Susan Westfall

Human Serum Albumin Nanoparticles for Use in Cancer Drug Delivery: Process Optimization and In Vitro Characterization

Human serum albumin nanoparticles (HSA-NPs) are widely-used drug delivery systems with applications in various diseases, like cancer. For intravenous administration of HSA-NPs, the particle size, surface charge, drug loading and in vitro release kinetics are important parameters for consideration. This study focuses on the development of stable HSA-NPs containing the anti-cancer drug paclitaxel (PTX) via the emulsion-solvent evaporation method using a high-pressure homogenizer. The key parameters for the preparation of PTX-HSA-NPs are: the starting concentrations of HSA, PTX and the organic solvent, including the homogenization pressure and its number cycles, were optimized. Results indicate a size of 143.4 ± 0.7 nm and 170.2 ± 1.4 nm with a surface charge of −5.6 ± 0.8 mV and −17.4 ± 0.5 mV for HSA-NPs and PTX-HSA-NPs (0.5 mg/mL of PTX), respectively. The yield of the PTX-HSA-NPs was ~93% with an encapsulation efficiency of ~82%. To investigate the safety and effectiveness of the PTX-HSA-NPs, an in vitro drug release and cytotoxicity assay was performed on human breast cancer cell line (MCF-7). The PTX-HSA-NPs showed dose-dependent toxicity on cells of 52%, 39.3% and 22.6% with increasing concentrations of PTX at 8, 20.2 and 31.4 μg/mL, respectively. In summary, all parameters involved in HSA-NPs’ preparation, its anticancer efficacy and scale-up are outlined in this research article.

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.

Circadian Clocks and Inflammation: Reciprocal Regulation and Shared Mediators

The immune system is deeply interconnected with the endogenous 24-h oscillators of the circadian system. Indeed, the connection between these two physiological systems occurs at multiple levels and in both directions. On one hand, various aspects of the immune system show daily rhythms, which appear to be essential for healthy immune maintenance and proper immune response. On the other hand, immune responses cause changes in circadian rhythms, disrupting their delicate balance and manifesting in disease. Indeed, immune challenges cause various time-, gene-, and tissue-specific effects on circadian-regulated factors. This article reviews the possible mediators of the cross talk between the circadian clock and the immune system, in particular the inflammatory pathways. The rhythmic expression of cytokines and their receptors, as well as other rhythmically regulated humoral factors such as glucocorticoids, melatonin, leptin, or prostaglandins, could gate the effects of the immune response on the circadian system. In addition, systemic cues such as body temperature and neuronal connections between the brain and peripheral tissues may underlie the immune–circadian communication.

Time-Dependent Effects of Localized Inflammation on Peripheral Clock Gene Expression in Rats

Many aspects of the immune system, including circulating cytokine levels as well as counts and function of various immune cell types, present circadian rhythms. Notably, the mortality rate of animals subjected to high doses of lipopolysaccharide is dependent on the time of treatment. In addition, the severity of symptoms of various inflammatory conditions follows a daily rhythmic pattern. The mechanisms behind the crosstalk between the circadian and immune systems remain elusive. Here we demonstrate that localized inflammation induced by turpentine oil (TURP) causes a time-dependent induction of interleukin (IL)-6 and has time-, gene- and tissue-specific effects on clock gene expression. More precisely, TURP blunts the peak of Per1 and Per2 expression in the liver while in other tissues, the expression nadir is elevated. In contrast, Rev-erbα expression remains relatively unaffected by TURP treatment. Co-treatment with the anti-inflammatory agent IL-1 receptor antagonist (IL-1Ra) did not alter the response of Per2 to TURP treatment in liver, despite the reduced induction of fever and IL-6 serum levels. This indicates that the TURP-mediated changes of Per2 in the liver might be due to factors other than systemic IL-6 and fever. Accordingly, IL-6 treatment had no effect on clock gene expression in HepG2 liver carcinoma cells. Altogether, we show that localized inflammation causes significant time-dependent changes in peripheral circadian clock gene expression, via a mechanism likely involving mediators independent from IL-6 and fever.

Longevity extension in Drosophila through gut-brain communication

Aging and chronic disease development are multifactorial processes involving the cumulative effects of metabolic distress, inflammation, oxidative stress and mitochondrial dynamics. Recently, variations in the gut microbiota have been associated with age-related phenotypes and probiotics have shown promise in managing chronic disease progression. In this study, novel probiotic and synbiotic formulations are shown to combinatorially extend longevity in male Drosophila melanogaster through mechanisms of gut-brain-axis communication with implications in chronic disease management. Both the probiotic and synbiotic formulations rescued markers of metabolic stress by managing insulin resistance and energy regulatory pathways. Both formulations also ameliorated elevations in inflammation, oxidative stress and the loss of mitochondrial complex integrity. In almost all the measured pathways, the synbiotic formulation has a more robust impact than its individual components insinuating its combinatorial effect. The concomitant action of the gut microbiota on each of the key risk factors of aging and makes it a powerful therapeutic tool against neurodegeneration, diabetes, obesity, cardiovascular disease and other age-related chronic diseases.

