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. Author manuscript; available in PMC: 2021 Mar 25.
Published in final edited form as: Curr Pharm Des. 2020;26(26):3134–3140. doi: 10.2174/1381612826666200210111925

Development of a Novel Oral Delivery Vehicle for Probiotics

Kevin Enck 1,2,3, Surya Banks 3,4, Hariom Yadav 5, Mark E Welker 3,4, Emmanuel C Opara 1,2,3,*
PMCID: PMC7992870  NIHMSID: NIHMS1680714  PMID: 32039674

Abstract

Background:

There is a significant interest in effective oral drug delivery of therapeutic substances. For probiotics, there is a particular need for a delivery platform that protects the bacteria from destruction by the acidic stomach while enabling targeted delivery to the intestine where microbiota naturally reside. The use of probiotics and how they impact the gut microbiota is a growing field and holds promise for the treatment of a variety of gastrointestinal diseases, including irritable bowel disease Crohn’s disease and C. diff and other diseases, such as obesity, diabetes, Parkinson’s, and Alzheimer’s diseases.

Objective:

The aim of this research was to use our newly developed chemically-modified alginate hydrogel with the characteristic feature of stability in acidic environments but disintegration under neutral-basic pH conditions to design a novel system for effective targeted delivery of ingested probiotics.

Methods and Results:

We have used the approach of encapsulation of bacterial cells in the hydrogel of the modified alginate with in vitro studies in both simulated stomach acid and intestinal fluid conditions to demonstrate the potential application of this novel platform in oral delivery of probiotics. Our data provide a proof-of-concept that enables further studies in vivo with this delivery platform.

Conclusion:

We have demonstrated in the present study that our chemically modified alginate hydrogel is resistant to acidic conditions and protects bacterial cells encapsulated in it, but it is sensitive to neutral-basic pH conditions under which it disintegrates and releases its viable bacteria cell payload. Our data provide a proof-of-concept that enables further studies in vivo with this delivery platform for the efficacy of therapeutic bacteria in various disease conditions.

Keywords: Alginate, chemical modification, hydrogel, oral, drug delivery, probiotics

1. INTRODUCTION

Emerging data indicate that the gut microflora may play a significant role in the pathogenesis and treatment of a variety of human diseases including some auto-immune diseases such as type 1 diabetes [1-8], gastrointestinal diseases including irritable bowel disease Crohn’s disease and C. diff as well as other diseases, such as obesity, Parkinson’s, and Alzheimer’s disease. The major challenge in the use of oral ingestion of probiotics to treat these diseases has been how to overcome the hostile conditions of the gastrointestinal tract against bacteria that include the harsh stomach acidity and enzymatic degradation barriers [9-11]. Consequently, fecal transplantation emerged as an alternative procedure for the effective delivery of therapeutic bacteria as it avoids the gastric environment [12-14].

However, oral drug delivery has been the preferred route for drug administration because it promotes patient compliance as well as solid-based delivery systems not requiring sterile conditions, which leads to less expensive manufacturing [15]. For most therapeutics, the desired goal is to safely deliver the therapeutic agent to the gut from where it would be absorbed to enhance bioavailability [16, 17]. When ingested drugs enter the gastrointestinal tract (GIT), some have the potential to harm the tissue by disrupting the biochemical environment and leading to ulceration and physiologic dysfunction [18]. On the other hand, gastric acid and enzymes may degrade pharmacologic agents such as probiotics, preventing them from reaching their intended site [10, 19]. Specifically, probiotics need to be safely delivered to the intestine where microbiota reside and influence human health [20-26]. Safe delivery of probiotics across the gastric acidic and enzymatic barriers to the intestines has been quite a challenge, which hitherto has been addressed by ingestion of large quantities of probiotics in the hope that a significant proportion of the ingested bacteria would escape the gastric barriers and successfully inhabit the gut [9-11, 23, 27, 28]. Consequently, developing an effective delivery vehicle that is protective of the bacteria during transit through the stomach and delivered safely to the intestines is not only beneficial to the health but also economically advantageous for the patient since it would imply the use of reduced amounts of bacteria with concomitant cost savings. In addition, such a delivery vehicle would be of interest to pharmaceutical companies who would benefit from enhanced efficacy in the use of their products [17].

