on the matrix.
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  The influence of coating materials on some properties of alginate beads and survivability of microencapsulated probiotic bacteria
  Krasaekoopt, Wunwisa; Bhandari, Bhesh; Deeth, Hilton
  International Dairy Journal (2004), 14 (8), 737-743CODEN: IDAJE6; ISSN:0958-6946. (Elsevier Science B.V)
  The probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, were encapsulated into uncoated calcium alginate beads and the same beads were coated with three types of material, chitosan, sodium alginate, and poly-L-lysine in combination with alginate. The thickness of the alginate beads increased with the addn. of coating materials. No differences were detectable in the bead strength by texture anal. or in the thickness of the beads with different types of coating materials by TEM. The survivability of three probiotics in uncoated beads, coated beads, and as free cells (unencapsulated) was conducted in 0.6% bile salt soln. and simulated gastric juice (pH 1.55) followed by incubation in simulated intestinal juice with and without 0.6% bile salt. Chitosan-coated alginate beads provided the best protection for L. acidophilus and L. casei in all treatments. However, B. bifidum did not survive the acidic conditions of gastric juice even when encapsulated in coated beads.
33. 33
  Chandramouli, V.; Kailasapathy, K.; Peiris, P.; Jones, M. J. Microbiol. Methods 2004, 56, 27– 35
  33
  An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions
  Chandramouli, V.; Kailasapathy, K.; Peiris, P.; Jones, M.
  Journal of Microbiological Methods (2004), 56 (1), 27-35CODEN: JMIMDQ; ISSN:0167-7012. (Elsevier Science B.V.)
  An improved method of microencapsulation was developed to increase the efficacy of capsules in protecting the encapsulated bacteria under simulated gastric conditions. Lactobacillus acidophilus CSCC 2400 was encapsulated in calcium alginate and tested for its survival in simulated gastric conditions. The effects of different capsule sizes (200, 450, 1000 μm), different sodium alginate concns. (0.75%, 1%, 1.5%, 1.8% and 2% w/v) and different concns. of calcium chloride (0.1, 0.2, 1.0 M) on the viability of encapsulated bacteria were investigated. The viability of the cells in the microcapsules increased with an increase in alginate capsule size and gel concn. There was no significant difference (p>0.05) in the viability of encapsulated cells when the concn. of calcium chloride was increased. Increase in cell load during encapsulation increased the no. of bacterial survivors at the end of 3-h incubation in simulated gastric conditions. Hardening the capsule in calcium chloride soln. for a longer time (8 h) had no impact on increasing the viability of encapsulated bacteria in a simulated gastric environment. The release of encapsulated cells at different phosphate buffer concns. was also studied. When encapsulated L. acidophilus CSCC 2400 and L. acidophilus CSCC 2409 were subjected to low pH (pH 2) and high bile concn. (1.0% bile) under optimal encapsulation conditions (1.8% (w/v) alginate, 109 CFU/mL, 30 min hardening in 0.1 M CaCl2 and capsule size 450 μm), there was a significant increase (p<0.05) in viable cell counts, compared to the free cells under similar conditions. Thus the encapsulation method described in this study may be effectively used to protect the lactobacillus from adverse gastric conditions.
34. 34
  Krasaekoopt, W.; Bhandari, B.; Deeth, H. C. Lebensm. Wiss. Technol. 2006, 39, 177– 183
  34
  Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated milk during storage
  Krasaekoopt, Wunwisa; Bhandari, Bhesh; Deeth, Hilton C.
  LWT--Food Science and Technology (2005), 39 (2), 177-183CODEN: LSTWB3 ISSN:. (Elsevier B.V.)
