Imen Kahouli

Development and characterization of chitosan- PEG-TAT nanoparticles for the intracellular delivery of siRNA

Recently, cell-penetrating peptides have been proposed to translocate antibodies, proteins, and other molecules in targeted drug delivery. The proposed study presents the synthesis and characterization of a peptide-based chitosan nanoparticle for small interfering RNA (siRNA) delivery, in-vitro. Specifically, the synthesis included polyethylene glycol (PEG), a hydrophilic polymer, and trans-activated transcription (TAT) peptide, which were chemically conjugated on the chitosan polymer. The conjugation was achieved using N-Hydroxysuccinimide-PEG-maleimide (heterobifunctional PEG) as a cross-linker, with the bifunctional PEG facilitating the amidation reaction through its N-Hydroxysuccinimide group and reacting with the amines on chitosan. At the other end of PEG, the maleimide group was chemically conjugated with the cysteine-modified TAT peptide. The degree of substitution on chitosan with PEG and on PEG with TAT was confirmed using colorimetric assays. The resultant polymer was used to form nanoparticles complexing siRNA, which were then characterized for particle size, morphology, cellular uptake, and cytotoxicity. The nanoparticles were tested in-vitro on mouse neuroblastoma cells (Neuro2a). Particle size and surface charge were characterized and an optimal pH condition and PEG molecular weight were determined to form sterically stable nanoparticles. Results indicate 7.5% of the amines in chitosan polymer were conjugated to the PEG and complete conjugation of TAT peptide was observed on the synthesized PEGylated chitosan polymer. Compared with unmodified chitosan nanoparticles, the nanoparticles formed at pH 6 were monodispersed and of <100 nm in size, exhibiting maximum cell transfection ability and very low cytotoxicity. Thus, this research may be of significance in translocating biotherapeutic molecules for intracellular delivery applications.

Probiotics in colorectal cancer (CRC) with emphasis on mechanisms of action and current perspectives

Colorectal cancer (CRC) is the third most common form of cancer. Diverse therapies such as chemotherapy, immunotherapy and radiation have shown beneficial effects, but are limited because of their safety and toxicity. Probiotic formulations have shown great promise in CRC as preventive and early stage therapeutics. This review highlights the importance of a balanced intestinal microbiota and summarizes the recent developments in probiotics for treating CRC. Specifically, this report describes evidence of the role of probiotics in modulating the microbiota, in improving the physico-chemical conditions of the gut and in reducing oxidative stress. It also discusses the mechanisms of probiotics in inhibiting tumour progression, in producing anticancer compounds and in modulating the host immune response. Even though some of these effects were observed in several clinical trials, when probiotic formulations were used as a supplement to CRC therapies, the application of probiotics as biotherapeutics against CRC still needs further investigation.

Microbiome, probiotics and neurodegenerative diseases: deciphering the gut brain axis

The gut microbiota is essential to health and has recently become a target for live bacterial cell biotherapies for various chronic diseases including metabolic syndrome, diabetes, obesity and neurodegenerative disease. Probiotic biotherapies are known to create a healthy gut environment by balancing bacterial populations and promoting their favorable metabolic action. The microbiota and its respective metabolites communicate to the host through a series of biochemical and functional links thereby affecting host homeostasis and health. In particular, the gastrointestinal tract communicates with the central nervous system through the gut–brain axis to support neuronal development and maintenance while gut dysbiosis manifests in neurological disease. There are three basic mechanisms that mediate the communication between the gut and the brain: direct neuronal communication, endocrine signaling mediators and the immune system. Together, these systems create a highly integrated molecular communication network that link systemic imbalances with the development of neurodegeneration including insulin regulation, fat metabolism, oxidative markers and immune signaling. Age is a common factor in the development of neurodegenerative disease and probiotics prevent many harmful effects of aging such as decreased neurotransmitter levels, chronic inflammation, oxidative stress and apoptosis—all factors that are proven aggravators of neurodegenerative disease. Indeed patients with Parkinson’s and Alzheimer’s diseases have a high rate of gastrointestinal comorbidities and it has be proposed by some the management of the gut microbiota may prevent or alleviate the symptoms of these chronic diseases.

