Toward a new generation of therapeutics: artificial cell targeted delivery of live cells for therapy

Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and kappa-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in "artificial cells," builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.     

Role of Gut Microbiota,Probiotics and Prebiotics in the Cardiovascular Diseases

In recent years, there has been a growing interest in identifying and applying new, naturally occurring molecules that promote health. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer health benefits on the host”. Quite a few fermented products serve as the source of probiotic strains, with many factors influencing the effectiveness of probiotics, including interactions of probiotic bacteria with the host’s microbiome. Prebiotics contain no microorganisms, only substances which stimulate their growth. Prebiotics can be obtained from various sources, including breast milk, soybeans, and raw oats, however, the most popular prebiotics are the oligosaccharides contained in plants. Recent research increasingly claims that probiotics and prebiotics alleviate many disorders related to the immune system, cancer metastasis, type 2 diabetes, and obesity. However, little is known about the role of these supplements as important dietary components in preventing or treating cardiovascular disease. Still, some reports and clinical studies were conducted, offering new ways of treatment. Therefore, the aim of this review is to discuss the roles of gut microbiota, probiotics, and prebiotics interventions in the prevention and treatment of cardiovascular disease.     

Probiotics—Interactions with Bile Acids and Impact on Cholesterol Metabolism

The use of probiotics, alone or in interaction with bile acids, is a modern strategy in the prevention and treatment of hypercholesterolemia. Numerous mechanisms for hypocholesterolemic effect of probiotics have been hypothesized, based mostly on in vitro evidence. Interaction with bile acids through reaction of deconjugation catalyzed by bile salt hydrolase enzymes (BSH) is considered as the main mechanism of cholesterol-lowering effects of probiotic bacteria, but it has been reported that microbial BSH activity could be potentially detrimental to the human host. There are several approaches for prevention of possible side effects associated with BSH activity, which at the same time increase the viability of probiotics in the intestines and also in food matrices. The aim of our study was to summarize present knowledge of probiotics??bile acids interactions, with special reference to cholesterol-lowering mechanisms of probiotics, and to report novel biotechnological approaches for increasing the pharmacological benefits of probiotics.     

Catechol determination in compost bioremediation using a laccase sensor and artificial neural networks

An electrochemical biosensor based on the immobilization of laccase on magnetic core-shell (Fe₃O₄–SiO₂) nanoparticles was combined with artificial neural networks (ANNs) for the determination of catechol concentration in compost bioremediation of municipal solid waste. The immobilization matrix provided a good microenvironment for retaining laccase bioactivity, and the combination with ANNs offered a good chemometric tool for data analysis in respect to the dynamic, nonlinear, and uncertain characteristics of the complex composting system. Catechol concentrations in compost samples were determined by using both the laccase sensor and HPLC for calibration. The detection range varied from 7.5 × 10^–7 to 4.4 × 10^–4 M, and the amperometric response current reached 95% of the steady-state current within about 70 s. The performance of the ANN model was compared with the linear regression model in respect to simulation accuracy, adaptability to uncertainty, etc. All the results showed that the combination of amperometric enzyme sensor and artificial neural networks was a rapid, sensitive, and robust method in the quantitative study of the composting system. Figure Structure of the magnetic carbon paste electrode used in the electrochemical biosensor     

Probiotics in Cosmetic and Personal Care Products: Trends and Challenges

Probiotics, defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host,” are becoming increasingly popular and marketable. However, too many of the products currently labelled as probiotics fail to comply with the defining characteristics. In recent years, the cosmetic industry has increased the number of products classified as probiotics. While there are several potential applications for probiotics in personal care products, specifically for oral, skin, and intimate care, proper regulation of the labelling and marketing standards is still required to guarantee that consumers are indeed purchasing a probiotic product. This review explores the current market, regulatory aspects, and potential applications of probiotics in the personal care industry.     

Selenium methylselenocysteine protects human hepatoma HepG2 cells against oxidative stress induced by tert-butyl hydroperoxide

Selenium methylselenocysteine (Se-MeSeCys) is a common selenocompound in the diet with a tested chemopreventive effect. This study investigated the potential protective effect of Se-MeSeCys against a chemical oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) on human hepatoma HepG2 cells. Speciation of selenium derivatives by liquid chromatography–inductively coupled plasma mass spectrometry depicts Se-MeSeCys as the only selenocompound in the cell culture. Cell viability (lactate dehydrogenase) and markers of oxidative status—concentration of reduced glutathione (GSH) and malondialdehyde (MDA), generation of reactive oxygen species (ROS) and activity of the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR)—were evaluated. Pretreatment of cells with Se-MeSeCys for 20 h completely prevented the enhanced cell damage, MDA concentration and GR and GPx activity and the decreased GSH induced by t-BOOH but did not prevent increased ROS generation. The results show that treatment of HepG2 cells with concentrations of Se-MeSeCys in the nanomolar to micromolar range confers a significant protection against an oxidative insult.     