Biopolymers are frequently used as drug delivery vehicles because they are biocompatible or bioinert, easily form stable hydrogels, and can typically be tuned for application to a wide range of therapeutics regardless of their solubility properties [17, 29-35]. No one polymer is universally suited for encapsulation of all drugs as many therapeutics have different molecular characteristics, such as size and charge that can affect how they interact with the polymer. Consequently, this has spurned the modification and combination of polymeric biomaterials to generate a vast library of compounds which possess unique advantages as encapsulation materials [36-43]. Based on the drug being encapsulated and the intended delivery mechanism, polymers can be selected and further modified to facilitate improved pharmacologic uptake and action. One of the most commonly used hydrogels is alginate. As a bioinert, rapidly crosslinking, and relatively inexpensive polymer, it has been used frequently in the food industry [21, 44, 45], for cell encapsulation (especially for islet encapsulation in type 1 diabetes treatment [46-49]), and for oral drug delivery [15, 16, 50-52]. While alginate has been shown to be protective in the stomach, it degrades slowly in the gut, which can lead to undesirable drug release kinetics [29, 50, 51, 53-56]. We have recently described chemical modifications of alginate that render the hydrogel more sensitive to neutral-basic pH conditions with the degradation kinetics. We concluded that hydrogels made with the modified alginates provide a potential platform for the controlled release of payloads in the gut [57].

The purpose of the present study was to examine the viability of bacteria cells incubated and/or released after encapsulation in the modified alginate hydrogels under simulated pH conditions of the gastrointestinal tract for potential application in oral probiotics delivery.

2. MATERIALS AND METHODS

2.1. Materials

Ultra-Pure low viscosity (20-200 mPa·s) high guluronic acid sodium alginate (LVG) (Nova-Matrix, Sandvika Norway) was prepared sterilely by mixing with Hanks Balanced Salt Solution (HBSS) (H6648, Sigma-Aldrich) at a 2% (w/v) concentration and stirred overnight at 4°C. Simulated gastric fluid (SGF) was prepared by mixing 2 g/L of NaCl with DiH2O and adjusting the pH to 2.0. Simulated intestinal fluid (SIF) was prepared by mixing 6.8 g/L monobasic KH2PO4 (60221, Sigma-Aldrich) with diH2O and adjusting the pH to 6.8. MRS broth was prepared by mixing MRS powder (M369, HiMedia) with diH2O (55 g/mL) MRS agar plates were prepared by mixing MRS broth with 2% (w/v) Agar (12177, Millipore) and autoclaving for 25 min. While warm, the agar was plated onto Falcon petri dishes (08-757-100, Fisher Scientific) and allowed to cool and solidify before storing upside down at 4°C.

2.2. Chemical Modification of Alginate

The alginate underwent 0.5%, 1% and 2% oxidation with aminoethyl benzoic acid (BA) reductive amination, as we have recently described [57]. Following Dalheim et al’s protocol, oxidation of the vicinal dialcohol was carried out using a desired molar percent ratio of NaIO4 and the generated dialdehyde was then reacted with 4-(2 aminoethyl)benzoic acid followed by reduction to obtain the desired percentage of the covalently linked small molecule in the alginate backbone [58].

2.3. Alginate Preparation

Alginate (modified and unmodified) solutions were prepared by mixing 1.5% (w/v) with Hanks Balanced Salt Solution (HBSS) (H6648, Sigma) and stirring overnight at 4°C.

2.4. Probiotic Culture and Encapsulation

Lactobaccillus casei NCDC 298 (L. casei) was obtained from American Type Culture Collection (ATCC; 39392) and cultured in MRS broth (55 g/L) at 37°C for 24 hours. After incubation, 5 mL of L. casei was centrifuged at 2000 x g for 10 min at 4°C. The MRS broth was aspirated and then 1 mL of alginate solution was added to the remaining bacteria pellet and mixed thoroughly with a syringe. The alginate-probiotic mixture was then drawn into the syringe and extruded through a 15 gauge blunt tip needle into a bath of 100 mM CaCl2 and allowed to crosslink for 10 minutes. The crosslinking solution was removed by allowing the beads to settle to the bottom of a 50 mL conical tube and aspirating the excess crosslinking solution. The beads were then washed with HBSS supplemented with 25 mM CaCl2 three times before testing. The crosslinked capsules were either tested in simulated gastric fluid (SGF), simulated intestinal fluid (SIF), or freeze-dried to permit the assessment of their drug delivery capabilities after long-term storage.