The Gut Microflora and its Metabolites Regulate the Molecular Crosstalk between Diabetes and Neurodegeneration

The gut microflora is a community of trillions of bacterial cells synergistically inhabiting the human gastrointestinal tract. These microbes contact everything that is consumed and release regulatory factors that affect host energy homeostasis, lipid and carbohydrate metabolism, activation of immune cells, oxidative state, epithelial cell wall integrity and even neurological signals. The gut microflora is essentially an independent organ supporting human health where imbalances in the gut community populations (dysbiosis) manifest in disease. Diabetes and neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease share a similar molecular pathology rooted in gut microflora activity. Both of these conditions are associated with a dysbiosis characterized by low species diversity, a higher proportion of pathobionts at the expense of symbionts, an abundance of proinflammatory microbes and fewer butyrate-producing strains. Many of these factors can be ameliorated with Lactobacillus spp. and Bifidobacterium spp. probiotic treatment aimed to reestablish healthy gut microflora diversity. Indeed, certain commensal and pathogenic strains promote chronic low-grade inflammation that stresses cellular infrastructure eventually leading to apoptosis in both the pancreas and the brain. Also, lack of some beneficial fermentation products such as butyrate and ferulic acid initiates a cascade of events disrupting metabolic homeostasis. Finally, signaling initiated by the microflora and its metabolites has been shown to disrupt the delicate intracellular balance of PI3K/Akt/mTOR signaling, which fundamentally regulates events leading up to diabetes and neurodegenerative disease pathogenesis. The following review investigates the relationship between the manifestation and molecular signaling of diabetes and neurodegenerative disorders and how the balance of gut microflora populations is critical to both prevent and possibly treat these diseases.

Ferulic Acid Produced by Lactobacillus fermentum NCIMB 5221 ReducesSymptoms of Metabolic Syndrome in Drosophila melanogaster

Abstract The gut microbiota is an intricate community of thousands of bacterial species living in the gastrointestinal tract critical for vitamin synthesis, mineral absorption, digestion of otherwise indigestible fibers and the extraction of energy from food. Recently, the health of the gut microbiota’s community architecture has become linked to energy-regulatory diseases including metabolic syndrome: a collection of symptoms including hyperglycemia, hypercholesterolemia, high blood pressure, increased abdominal fat and triglyceride levels. However, the mechanism of communication between the gut microbiota and the host energy metabolism remains elusive. The current study shows that ferulic acid (FA) produced by the intrinsic ferulic acid esterase activity of the probiotic bacteria Lactobacillus fermentum NCIMB 5221 (Lf5221) can dose-dependently rescue the phenotypic markers of diet-induced diabetes and obesity in Drosophila melanogaster. In Drosophila exposed to either a high-sugar or high-fat diet, living Lf5221 at 2.5 or 7.5 × 109 CFU/ml media effectively rescued whole-body weight, glucose, trehalose and triglyceride levels. All of the aforementioned effects were lost in heat-inactivated bacteria indicating that a metabolic product is responsible. Likewise, FA at 0.5 mM in the metabolically challenged Drosophila models reared similar effects on the physiological markers while also reducing hyperglycemia in the circulating hemolymph. On the signaling level, the high-sugar diet predictably had an elevated expression of the Drosophila insulin-like peptides 2, 3 and 5 and in the high-fat diet, an increase in fatty acid synthase, acetyl-CoA carboxylase and phosphoenolpyruvate carboxykinase expression. On both diets, Lf5221 and FA rescued gene expression, at different concentrations, to the level of controls. Examining the mechanistic gene expression, both Lf5221 and FA rescued expression of dFOXO and dTOR, but not dAkt indicating that FA produced by Lf5221 is acting on one of the downstream-signaling molecules from the insulin receptor, possibly dTOR: an overall energy regulator in flies and humans alike. The present study for the first time outlines a streamlined mechanism for how the gut microbiota communicates with the host’s energy-regulating metabolism. Proper supplementation with ferulic acid esterase active probiotics such as Lf5221 could potentially prevent or alleviate symptoms of metabolic syndrome and other energy-regulating diseases including diabetes, obesity and neurodegeneration.