Ferulated Pectins and Ferulated Arabinoxylans Mixed Gel for Saccharomyces boulardii Entrapment in Electrosprayed Microbeads

Ferulated polysaccharides such as pectin and arabinoxylan form covalent gels which are attractive for drug delivery or cell immobilization. Saccharomyces boulardii is a probiotic yeast known for providing humans with health benefits; however, its application is limited by viability loss under environmental stress. In this study, ferulated pectin from sugar beet solid waste (SBWP) and ferulated arabinoxylan from maize bioethanol waste (AX) were used to form a covalent mixed gel, which was in turn used to entrap S. boulardii (2.08 × 10⁸ cells/mL) in microbeads using electrospray. SBWP presented a low degree of esterification (30%), which allowed gelation through Ca²⁺, making it possible to reduce microbead aggregation and coalescence by curing the particles in a 2% CaCl₂ cross-linking solution. SBWP/AX and SBWP/AX+ S. boulardii microbeads presented a diameter of 214 and 344 µm, respectively, and a covalent cross-linking content (dimers di-FA and trimer tri-FA of ferulic acid) of 1.15 mg/g polysaccharide. The 8-5′, 8-O-4′and 5-5′di-FA isomers proportions were 79%, 18%, and 3%, respectively. Confocal laser scanning microscopy images of propidium iodide-stained yeasts confirmed cell viability before and after microbeads preparation by electrospray. SBWP/AX capability to entrap S. boulardii would represent an alternative for probiotic immobilization in tailored biomaterials and an opportunity for sustainable waste upcycling to value-added products.     

Stronger together for in-cell translation: natural and unnatural base modified mRNA

The preparation of highly modified mRNAs and visualization of their cellular distribution are challenging. We report in-cell application of in vitro transcribed mRNA containing natural base modifications and site-specifically introduced artificial nucleotides. Click chemistry on mRNA allows visualization in cells with excellent signal intensities. While non-specific introduction of reporter groups often leads to loss in mRNA functionality, we combined the benefits from site-specificity in the 3′-UTR incorporated unnatural nucleotides with the improved translation efficiency of the natural base modifications Ψ and 5mC. A series of experiments is described to observe, quantify and verify mRNA functionality. This approach represents a new way to visualize mRNA delivery into cells and monitor its spread on a cellular level and translation efficiency. We observed increased protein expression from this twofold chemically modified, artificial mRNA counterbalancing a reduced transfection rate. This synergetic effect can be exploited as a powerful tool for future research on mRNA therapeutics.

Introducing unnatural base modifications site-specifically into the 3′-UTR of an mRNA bearing natural base modifications allows efficient visualization in cells by click chemistry. An enhanced protein expression in cells is observed from this twofold modified mRNA.     

Epicatechin-copper 9II) complexes: Damage of small intestinal epithelium

Four epicatechins [(−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCg)] and their corresponding copper complexes were compared with regard to their effect on the viability of Caco-2 colon cancer cells in vitro, measured by 3-(4,5-dimethylthyazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. The viability of Caco-2 cells exposed to EC (1 mM), ECg (1 mM) or EGC (1mM) respectively, for 30 min, was comparable to that of the saline control group, while EGCg (1 mM) apparently enhanced cellular activity. in contrast, the cells treated with epicatechin-copper complexes were killed. Bivalent copper 91 mM), in similar conditions, did not affect the cells. No cell leakage or other histological differences were observed, implying a rapid cell death. The suggested mechanism of killing is by OH radical attack, produced in the presence of epicatechin-copper complexes, but not in the presence of either of the epicatechins or copper alone. The reaction sites are discussed.     

Thermal Drying of <Emphasis Type="BoldItalic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="BoldItalic">bulgaricus</Emphasis> and its Efficient Use as Starter for Whey Fermentation and Unsalted Cheese Making

Lactobacillus bulgaricus grown on whey was dried by a simple thermal drying method at the range 35–55°C and its efficiency for lactic acid fermentation of whey was evaluated. Drying of cells in whey suspension in the examined temperature range did not affect significantly their viability (82–87% survival), indicating a protective effect of whey as both growth and drying medium. The kinetics of fermentation of whey and mixtures of whey/molasses using the dried culture were comparable to those of non-dried cells, and only low pH had a detrimental effect on the fermentation ability of the dried cells. Furthermore, dried L. bulgaricus, free or immobilized on casein coagulates, was used as starter for the production of unsalted hard-type cheese. The effects of the amount of starter culture and the immobilization technique, the evolution of microbial counts, and the sensory properties of the produced cheeses were evaluated during ripening at various temperatures.     