2.5. SGF Viability Assessment

A portion of alginate capsules from each group was first liquefied with 55 mM sodium citrate (BP327-1, Fisher Scientific) then serially diluted in HBSS. Once diluted to the desired concentration, the bacterial cells were plated onto the MRS agar plate, spread evenly across the plate, and incubated at 37°C for 24 hours to determine the initial cell concentration in each capsule. The remaining alginate capsules from each group were placed in 6 well non-tissue culture plates (08-772-49, Fisher Scientific) with 1-2 mL of SGF. For physiological relevance, a concentration of 10E10 Colony Forming Units (CFU)/mL SGF was used. These capsules were shaken at 60 RPM for 1 or 2 hours, removed and liquefied with 55 mM sodium citrate before serially diluting in HBSS and plating onto MRS agar plates as described above. After 24 hour incubation, the number of CFU were counted, and based on the dilution, the total CFU/bead was determined at 0, 1, and 2-hour time points. For the unencapsulated group, the bacteria were still centrifuged, but after aspiration, they were not mixed with alginate or encapsulated. Instead, they were directly added to the SGF solution.

2.6. SIF Release Assessment

Alginate capsules from each group were placed in Petri dishes with 15 mL of SIF at 37°C and shaken at 60 RPM for 3 hours. At predetermined intervals, 1 mL of the SIF solution was collected and measured in a Spectrophotometer (Thermo Spectronic Biomate 3 UV-visible Spectrophotometer, Thermo Fisher) at 600 nm wave-length to determine bacteria concentration. SIF with no beads was used as a blank while beads that were immediately liquefied with 55 mM sodium citrate were used as maximum concentrations for each group. The corresponding absorbance readings were then compared to both the blanks and maximum concentration to determine the percentage of bacterial cells released.

2.7. Freeze Drying and Long-term Storage of Capsules

Alginate capsules were placed in liquid nitrogen within 15 minutes of encapsulation to preserve them for long-term storage. Once frozen, the capsules were placed in a 50 mL conical tube with proper ventilation and lyophilized overnight. The capsules were then removed and stored at room temperature for up to 60 days. At predetermined intervals, capsules from each of the groups were liquefied in 55 mM sodium citrate, serially diluted, and spread onto a MRS agar plate similar to above. The CFU were then counted after 24 hour incubation at 37°C.

2.8. Staistical Analysis

Data in Figs. (1-4) are expressed as mean ± standard deviation and a 2-Way ANOVA for multiple comparisons was performed using Prism 6 software to determine the difference between groups. Differences were considered significant if p<0.05.

Fig. (1).

Fig. (1).

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Alginate capsules (~3 mm diameter) in CaCl2 containing L. casei, which gives a white color to the beads denoted by the arrows. Beads were formed through extrusion through a 15 gauge blunt tip needle.

Fig. (4).

Fig. (4).

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Release of L. casei from alginate in SIF over time. Values are expressed in percentage of total encapsulated bacteria and were measured with a spectrophotometer. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001, n = 4.

3. RESULTS

3.1. Encapsulated Bacteria

L. casei was encapsulated in alginate microbeads, as shown in Fig. (1). The microbeads were 2.73 ± 0.05 mm in diameter and contained between 1E9 and 1E10 CFU.

3.2. SGF Viability Assessment

The viability of bacteria in SGF was assessed as a percent of initial viability since the total concentration of bacteria in each group varied such that comparisons could not be drawn between groups (Fig. 2). After 1 hour in SGF, bacteria encapsulated in 2%, 1%, and 0.5% oxidized alginate had a viability of 0.483% ± 0.074, 0.210% ± 0.006, 0.115% ± 0.002, and 0.061% ± 0.008, respectively while the unencapsulated bacteria had a viability of 0.004% ± 2.95e-004 (n=3). Each difference between the groups was significant, meaning that the alginate capsules offered protection from the acidic environment of the stomach. Increasing the modification on the alginate also caused an increase in bacteria protection. This could be due to the amine group attached to the alginate providing a neutralizing effect to the surrounding environment. At 2 hours in SGF, bacteria encapsulated in 2%, 1%, 0.5%, and unmodified alginate had a viability of 0.053% ± 0.007, 0.027% ± 0.001, 0.018% ± 0.003, and 0.018% ± 0.006, respectively while the unencapsulated bacteria had a viability of 0.002% ± 3.02e-004 (n=3). At 2 hours, only cells encapsulated in 2% modified alginate were significantly higher in number than those that were unencapsulated (p<0.05) indicating that the high acidity overtime was capable of overcoming the protective effect of the alginate on encapsulated bacteria.