  Survival of the microencapsulated probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, in stirred yoghurt from UHT- and conventionally treated milk during low temp. storage was investigated. The probiotic cells both as free cells and microencapsulated cells (in alginate beads coated with chitosan) were added into 20 g/100 g total solids stirred yoghurt from UHT-treated milk and 16 g/100 g total solids yoghurt from conventionally treated milk after 3.5 h of fermn. The products were kept at 4° for 4 wk. The survival of encapsulated probiotic bacteria was higher than free cells by approx. 1 log cycle. The no. of probiotic bacteria was maintained above the recommended therapeutic min. (107 cfu g-1) throughout the storage except for B. bifidum. The viabilities of probiotic bacteria in yoghurts from both UHT- and conventionally treated milks were not significantly (P > 0.05) different.
35. 35
  Dressman, J. B.; Berardi, R. R.; Dermentzoglou, L. C.; Russell, T. L.; Schmaltz, S. P.; Barnett, J. L.; Jarvenpaa, K. M. Pharm. Res. 1990, 7, 756– 61
  There is no corresponding record for this reference.
36. 36
  Mojaverian, P.; Vlasses, P. H.; Kellner, P. E.; Rocci, M. L. Pharm. Res. 1988, 5, 639– 644
  There is no corresponding record for this reference.
37. 37
  Naguib, M.; Samarkandimb, A. H.; Al-Hattab, Y.; Turkistani, A.; Delvi, M. B.; Riad, W.; Attia, M. Can. J. Anaesth. 2001, 48, 344– 50
  37
  Metabolic, hormonal and gastric fluid and pH changes after different preoperative feeding regimens
  Naguib M; Samarkandimb A H; Al-Hattab Y; Turkistani A; Delvi M B; Riad W; Attia M
  Canadian journal of anaesthesia = Journal canadien d'anesthesie (2001), 48 (4), 344-50 ISSN:0832-610X.
  PURPOSE: To evaluate the metabolic, hormonal and gastric fluid and pH changes after administration of a small volume of different preoperative feeding regimens. METHODS: In a prospective, randomized, double-blind study 375 adult patients were allocated to one of five groups. Patients ingested 60 ml honey, glucose-fructose-sucrose-maltose mixture (GFSM), apple juice or water two hours before surgery or continued their overnight fast (controls). Blood samples were obtained from an indwelling venous catheter before the administration of feeding regimens and before induction of anesthesia for determination of glucose, triglycerides, insulin, epinephrine and norepinephrine concentrations. Before anesthesia induction, patients were asked to grade the degree of thirst and hunger. After tracheal intubation residual gastric volume (RGV) was suctioned through an orogastric tube. RESULTS: Administration of honey, GFSM, apple juice or water resulted in increases in RGV without changes in the gastric pH. The median RGV values were 15 ml in controls and 20-25 ml in other groups. Thirst was noted after administration of fluids containing sugars. Hunger was noted in the apple juice group. Plasma concentrations of glucose increased and triglycerides decreased after ingestion of fluids containing sugars. Plasma insulin concentrations decreased in GFSM and apple juice groups. Norepinephrine concentrations increased in the control, apple juice and water groups. CONCLUSIONS: Small volumes of fluid increased RGV (P < 0.05). Apple juice resulted in increased incidence of thirst and hunger and plasma glucose and norepinephrine concentrations. Compared with GFSM or apple juice, honey had a gentler effect on plasma glucose and insulin concentrations.
38. 38
  Locatelli, I.; Mrhar, A.; Bogataj, M. Pharm. Res. 2009, 26 (7) 1607– 17
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39. 39
  Corcoran, B. M.; Stanton, C.; Fitzgerald, G. F.; Ross, R. P. Appl. Environ. Microbiol. 2005, 71, 3060– 7
  There is no corresponding record for this reference.
40. 40
  Ding, W. K.; Shah, N. P. J. Food Sci. 2009, 74, M100– M107
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41. 41
  Hansen, L. T.; Allan-Wojtas, P. M.; Jin, Y. L.; Paulson, A. T. Food Microbiol. 2002, 19, 35– 45
  41
  Survival of Ca-alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions
  Hansen, L. Truelstrup; Allan-Wojtas, P. M.; Jin, Y.-L.; Paulson, A. T.
  Food Microbiology (2002), 19 (1), 35-45CODEN: FOMIE5; ISSN:0740-0020. (Elsevier Science)