Microencapsulation for the Therapeutic Delivery of Drugs, Live Mammalian and Bacterial Cells, and Other Biopharmaceutics: Current Status and Future Directions

Microencapsulation is a technology that has shown significant promise in biotherapeutics, and other applications. It has been proven useful in the immobilization of drugs, live mammalian and bacterial cells and other cells, and other biopharmaceutics molecules, as it can provide material structuration, protection of the enclosed product, and controlled release of the encapsulated contents, all of which can ensure efficient and safe therapeutic effects. This paper is a comprehensive review of microencapsulation and its latest developments in the field. It provides a comprehensive overview of the technology and primary goals of microencapsulation and discusses various processes and techniques involved in microencapsulation including physical, chemical, physicochemical, and other methods involved. It also summarizes the state-of-the-art successes of microencapsulation, specifically with regard to the encapsulation of microorganisms, mammalian cells, drugs, and other biopharmaceutics in various diseases. The limitations and future directions of microencapsulation technologies are also discussed.

Probiotic Ferulic Acid Esterase Active Lactobacillus fermentum NCIMB 5221 APA Microcapsules for Oral Delivery: Preparation and in Vitro Characterization

Probiotics possess potential therapeutic and preventative effects for various diseases and metabolic disorders. One important limitation for the oral delivery of probiotics is the harsh conditions of the upper gastrointestinal tract (GIT) which challenge bacterial viability and activity. One proposed method to surpass this obstacle is the use of microencapsulation to improve the delivery of bacterial cells to the lower GIT. The aim of this study is to use alginate-poly-L-lysine-alginate (APA) microcapsules to encapsulate Lactobacillus fermentum NCIMB 5221 and characterize its enzymatic activity and viability through a simulated GIT. This specific strain, in previous research, was characterized for its inherent ferulic acid esterase (FAE) activity which could prove beneficial in the development of a therapeutic for the treatment and prevention of cancers and metabolic disorders. Our findings demonstrate that the APA microcapsule does not slow the mass transfer of substrate into and that of the FA product out of the microcapsule, while also not impairing bacterial cell viability. The use of simulated gastrointestinal conditions led to a significant 2.5 log difference in viability between the free (1.10 × 104 ± 1.00 × 103 cfu/mL) and the microencapsulated (5.50 × 106 ± 1.00 × 105 cfu/mL) L. fermentum NCIMB 5221 following exposure. The work presented here suggests that APA microencapsulation can be used as an effective oral delivery method for L. fermentum NCIMB 5221, a FAE-active probiotic strain.

Use of Artificial Cell Microcapsule Containing Thalidomide for Treating TNBS-induced Crohn's Disease in Mice

In this study, we examined the in-vivo characteristics of a novel microencapsulated thalidomide formulation in a murine model of experimental Crohns disease. Crohns disease was induced with a single intra-colonic injection of 120 mg/kg of bodyweight of 2,5,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol in Balb/c mice. Level of tumor necrosis factor alpha (TNF-α), interleukin one beta (IL-1β), interleukin 6 (IL-6) and nitric oxide (NO) were measured in tissue homogenate. Moreover, myeloperoxidase (MPO) activity was determined to assess the extent of neutrophil infiltration. Dose response study showed that treating the mice with microencapsulated thalidomide (100 mg/kg of bodyweight) for two weeks significantly decreased the degree of intestinal inflammation related to Crohns disease. Higher and lower doses (0, 25, 50 and 200 mg/kg of bodyweight) did not exhibit comparable effects. The present study validates the success of alginate-poly-L-lysine-alginate (APA) microcapsules containing thalidomide in reducing colonic inflammation, and proposes a potential remedy for Crohns disease.