Neuroprotective Effects of Curcumin in Methamphetamine-Induced Toxicity

Curcumin (CUR), a natural polyphenol extracted from rhizome of the Curcuma longa L, has received great attention for its multiple potential health benefits as well as disease prevention. For instance, CUR protects against toxic agents acting on the human body, including the nervous system. In detail, CUR possesses, among others, strong effects as an autophagy activator. The present study indicates that CUR counteracts methamphetamine (METH) toxicity. Such a drug of abuse is toxic by disturbing the autophagy machinery. We profited from an unbiased, low variable cell context by using rat pheochromocytoma PC12 cell line. In such a system, a strong protection was exerted by CUR against METH toxicity. This was associated with increased autophagy flux, merging of autophagosomes with lysosomes and replenishment of autophagy vacuoles with LC3, which instead is moved out from the vacuoles by METH. This is expected to enable the autophagy machinery. In fact, while in METH-treated cells the autophagy substrates α-synuclein accumulates in the cytosol, CUR speeds up α-synuclein clearance. Under the effects of CUR LC3 penetrate in autophagy vacuoles to commit them to cell clearance and promotes the autophagy flux. The present data provide evidence that CUR counteracts the neurotoxic effects induced by METH by promoting autophagy.     

Efficiency of DNA Transfection of Rat Heart Myoblast Cells H9c2(2-1) by Either Polyethyleneimine or Electroporation

Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210 ± 12 RFU) and continued to increase after 72 h (243 ± 14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 μF, and one pulse), low level EGFP expression was observed after 48 h (49 ± 4 RFU) or 72 h (21 ± 14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6 ± 2.2% of PEI and 10.1 ± 1.5% of electroporation (250 V, 500 μF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50 Ω, and 50 μF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.     

Immobilization of isolated and cellular hydrogenase ofD. desulfuricans in radiation polymerized polyacrylamides

Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells.     

Microtable arrays for culture and isolation of cell colonies

Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new three-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10-μJ pulses to each of the four support structures. In contrast, microstructures with a 150 × 150-μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie–Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array.     

Resveratrol Enhances Inhibition Effects of Cisplatin on Cell Migration and Invasion and Tumor Growth in Breast Cancer MDA-MB-231 Cell Models In Vivo and In Vitro

Triple-negative breast cancer (TNBC) is a refractory type of breast cancer that does not yet have clinically effective drugs. The aim of this study is to investigate the synergistic effects and mechanisms of resveratrol combined with cisplatin on human breast cancer MDA-MB-231 (MDA231) cell viability, migration, and invasion in vivo and in vitro. In vitro, MTS assays showed that resveratrol combined with cisplatin inhibits cell viability as a concentration-dependent manner, and produced synergistic effects (CI < 1). Transwell assay showed that the combined treatment inhibits TGF-β1-induced cell migration and invasion. Immunofluorescence assays confirmed that resveratrol upregulated E-cadherin expression and downregulated vimentin expression. Western blot assay demonstrated that resveratrol combined with cisplatin significantly reduced the expression of fibronectin, vimentin, P-AKT, P-PI3K, P-JNK, P-ERK, Sma2, and Smad3 induced by TGF-β1 (p < 0.05), and increased the expression of E-cadherin (p < 0.05), respectively. In vivo, resveratrol enhanced tumor growth inhibition and reduced body weight loss and kidney function impairment by cisplatin in MDA231 xenografts, and significantly reduced the expressions of P-AKT, P-PI3K, Smad2, Smad3, P-JNK, P-ERK, and NF-κB in tumor tissues (p < 0.05). These results indicated that resveratrol combined with cisplatin inhibits the viability of breast cancer MDA231 cells synergistically, and inhibits MDA231 cells invasion and migration through Epithelial-mesenchymal transition (EMT) approach, and resveratrol enhanced anti-tumor effect and reduced side of cisplatin in MDA231 xenografts. The mechanism may be involved in the regulations of PI3K/AKT, JNK, ERK and NF-κB expressions.     