  Nine bifidobacteria were tested for their tolerance to simulated gastrointestinal tract conditions at 37°C. Resistance to simulated gastric juice (pH 2·0 and 3·0) and bile salts (5 and 10 g l-1) varied among test strains. Bifidobacteria lactis Bb-12 was significantly more resistant to low pH and bile than any other test strain. Bifidobacteria adolescentis 15703, Bifidobacteria breve 15700, Bifidobacteria lactis Bb-12 and Bifidobacteria longum Bb-46, with the best overall resistance, were encapsulated in alginate microspheres (mean diams. of 20 and 70 μ m). Cold-stage SEM revealed groups of encapsulated bacteria in the porous alginate microspheres. The alginate microspheres retained their integrity at pH 2·0. Microencapsulation of Bifidobacteria did not significantly improve survival over free cells when exposed to simulated gastric juice. Survival was improved for microencapsulated Bifidobacteria longum Bb-46 over that of free cells during refrigerated storage in milk with 2% fat. However, the presence of microencapsulated Bifidobacteria longum and Bifidobacteria lactis in milk resulted in off-flavors not found in samples with free cells, indicating the metab. of entrapped cells had been altered.
42. 42
  Gombotz, W. R.; Wee, S. F. Adv. Drug Delivery Rev. 1998, 31, 267– 285
  42
  Protein release from alginate matrixes
  Gombotz, Wayne R.; Wee, SiowFong
  Advanced Drug Delivery Reviews (1998), 31 (3), 267-285CODEN: ADDREP; ISSN:0169-409X. (Elsevier Science B.V.)
  A review with 129 refs. There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins. Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent. Alginate, a naturally occurring biopolymer extd. from brown algae, has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biol. agents. Alginate polymers are a family of linear unbranched polysaccharides which contain varying amts. of 1,4'-linked β-d-mannuronic acid and α-l-guluronic acid residues. The residues may vary widely in compn. and sequence and are arranged in a pattern of blocks along the chain. Alginate can be ionically crosslinked by the addn. of divalent cations in aq. soln. The relatively mild gelation process has enabled not only proteins, but cells and DNA to be incorporated into alginate matrixes with retention of full biol. activity. Furthermore, by selection of the type of alginate and coating agent, the pore size, degrdn. rate, and ultimately release kinetics can be controlled. Gels of different morphologies can be prepd. including large block matrixes, large beads (>1 mm in diam.) and microbeads (<0.2 mm in diam.). In situ gelling systems have also been made by the application of alginate to the cornea, or on the surfaces of wounds. Alginate is a bioadhesive polymer which can be advantageous for the site specific delivery to mucosal tissues. All of these properties, in addn. to the nonimmunogenicity of alginate, have led to an increased use of this polymer as a protein delivery system. This review will discuss the chem. of alginate, its gelation mechanisms, and the phys. properties of alginate gels. Emphasis will be placed on applications in which biomols. have been incorporated into and released from alginate systems.
43. 43
  Hanson, M. A.; Ge, X.; Kostov, Y.; Brorson, K. A.; Moreira, A. R.; Rao, G. Biotechnol. Bioeng. 2007, 97, 833– 841
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Supporting Information
Supporting Information
The calibration curve determined for the fluorescence ratio of pHrodo/FITC with pH. This material is available free of charge via the Internet at http://pubs.acs.org.
- bm301569r_si_001.pdf (107.88 kb)
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CLSM Method for the Dynamic Observation of pH Change within Polymer Matrices for Oral DeliveryClick to copy article linkArticle link copied!

Biomacromolecules

Abstract

Introduction

Materials

Bifidobacterium breve

Methods

Viability of B. breve at pH 2–7

Survival of Alginate and Alginate-Chitosan Encapsulated B. breve During Simulated Gastrointestinal Passage

Preparation of pHrodo/FITC Labeled B. breve

Preparation of Alginate-Chitosan Microcapsules Containing pHrodo/FITC Labeled B. breve

Calibration of Microscope

Measurement of pH within Alginate-Chitosan Microcapsules

Figure 1

Results and Discussion

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Concluding Remarks

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CLSM Method for the Dynamic Observation of pH Change within Polymer Matrices for Oral Delivery
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