In-Vitro Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus Fermentum NCIMB 5221 and Potential against Colorectal Cancer

Objective: Lactic acid bacteria such as Lactobacillus fermentum, have shown to increase the levels of fecal short chain fatty acids known with its beneficial role in colonic health and were found to produce anti-carcinogenic compounds, suggesting a potential in colorectal cancer prevention. The aim of this study is to characterize the metabolic and anti-cancer features of L. fermentum NCIMB 5221 compared to two other Lactobacillus species. Methods: A free fatty acid (FFA) profile was determined and the anti-proliferative and apoptotic effects of bacterial cell free extracts were investigated. The effect on the growth of colon cancer cells compared to nonneoplastic colon cells was determined. The production of different SCFAs by the probiotic bacteria and the efficacy of their composition were analyzed. Results: The FFA profile of L. fermentum is distinctive FFA profile (~ 368 MAE, 16 h, p < 0.01) compared to L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103. L. fermentum extracts significantly inhibited cancer cell growth up to ~ 40% and induced apoptosis up to ~ 30% in SW-480 colon cancer cells (24 h, p < 0.05) compared to the untreated cells. However, L. fermentum did not inhibit CRL-1831 non-neoplastic colon cell growth but had a significant anti-proliferative effect against Caco-2 cancer cells (~ 60%, 72 h, p < 0.001) compared to control, which was related to the higher levels of SCFAs produced (~ 377 mg/L). Similar concentrations of SCFA formulations (that correspond to what was produced by L. fermentum) have shown the same inhibitory effect on Caco-2 cells. Conclusion: L. fermentum NCIMB 5221 was more potent in suppressing colon cancer cells and promoting normal epithelial colon cell growth by the production of SCFAs and could be considered as biotherapeutic agent for the support of colonic health and the prevention of colorectal cancer.

Characterization of L. reuteri NCIMB 701359 Probiotic Features forPotential Use as a Colorectal Cancer Biotherapeutic by Identifying FattyAcid Profile and Anti-Proliferative Action against Colorectal Cancer Cells

Colorectal cancer (CRC), the third cause of cancer deaths worldwide, presents a condition with preventable aspects related to diet, lifestyle, and, in particular host gut microflora. Probiotic regimens have been proposed to diminish CRC risk and complications. Among L. reuteri strains previously screened some are still in need for further characterization to understand the connection between the probiotic metabolic activity and the potential anti-cancer features. Here, L. reuteri NCIMB 701359 was characterized for growth and fatty acid profile. The apoptotic and antiproliferative capacities of the bacterial extracts (supernatant and conditioned medium) against cCRC cells have been assessed. To investigate a potential anti-cancer activity, the effect of L. reuteri on the proliferation of Caco-2 CRC cells compared with CRL-1831 normal coloretcal cells was analyzed. Later, short chain fatty acids that L. reuteri produced, were measured and the inhibitory action of short chain fatty acids against Caco-2 cells was investigated using short chain fatty acid synthetic formulations. Results revealed a significantly higher fatty acid production for L. reuteri during growth compared with other Lactobacilli. Also, both L. reuteri extracts, especially the conditioned cell culture medium, exhibited significant inhibitory effects against SW-480 cancerous cells and induced apoptosis. L. reuteri suppressed Caco-2 (cancer) but not CRL-1831 (non-neoplastic). Caco-2 inhibition strongly correlated with the concentration of bacterial short chain fatty acids and was confirmed to be partially but not totally because of short chain fatty acid production. This suggests the potential of L. reuteri NCIMB 701359 in suppressing colorectal cancer cell growth and survival, which may assist in the formulation of probiotic-based interventions to limit colorectal cancer development.

