Orally delivered microencapsulated probiotic formulation favorably impacts polyp formation in APC (Min/+) model of intestinal carcinogenesis.

The development of intestinal polyps in an orthotopic colorectal mouse model, receiving a probiotic yogurt formulation containing microencapsulated live Lactobacillus acidophilus cells was investigated. The expression of various immunohistochemical markers namely CD8, Mac-1, Ki-67, and cleaved caspase-3, was evaluated. Results suggest that the probiotic formulation decreases overall intestinal inflammation. Mice receiving the probiotic formulation were found to develop almost two-fold fewer tumors in the small intestines. In the large intestine, however, there was no significant difference observed among polyp numbers. The formulation appears to have potential application in the prevention of various GI pathological conditions.

Bioengineered Colorectal Cancer Drugs: Orally Delivered Anti-Inflammatory Agents.

Intestinal inflammation is one of the major factors that increase colorectal cancer (CRC) incidence worldwide. Inflammation in the gastrointestinal tract is directly linked to tumor development at the early stages of the disease, thus a key issue toward the prevention and the treatment of colonic neoplasia. Thus, the use of anti-inflammatory drugs has emerged first as a strategy to reduce chronic inflammation in case of many inflammatory bowel diseases (IBD), but it has proven its efficacy by reducing the risk of colonic neoplasia. This comprehensive review highlights the role of chronic inflammation, mainly in IBD, in the development of CRC including molecular and immune mechanisms that have tumorigenic effects. Multiple lines of evidence indicate that several bioactive and phytochemical compounds used as anti-inflammatory drugs have also antitumoral attributes. The uses of orally delivered cytokines and small molecules, as well as key dietary supplementation as anti-inflammatory therapeutics are discussed. In addition, comprehensive knowledge about CRC and intestinal inflammation, and the importance of the intestinal mucosal wall as a mucosal immunological barrier that comes into play during interactions with gut microbiota (pathogens and commensal), luminal secretions (bile acids, and bacterial and epithelial metabolites), and ingested chemicals (food components, high fat content, heterocyclic amines, and low intake of dietary fiber) are underscored. The multifunctionality of several anti-inflammatory drugs opens a line for their application in the treatment and prevention not only in IBD but also in CRC. Current bioengineering approaches for oral delivery of anti-inflammatory agents including cytokines, genetically modified bacteria, or small molecule inhibitors of inflammation directly contribute to the early management of CRC. Limitations of the current therapeutics, which stem from the lack of complete understanding of the complex molecular interactions between the intestinal microbiota, colonic epithelial barrier, and host immune system, are also discussed.

Oral probiotic microcapsule formulation ameliorates non-alcoholic fatty liver disease in Bio F1B Golden Syrian hamsters.

The beneficial effect of a microencapsulated feruloyl esterase producing Lactobacillus fermentum ATCC 11976 formulation for use in non-alcoholic fatty liver disease (NAFLD) was investigated. For which Bio F1B Golden Syrian hamsters were fed a methionine deficient/choline devoid diet to induce non-alcoholic fatty liver disease. Results, for the first time, show significant clinical benefits in experimental animals. Examination of lipids show that concentrations of hepatic free cholesterol, esterified cholesterol, triglycerides and phospholipids were significantly lowered in treated animals. In addition, serum total cholesterol, triglycerides, uric acid and insulin resistance were found to decrease in treated animals. Liver histology evaluations showed reduced fat deposits. Western blot analysis shows significant differences in expression levels of key liver enzymes in treated animals. In conclusion, these findings suggest the excellent potential of using an oral probiotic formulation to ameliorate NAFLD.

Diet-induced metabolic hamster model of nonalcoholic fatty liver disease.