Nanoparticle-Decorated Biomimetic Extracellular Matrix for Cell Nanoencapsulation and Regulation

Cell encapsulation has been studied for various applications ranging from cell transplantation to biological production. However, current encapsulation technologies focus on cell protection rather than cell regulation that is essential to most if not all cell-based applications. Here we report a method for cell nanoencapsulation and regulation using an ultrathin biomimetic extracellular matrix as a cell nanocapsule to carry nanoparticles (CN²). This method allows high-capacity nanoparticle retention at the vicinity of cell surfaces. The encapsulated cells maintain high viability and normal metabolism. When gold nanoparticles (AuNPs) are used as a model to decorate the nanocapsule, light irradiation transiently increases the temperature, leading to the activation of the heat shock protein 70 (HSP70) promoter and the regulation of reporter gene expression. As the biomimetic nanocapsule can be decorated with any or multiple NPs, CN² is a promising platform for advancing cell-based applications.     

Human cardiac stem cells rejuvenated by modulating autophagy with MHY-1685 enhance the therapeutic potential for cardiac repair

Stem cell-based therapies with clinical applications require millions of cells. Therefore, repeated subculture is essential for cellular expansion, which is often complicated by replicative senescence. Cellular senescence contributes to reduced stem cell regenerative potential as it inhibits stem cell proliferation and differentiation as well as the activation of the senescence-associated secretory phenotype (SASP). In this study, we employed MHY-1685, a novel mammalian target of rapamycin (mTOR) inhibitor, and examined its long-term priming effect on the activities of senile human cardiac stem cells (hCSCs) and the functional benefits of primed hCSCs after transplantation. In vitro experiments showed that the MHY-1685‒primed hCSCs exhibited higher viability in response to oxidative stress and an enhanced proliferation potential compared to that of the unprimed senile hCSCs. Interestingly, priming MHY-1685 enhanced the expression of stemness-related markers in senile hCSCs and provided the differentiation potential of hCSCs into vascular lineages. In vivo experiment with echocardiography showed that transplantation of MHY-1685‒primed hCSCs improved cardiac function than that of the unprimed senile hCSCs at 4 weeks post-MI. In addition, hearts transplanted with MHY-1685-primed hCSCs exhibited significantly lower cardiac fibrosis and higher capillary density than that of the unprimed senile hCSCs. In confocal fluorescence imaging, MHY-1685‒primed hCSCs survived for longer durations than that of the unprimed senile hCSCs and had a higher potential to differentiate into endothelial cells (ECs) within the infarcted hearts. These findings suggest that MHY-1685 can rejuvenate senile hCSCs by modulating autophagy and that as a senescence inhibitor, MHY-1685 can provide opportunities to improve hCSC-based myocardial regeneration.Subject terms: Heart stem cells, Stem-cell therapies     

Natural Hemp-Ginger Extract and Its Biological and Therapeutic Efficacy

The prevention and treatment of skin diseases remains a major challenge in medicine. The search for natural active ingredients that can be used to prevent the development of the disease and complement treatment is on the rise. Natural extracts of ginger and hemp offer a wide range of bioactive compounds with potential health benefits. This study evaluates the effectiveness of hemp and ginger extract as a supportive treatment for skin diseases. It reports a synergistic effect of hemp and ginger extract. The contents of cannabinoids and components of ginger are determined, with the highest being CBD (587.17 ± 8.32 µg/g) and 6-gingerol (60.07 ± 0.40 µg/g). The minimum inhibitory concentration for Staphylococcus aureus (156.5 µg/mL), Escherichia coli (625.2 µg/mL) and Candida albicans (78.3 µg/mL) was also analyzed. Analysis of WM-266-4 cells revealed the greatest decrease in metabolic activity in cells exposed to the extract at a concentration of 1.00 µg/mL. Regarding the expression of genes associated with cellular processes, melanoma aggressiveness, resistance and cell survival, a significant difference was found in the expression of ABCB5, CAV1 and S100A9 compared with the control (cells not exposed to the extract).     

Ethanol Production Using Immobilized <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in Lyophilized Cellulose Gel

A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12–216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.     

Electroenzymatic reactions with sorbitol dehydrogenase on gold electrodes

Sorbitol dehydrogenase (SDH) originating from recombinant Escherichia coli cells is immobilized on gold electrodes. First of all, (4-carboxy-2,5,7-trinitrofluorenyliden)malon-nitrile (CTFM) is adsorbed on the surface as mediator. In a second step, the cofactor β-nicotinamide adenine dinucleotide (NAD⁺) is immobilized on the gold electrode. Due to the formation of a complex between the mediator and the cofactor, the electron transfer rate can be enhanced by adding calcium ions to the buffer. The immobilization of NAD⁺ and SDH on the surface has been achieved by cross-linking with the glutaraldehyde/bovine serum albumin system. The successful biofunctionalization is monitored by cyclic voltammetry.Paper presented at the “Jahrestagung der Fachgruppe Angewandte Electrochemie der Gesellschaft Deutscher Chemiker, Düsseldorf, 11.-14.09.2005”.