Fig. (2).

Fig. (2).

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Viability of L. casei in SGF over time. Viability was assessed by counting CFUs at times 0, 1 and 2 hours and the percent change was based on the initial CFUs. Modified alginate comprised of alginate oxidized to varying degrees and all contained benzoic acid. Percent viability is displayed in log10. Flat lines (—) represent p value differences between connecting groups. Capped lines (┌—┐) represent p value differences between the furthest left group and all other groups within the line. (*) p<0.05, (****) p<0.0001, n = 3.

Next, we examined if there was any difference in bacterial viability between our oxidized alginates with and without benzoic acid amination (Fig. 3). We had previously determined that there was no difference in bead degradation of oxidized alginate hydrogel with or without benzoic acid amination but the present study provides an opportunity to examine the effect of benzoic acid amination on the protection of bacteria from the acidic stomach. At 1 hour, the bacteria encapsulated in oxidized alginate with benzoic acid amination were significantly more viable than the ones encapsulated in oxidized alginate without benzoic acid. After 2 hours, there was no difference between the viabilities of the bacteria cells encapsulated in the two forms of oxidized alginate consistent with observations in Fig. (2).

Fig. (3).

Fig. (3).

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Comparison of two alginate modification methods on their protective effects on encapsulated L. casei in SGF. CFUs are displayed in log10. Viability was assessed by counting CFUs at 0, 1, and 2 hours. (***) p<0.001, n = 3.

3.3. SIF Viability Assessment

Bacteria release in SIF was examined over a 3-hour period (Fig. 4). We had previously shown that increasing the percent modification of the alginate caused an increase in the hydrogel degradation rate [57]. We, therefore, predicted that the release of bacteria encapsulated in microbeads made with different degrees of modified alginate would follow a similar trend. We found that within 45 minutes, 63.7% ± 5.1 of the encapsulated bacteria had been released into the SIF from the 2% oxidized group while only 38.3% ± 6.7, 27.8% ± 4.5, and 3.23% ± 2.1 of bacteria had been released from the 1% oxidized, 0.5% oxidized, and unmodified groups, respectively. This pattern of release continued out to 3 hours. By 1.5 hours, all of the bacteria encapsulated in the 2% oxidized alginate had been released whereas only 72.6% ± 1.2 and 49.1% ± 6.8 of bacteria had been released by the 1% and 0.5% oxidized alginate even after 3 hours. However, only 33.7% ± 4.1 encapsulated bacteria from the unmodified group had been released after 3 hours, consistent with observations in our previous report [57].

3.4. Freeze Drying Storage and Viability

Lastly, we examined the stability of the probiotics during long-term storage after freeze-drying and lyophilization. While the viability of bacteria cells decreased during the lyophilization process and gradually decreased over time, there was no significant difference in the viability of encapsulated cells between the modified and unmodified alginate groups (Fig. 5).

Fig. (5).

Fig. (5).

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Viability of L. casei in long term storage. Beads were frozen in liquid N2 then lyophilized and kept at room temperature for up to 60 days. Comparisons were made from modified and unmodified alginate to determine any long-term cytotoxic effects of the modification. P>0.05 for each time point, n = 3.