BACKGROUND: Obesity, hypercholesterolemia, elevated triglycerides, and type 2 diabetes are major risk factors for metabolic syndrome. Hamsters, unlike rats or mice, respond well to diet-induced obesity, increase body mass and adiposity on group housing, and increase food intake due to social confrontation-induced stress. They have a cardiovascular and hepatic system similar to that of humans, and can thus be a useful model for human pathophysiology. METHODS: Experiments were planned to develop a diet-induced Bio F(1)B Golden Syrian hamster model of dyslipidemia and associated nonalcoholic fatty liver disease in the metabolic syndrome. Hamsters were fed a normal control diet, a high-fat/high-cholesterol diet, a high-fat/high-cholesterol/methionine-deficient/choline-devoid diet, and a high-fat/high-cholesterol/choline-deficient diet. Serum total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glucose, atherogenic index, and body weight were quantified biweekly. Fat deposition in the liver was observed and assessed following lipid staining with hematoxylin and eosin and with oil red O. RESULTS: In this study, we established a diet-induced Bio F(1)B Golden Syrian hamster model for studying dyslipidemia and associated nonalcoholic fatty liver disease in the metabolic syndrome. Hyperlipidemia and elevated serum glucose concentrations were induced using this diet. Atherogenic index was elevated, increasing the risk for a cardiovascular event. Histological analysis of liver specimens at the end of four weeks showed increased fat deposition in the liver of animals fed with a high-fat/high cholesterol diet, as compared to animals fed with the control diet. CONCLUSION: Our study established that hamsters fed with a high-fat/high-cholesterol diet developed fatty liver and mild diabetes. Bio F(1)B hamsters fed with a high-fat/high-cholesterol diet may thus be a good animal model for research on the treatment of diet-induced metabolic syndrome complicated by nonalcoholic fatty liver disease.

Suppression of tumorigenesis: modulation of inflammatory cytokines by oral administration of microencapsulated probiotic yogurt formulation.

The objective of this study was to examine the ability of a novel microencapsulated probiotic yogurt formulation to suppress the intestinal inflammation. We assessed its anticancer activity by screening interleukin-1, 6, and 12 (IL-1, 6, 12), secretory levels of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), prostaglandin E(2) (PGE(2)), and thromboxane B2 in the digesta obtained from the duodenum, jejunum, proximal, and distal segments of the ileum of C57BL/6J-Apc(Min)/J mice. Formulation-receiving animals showed consistently lower proinflammatory cytokines' levels when compared to control group animals receiving empty alginate-poly-L-lysine-alginate (APA) microcapsules suspended in saline. The concentrations of IL-12 found in serum in control and treatment group animals were significant: 46.58 ± 16.96 pg/mL and 158.58 ± 28.56 pg/mL for control and treatment animals, respectively. We determined a significant change in plasma C-reactive protein: 81.04 ± 23.73 ng/mL in control group and 64.21 ± 16.64 ng/mL in treatment group. Western blots showed a 71% downregulation of cyclooxygenase-2 (COX-2) protein in treatment group animals compared to control. These results point to the possibility of using this yogurt formulation in anticancer therapies, in addition to chronic gut diseases such as Crohn's disease, irritable bowel syndrome (IBS), and inflammatory bowel disease (IBD) thanks to its inflammation lowering properties.

Oral microencapsulated live Saccharomyces cerevisiae cells for use in renal failure uremia: preparation and in vivo analysis.

Orally administrable alginate-poly-L-lysine-alginate (APA) microcapsules containing live yeast cells was investigated for use in renal failure. At all times, yeast cells remain inside the microcapsules, which are then excreted in the stool. During their gastrointestinal passage, small molecules, like urea, diffuse into the yeast microcapsules where they are hydrolyzed. Orally administrating these microcapsules to uremic rats was found to decrease urea concentrations from 7.29 +/- 0.89 mmol/L to 6.12 +/- 0.90 mmol/L over a treatment period of eight weeks. After stopping the treatment, the urea concentrations increased back to uremic levels of 7.64 +/- 0.77 mmol/L. The analysis of creatinine concentrations averaged 39.19 +/- 4.33 micromol/L, 50.83 +/- 5.55 micromol/L, and 50.28 +/- 7.10 micromol/L for the normal-control, uremic-control and uremic-treatment groups, respectively. While creatinine concentrations for both uremic-control and uremic-treatment groups did not differ among each other (P > .05), they were, however, significantly higher than those of the normal control group (P < .05). Uric acid concentrations averaged 80.08 +/- 26.49 micromol/L, 99.92 +/- 26.55 micromol/L, and 86.49 +/- 28.42 micromol/L for the normal-control, uremic-control and uremic-treatment groups, respectively. There were no significant differences in both calcium and phosphate concentrations among all three groups (P > .05). The microbial populations of five tested types of bacteria were not substantially altered by the presence of the yeast APA encapsulated yeast (P > .05).

Estimation of the potential antitumor activity of microencapsulated Lactobacillus acidophilus yogurt formulation in the attenuation of tumorigenesis in Apc(Min/+) mice.