A novel polyphenolic prebiotic and probiotic formulation have synergistic effects on the gut microbiota influencing Drosophila melanogaster physiology

Abstract The gut microbiota is a vast community of synergistic bacterial species providing health benefits to the host. Imbalances in the gut microbiota (dysbiosis) due to diet, antibiotic use, age and stress contribute to disease development including diabetes, obesity, colon cancer, inflammatory bowel disease, inflammaging and neurodegeneration. Fortunately, a probiotic regime with a diet rich in prebiotics may reverse dysbiosis promoting health and wellness in age. The current study designs, optimizes and tests a novel probiotic and synbiotic formulation consisting of three metabolically active probiotics Lactobacillus plantarum, Lactobacillus fermentum and Bifidobacteria infantis together with a novel polyphenol-rich prebiotic, Triphala. The prebiotic action of Triphala was characterized using in vitro batch cultures, Drosophila melanogaster and a simulated model of the human gastrointestinal tract (SHIME) where in each model, Triphala supported growth of beneficial bacteria while inhibiting pathogenic species. Neither Triphala at 0.5% w/v nor the individual probiotics at 5.0 × 108 to 7.5 × 109 CFU/ml demonstrated toxicity in Drosophila. Interestingly, motility was combinatorially enhanced by the probiotic and synbiotic formulations reflecting the beneficial variations in the gut microbiota. Altogether, the present study shows that probiotics and synbiotics in combination are more effective at modulating the gut microbiota and eliciting biological effects than their components.

A novel milrinone nanoformulation for use in cardiovascular diseases: preparation and in vitro characterization.

Cardiovascular diseases are the leading causes of mortality across the globe. Over the years, various drug formulations and delivery methods have been tested for cardiac repair. Milrinone (MRN) is a widely known cardiac inotrope drug used for the treatment of congestive heart failure in patients, however, its efficacy is limited. This study is the first to report the design of a novel MRN-nanoformulation using human serum albumin nanoparticles (HSA-NPs). The HSA-NPs exhibit promising drug delivery characteristics, such as target specificity, nonimmunogenicity, biocompatibility, and enhanced bioavailability. This article describes a MRN-nanoformulation design for in vitro drug release, cellular uptake, biocompatibility, and other features. The MRN-nanoformulation was prepared by the ethanol desolvation technique and key parameters were optimized to obtain a desired particle size of 154.2 ± 5.8 nm, zeta potential of -29.5 ± 2.9 mV, and a drug encapsulation efficiency of 41.1 ± 1.7%. Molecular docking studies have revealed that MRN binds in the hydrophobic cavity of HSA, which has also been indicated by circular dichroism and enzyme-mediated drug release studies in the presence of trypsin, pepsin, proteinase K, protease, and cathepsin D. The intracellular uptake of fluorescently tagged MRN-HSA-NPs using HUVEC and H9c2 cells was evaluated by flow cytometry. The nanoparticle toxicity results indicated that MRN-HSA-NPs show significantly lower cytotoxicity and higher cell viability ( P < 0.0001) as compared to the MRN-lactate drug in HUVEC (61.6 ± 3.7% vs 36.2 ± 2.9%) and H9c2 (58.8 ± 5.7% vs 18.8 ± 4.9%) cells. These studies indicate that the novel MRN-nanoformulation offers better drug delivery procedures than currently used methods and has potential in treatment of congestive heart failure and other cardiovascular diseases.

Novel microencapsulated probiotic blend for use in metabolic syndrome: design and in-vivo analysis

Abstract The increasing prevalence of the metabolic syndrome has made it a medical issue that currently affects 1 in 5 Canadians. The metabolic syndrome is defined by risk factors that predispose an individual to diabetes and cardiovascular disease. Current forms of interventions have been inadequate as substantiated by the fact that the prevalence of metabolic syndrome has not reduced over the years. The objective of this study was to investigate the therapeutic benefits of a novel microencapsulated probiotic blend in treating the metabolic syndrome. Three probiotic strains were microencapsulated into alginate-polylysine-alginate (APA) microcapsules: L. rhamnosus NCIMB 6375, L. plantarum NCIMB 8826 and L. fermentum NCIMB 5221. From the results, it was observed that the microencapsulated probiotic blend significantly reduced serum total cholesterol, LDL cholesterol and triglyceride levels (reducing from 516 mg/dL to 379 mg/dL, 314 mg/dL to 231 mg/dL and 580 mg/dL to 270 mg/dL, respectively). In addition, the administration of the microencapsulated probiotic blend was found to favourably influence the gut microbiota, decreasing Firmicutes levels and increasing Bacteroidetes levels. Overall, this work demonstrates the potential a microencapsulated probiotic blend could have in targeting multiple risk factors of the metabolic syndrome; however, greater research is still needed.