Identification of Lactobacillus Fermentum Strains with Potential against Colorectal Cancer by Characterizing Short Chain Fatty Acids Production, Anti-Proliferative Activity and Survival in an Intestinal Fluid: In Vitro Analysis

The use of probiotics as preventive agents in colorectal cancer (CRC), as widely suggested in many clinical and pre-clinical studies, was often linked to the potency of short chain fatty acids (SCFAs) in the gut. However, there remains an incomplete understanding of the fatty-acid-producing activity of certain probiotics and their cancer preventive potential. In the current study, L. fermentum strains were investigated for their potential use with CRC treatments. Using cell-free extracts, L. fermentum NCIMB -5221, - 2797, and -8829 were first compared based on their SCFAs production and anti-proliferative activity against Caco-2 colon cancer cells. The corresponding SCFAs synthetic formulations, similar to the ones produced by the bacteria, were prepared and compared with the latter to determine the role and efficacy of naturally produced SCFAs in inhibiting the proliferation of colon cancer cells. Subsequently, the bioactivity and stability of L. fermentum bacterial strains in a simulated intestinal fluid (SIF) was determined. Results showed that L. fermentum NCIMB -5221 and -8829 were the most potent in producing SCFAs, in particular, acetic (192.3 ± 4 mg/L minimum), propionic (69.2 ± 1.6 mg/L minimum), and butyric (35.4 ± 2.9 mg/L minimum) acids. They were also found to inhibit the growth of Caco-2 cells (53.4 ± 1.6%, 72 h, p = 0.021) in comparison with L. acidophilus ATCC 314. Additionally, they showed resistance to SIF (16.3 ± 1.9% minimum, 72 h, p = 0.006) and produced SCFAs in SIF at concentrations high enough to significantly inhibit Caco-2 proliferation (74.73 ± 2.1%, 72 h). Based on characteristics related to bacterial cell survival, SCFA production, and anti-proliferative activity, L. fermentum NCIMB -5221 and - 2797 could potentially be considered as biotherapeutic agents against CRC.

Enrichment of Bifidobacterium longum subsp. infantis ATCC 15697 within the human gut microbiota using alginate-poly-l-lysine-alginate microencapsulation oral delivery system: an in vitro analysis using a computer-controlled dynamic human gastrointestinal model

Abstract This study evaluates alginate-poly-l-lysine-alginate Bifidobacterium longum subsp. infantis ATCC 15697-loaded microcapsules to enrich the human gut microbiota. The cell survival of alginate-poly-l-lysine-alginate microencapsulated B. infantis ATCC 15697 in gastric acid, bile, and through human gastrointestinal transit was investigated, as well as the formulation’s effect on the gut microbiota. Results show that microencapsulation increases B. infantis ATCC 15697 cell survival at pH1.0 (33.54 ± 2.80% versus <1.00 ± 0.00%), pH1.5 (41.15 ± 2.06% versus <1.00 ± 0.00%), pH2.0 (60.88 ± 1.73% versus 36.01 ± 2.63%), pH3.0 (75.43 ± 1.23% versus 46.30 ± 1.43%), pH4.0 (71.40 ± 2.02% versus 47.75 ± 3.12%) and pH5.0 (73.88 ± 3.79% versus 58.93 ± 2.26%) (p < 0.05). In addition, microencapsulation increases cell survival at 0.5% (76.85 ± 0.80% versus 70.77 ± 0.64%), 1.0% (59.99 ± 0.97% versus 53.47 ± 0.58%) and 2.0% (53.10 ± 1.87% versus 44.59 ± 1.52%) (p < 0.05) (w/v) bile. Finally, daily administration of alginate-poly-l-lysine-alginate microencapsulated B. infantis ATCC 15697 in a human gastrointestinal model induces a significant enrichment of B. infantis within the ascending (184.51 ± 17.30% versus 53.83 ± 17.82%; p < 0.05), transverse (174.79 ± 25.32% versus 73.17 ± 15.30%; p < 0.05) and descending (94.90 ± 25.22% versus 46.37 ± 18.93%; p > 0.05) colonic microbiota.