There is a strong correlation between orally administered probiotics and suppression of the low-grade inflammation that can lead to restoration of normal local immune functions. We studied the potential immunomodulatory and antitumorigenic properties of microencapsulated probiotic bacterial cells in a yogurt formulation in Min mice carrying a germline APC mutation. Daily oral administration of microencapsulated Lactobacillus acidophilus bacterial cells in the yogurt formulation mice resulted in significant suppression of colon tumor incidence, tumor multiplicity, and reduced tumor size. Results show that oral administration of microencapsulated L. acidophilus contributed to the stabilization of animal body weight and decreased the release of bile acids. Histopathological analyses revealed fewer adenomas in treated versus untreated animals. Furthermore, treated animals exhibited fewer gastrointestinal intra-epithelial neoplasias with a lower grade of dysplasia in detected tumors. Results suggest that oral administration of microencapsulated probiotic L. acidophilus exerts anti-tumorous activity, which consequently leads to reduced tumor outcome.

Microencapsulated bile salt hydrolase producing Lactobacillus reuteri for oral targeted delivery in the gastrointestinal tract.

This is the first study of its kind to screen probiotic lactic acid bacteria for the purpose of microencapsulating a highly bile salt hydrolase (BSH)-active strain. A Lactobacillus reuteri strain and a Bifidobacterium longum strain were isolated as the highest BSH producers among the candidates. Microcapsules were prepared with a diameter of 619 +/- 31 mum and a cell load of 5 x 10(9) cfu/ml. Post de Man, Rogosa, and Sharpe broth-acid challenge, L. reuteri microcapsules metabolized glyco- and tauro-conjugated bile salts at rates of 10.16 +/- 0.46 and 1.85 +/- 0.33 micromol/g microcapsule per hour, respectively, over the first 2 h. Microencapsulated B. longum had minimal BSH activity and were significantly (P < 0.05) more susceptible to acid challenge. Further testing of L. reuteri microcapsules in a simulated human gastrointestinal (GI) model showed an improved rate, with 49.4 +/- 6.21% of glyco-conjugates depleted after 60 min and complete deconjugation after 4 h. Microcapsules protected the encased cells in the simulated stomach maintaining L. reuteri viability above 10(9), 10(8), and 10(6) cfu/ml after 2 h at pH 3.0, 2.5, and 2.0, respectively. Results show excellent potential for this highly BSH-active microencapsulation system in vitro, highlighted by improved viability and substrate utilization in simulated GI transit.

Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells.

There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review.

Preparation and in vitro analysis of microencapsulated live Lactobacillus fermentum 11976 for augmentation of feruloyl esterase in the gastrointestinal tract.

FA (ferulic acid) is a well-known phenolic phytochemical present in plant cell walls. Various studies have indicated that FA has many physiological functions in the prevention of chronic disease. It has been shown to play an important chemoprotective role in degenerative diseases. FA also shows strong antioxidant and nitrite-scavenging potential and anticarcinogenic and antiinflammatory properties. The in vivo physiological importance of FA depends on its availability for absorption. Dietary fibre-bound FA is partially released by gut micro-organisms; however, the concentration of the released FA is too low to act as a chemopreventive agent. Therefore it is important to augment the bioavailability of FA to appreciate more fully its real physiological effect. This paper evaluates the suitability of the alginate-poly(L-lysine)-alginate microcapsules for oral delivery of live feruloyl esterase-producing Lactobacillus fermentum 11976 cells, in vitro, by using a dynamic simulated human GI (gastrointestinal) model. The present study shows that microencapsulated L. fermentum 11976 cells can efficiently break down a FA-containing substrate, and establishes the biotechnological basis for their use in supplementing the bioavailability of dietary FA in the intestine.

Live encapsulated Lactobacillus acidophilus cells in yogurt for therapeutic oral delivery: preparation and in vitro analysis of alginate-chitosan microcapsules.