4. DISCUSSION

The chemically modified alginate we described previously degrades at a rate that depends on both pH and the degree of modification [57]. As a result, it can be used as an oral drug delivery vehicle for controlled drug release. In the present study, we encapsulated probiotics, specifically L. casei, in the chemically modified alginate and measured their viability and release in SGF and SIF, respectively. We found that encapsulating the bacteria in alginate beads protects them from the harsh stomach acidity much better than if they were unencapsulated. Losing over 99% of probiotics due to the gastric environment is expected and typical in the field. Since there are trillions of cells being encapsulated, protecting even a fraction of a percent more via encapsulation can greatly improve the likelihood of a therapeutic effect [10, 11, 19, 59]. The beads were made with either unmodified or modified alginate and the modification of alginate was performed by reductive amination of oxidized alginate using 4-(2-aminoethyl)benzoic acid [57]. Increasing the degree of modification of the alginate polymer resulted in significantly increased viability of encapsulated L. casei after 1 hour of incubation in the SGF medium. We hypothesize that this may be due to the benzoic acid providing a buffering effect around the beads. As a weak acid in a strong acid environment, the benzoate ions become protonated, causing a potential neutralizing effect. At 2 hours of incubation, this effect seems to diminish and the viability of the encapsulated bacteria drops significantly. Still, the proportion of encapsulated bacteria that are viable is significantly higher than that of the unencapsulated bacteria, thus demonstrating the protective effect of the alginate hydrogel on bacteria under acid conditions. Since the transit time of ingested substances through the stomach rarely reaches two hours [19], the duration of this protective effect may be sufficient for counteracting the destructive effects of gastric acid. With high levels of the probiotics protected by the modified alginate, we predict that there will be sufficient amounts of the bacteria available for targeted release in the small and large intestine, thus providing a more impactful therapeutic effect.

As expected, the 2% modified alginate released the bacteria in SIF the fastest, followed by the 1%, 0.5% and lastly, the unmodified alginate. This is promising from a drug delivery standpoint as we can control the release of therapeutic agents based on the degree of modification of the alginate. If an early release in the small intestine is desired, the 2% modified alginate may be used whereas a therapeutic aimed for the colon could be delayed by encapsulation with 0.5% or even 0.25% modified alginate. The wide range of achievable release rates is the main novelty of this delivery platform, making it useful for both site-specific drug delivery and staggered release of multiple therapeutics encapsulated in various degrees of modified alginate. Since this modified alginate material is designed for a wide range of therapeutic drug delivery, determining the shelf life of the beads is important from a product development standpoint. We found a significant drop in viability immediately after the probiotics underwent freezing and lyophilization, which was expected as standard in the field [19, 59]. After 60 days, only a slight decrease in the viability of the bacteria was observed. We predict that if the duration of storage of the encapsulated probiotics was extended, only a similar gradual decline in probiotic viability would occur. We found no difference in bacteria viability between the unmodified and modified alginates suggesting that the modification of alginate did not induce a cytotoxic effect on the encapsulated products.

In summary, we have shown that hydrogel beads made with our modified alginate material are capable of protecting potential probiotic therapeutics from the harsh gastric environment while also providing a controlled release of encapsulated agents based on the level of modification of the alginate. With no demonstrable long-term cytotoxic effects, this novel oral drug delivery vehicle may present specific advantages over the more commonly used unmodified alginates for therapeutic drug delivery. Since probiotics are comprised of bacteria cells, which are relatively larger than most therapeutics, they are ideal for determining the release rate of encapsulated substances based on the degrees of modification of the alginate hydrogel. Future work with this material needs to be performed to determine if the level of modification has an impact on more soluble drugs that tend to have a burst release due to concentration gradients. If enough therapeutic is retained in the hydrogel after the initial burst release, then the level of modification should dictate the release of such drugs in the small intestine for enhancement of drug bioavailability.

CONCLUSION

We have shown in the present study that our recently described modified alginate material has many potential applications in oral drug delivery while representing a unique platform for effective targeted delivery of probiotics since it protects bacteria in acidic environments and potentially shields them from enzymatic degradation.

Acknowledgments

FUNDING

This work was supported by the Office of Research and Sponsored Programs of Wake Forest University; the Center on Diabetes, Obesity and Metabolism of the Wake Forest School of Medicine Collaborative Pilot Grant; NIH Pre-doctoral Training Program: Studies in Translational Regenerative Medicine (T32 NIBIB Grant #1T32EB014836-01A1, A. Atala, PI); and the Center for Functional Materials of Wake Forest University.

Footnotes

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or other-wise.

AVAILABILITY OF DATA AND MATERIALS

Data are available from the authors upon reasonable request and with permission of Wake Forest University Health Sciences.

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