Targeted delivery of live microencapsulated bacterial cells has strong potential for application in treating various diseases, including diarrhea, kidney failure, liver failure, and high cholesterol, among others. This study investigates the potential of microcapsules composed of two natural polymers, alginate and chitosan (AC), and the use of these artificial cells in yogurt for delivery of probiotic Lactobacillus acidophilus bacterial live cells. Results show that the integrity of AC microcapsules was preserved after 76 h of mechanical shaking in MRS broth and after 12 h and 24 h in simulated gastric and intestinal fluids. Using an in vitro computer-controlled simulated human gastrointestinal (GI) model, we found 8.37 log CFU/mL of viable bacterial cells were present after 120 min of gastric exposure and 7.96 log CFU/mL after 360 min of intestinal exposure. In addition, AC microcapsules composed of chitosan 10 and 100 at various concentrations were subjected to 4-week storage in 2% milk fat yogurt or 0.85% physiological solution. It was found that 9.37 log CFU/mL of cells encapsulated with chitosan 10 and 8.24 log CFU/mL of cells encapsulated with chitosan 100 were alive after 4 weeks. The AC capsule composed of 0.5% chitosan 10 provided the highest bacterial survival of 9.11 log CFU/mL after 4 weeks. Finally, an investigation of bacterial viability over 72 h in different pH buffers yielded highest survival of 6.34 log CFU/mL and 10.34 log CFU/mL at pH 8 for free and AC-encapsulated cells, respectively. We conclude from these findings that encapsulation allows delivery of a higher number of bacteria to desired targets in the GI tract and that microcapsules containing bacterial cells are good candidates for oral artificial cells for bacterial cell therapy.

Microencapsulated bacterial cells can be used to produce the enzyme feruloyl esterase: preparation and in-vitro analysis.

Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.

Investigation of microencapsulated BSH active lactobacillus in the simulated human GI tract.

This study investigated the use of microencapsulated bile salt hydrolase (BSH) overproducing Lactobacillus plantarum 80 cells for oral delivery applications using a dynamic computer-controlled model simulating the human gastrointestinal (GI) tract. Bile salt deconjugation rates for microencapsulated BSH overproducing cells were 4.87 +/- 0.28 mumol/g microcapsule/h towards glycoconjugates and 0.79 +/- 0.15 mumol/g microcapsule/h towards tauroconjugates in the simulated intestine, a significant (P< .05) increase over microencapsulated wild-type cells. Microcapsules protected the encased cells in the simulated stomach prior to intestinal release, maintaining cell viability above 109 cfu/mL at pH 2.5 and 3.0 and above 106 cfu/mL at pH 2.0 after 2-hour residence times. In the simulated intestine, encased cell viability was maintained above 1010 cfu/mL after 3, 6, and 12-hour residence times in bile concentrations up to 1.0%. Results show that microencapsulation has potential in the oral delivery of live BSH active bacterial cells. However, in vivo testing is required.

Superior cell delivery features of poly(ethylene glycol) incorporated alginate, chitosan, and poly-L-lysine microcapsules.

Microencapsulation is an emerging technology in the development of bioartificial organs for drug, protein, and delivery systems. One of the advancements in establishing an appropriate membrane material for live cell and tissue encapsulation is the incorporation of poly(ethylene glycol) (PEG) to the widely studied alginate microcapsules. The current study investigates the properties of integrating PEG to microcapsules coated with poly-L-lysine (PLL) and chitosan as well as a novel microcapsule membrane which combines both PLL and chitosan. Results show that microcapsules containing PEG can support cell viability and protein secretion. The addition of PEG to PLL and chitosan-coated microcapsules improves the stability of microcapsules when exposed to a hypotonic solution. We also compared the novel microcapsule with two other previously used microcapsules including alginate-chitosan-PEG and alginate-PLL-PEG-alginate. Results show that all three membranes are capable of providing immunoprotection to the cells and have the potential for long-term storage at -80 degrees C. The novel membrane containing PEG, chitosan, and PLL, however, revealed the highest cell viability and mechanical strength when exposed to external rotational force, but it was unable to sustain osmotic pressure. The study revealed the potential of using PEG-incorporated alginate, chitosan, and PLL microcapsules for encapsulating live cells producing proteins and hormones for therapy.

In vitro study of alginate-chitosan microcapsules: an alternative to liver cell transplants for the treatment of liver failure.

The application of alginate-chitosan (AC) microcapsules to liver cell transplantation has not been previously investigated. In the current in vitro study, we have investigated the potential of AC microcapsules for the encapsulation of liver cells and show that the AC membrane supports the survival, proliferation and protein secretion by entrapped hepatocytes. The AC membrane provides cell immuno-isolation and has the potential for cell cryopreservation. The AC microcapsule has several advantages compared to more widely used alginate-poly-L-lysine (APA) microcapsules for the application of cell therapy.