Aloe arborescens: In Vitro Screening of Genotoxicity, Effective Inhibition of Enzyme Characteristics for Disease Etiology, and Microbiological Activity.

The present study assessed the genotoxicity, the possibility of inhibiting selected enzymes, and the microbial activity of lyophilisate from 3-year-old A. arborescens leaves obtained from controlled crops. The lyophilisate from 3-year-old A. arborescens leaves was standardized for aloin A and aloenin A content. Moreover, concentrations of polyphenolic compounds and phenolic acids were determined. The first stage of the research was to determine genotoxicity using the comet test, which confirmed the safety of A. arborescens. Assays of enzymatic inhibition were performed for hyaluronidase (IC50 = 713.24 ± 41.79 µg/mL), α-glucosidase (IC50 = 598.35 ± 12.58 µg/mL), acetylcholinesterase and butyrylcholinesterase (1.16 vs. 0.34 µM of eserine/g d.m., respectively). The next stage of the research was to determine the ability of the healing properties using the scratch test, which showed a positive response using the extract. Microbial activity was evaluated and obtained against Gram-negative and Gram-positive bacteria and yeasts. We concluded that A. arborescens leaf gel meets the important conditions for plant raw materials to obtain semi-solid forms of herbal medicinal products.

Enhancing the Furosemide Permeability by Papain Minitablets Through a Triple Co-culture In Vitro Intestinal Cell Model.

The administration of medicines by the oral route is the most used approach for being very convenient. Although it is the most popular, this route also has absorption, and consequently, bioavailability limitations. In this sense, several pharmacotechnical strategies have been used to improve drug absorption, one of which is the use of permeation promoters. Papain is a very versatile plant enzyme that can be used as a permeation promoter of various active compounds. This study aimed to evaluate the safety of papain and the formulation of native papain minitablets to promote in vitro permeation of furosemide through an innovative biomimetic triple co-culture model of Caco-2, HT29-MTX, and Raji cells. Regarding permeation, furosemide and metaprolol concentrations are determined with HPLC; those are used to calculate Papp. Monolayer integrity was evaluated using TEER and Lucifer Yellow. In the presence of papain, TEER decreased two-fold and the Papp of furosemide increased six-fold. The results suggest that native papain minitablets can be used as therapeutic adjuvants to enhance the permeation of drugs significantly improving bioavailability.

Modular and tunable alternative surfactants for biopharmaceuticals provide insights into Surfactant's Structure-Function relationship.

Surface-induced aggregation of protein therapeutics is opposed by employing surfactants, which are ubiquitously used in drug product development, with polysorbates being the gold standard. Since poloxamer 188 is currently the only generally accepted polysorbate alternative, but cannot be ubiquitously applied, there is a strong need to develop surfactant alternatives for protein biologics that would complement and possibly overcome known drawbacks of existing surfactants. Yet, a severe lack of structure-function relationship knowledge complicates the development of new surfactants. Herein, we perform a systematic analysis of the structure-function relationship of three classes of novel alternative surfactants. Firstly, the mode of action is thoroughly characterized through tensiometry, calorimetry and MD simulations. Secondly, the safety profiles are evaluated through cell-based in vitro assays. Ultimately, we could conclude that the alternative surfactants investigated possess a mode of action and safety profile comparable to polysorbates. Moreover, the biophysical patterns elucidated here can be exploited to precisely tune the features of future surfactant designs.

Antimicrobial Activity, Genetic Relatedness, and Safety Assessment of Potential Probiotic Lactic Acid Bacteria Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis) Used as Live Feed in Fish Larviculture.

Aquaculture is a rapidly expanding agri-food industry that faces substantial economic losses due to infectious disease outbreaks, such as bacterial infections. These outbreaks cause disruptions and high mortalities at various stages of the rearing process, especially in the larval stages. Probiotic bacteria are emerging as promising and sustainable alternative or complementary strategies to vaccination and the use of antibiotics in aquaculture. In this study, potential probiotic candidates for larviculture were isolated from a rotifer-rearing tank used as the first live feed for turbot larvae. Two Lacticaseibacillus paracasei and two Lactiplantibacillus plantarum isolates were selected for further characterization due to their wide and strong antimicrobial activity against several ichthyopathogens, both Gram-positive and Gram-negative. An extensive in vitro safety assessment of these four isolates revealed the absence of harmful traits, such as acquired antimicrobial resistance and other virulence factors (i.e., hemolytic and gelatinase activities, bile salt deconjugation, and mucin degradation, as well as PCR detection of biogenic amine production). Moreover, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) analyses unveiled their genetic relatedness, revealing two divergent clusters within each species. To our knowledge, this work reports for the first time the isolation and characterization of Lactic Acid Bacteria (LAB) with potential use as probiotics in aquaculture from rotifer-rearing tanks, which have the potential to optimize turbot larviculture and to introduce novel microbial management approaches for a sustainable aquaculture.

In vitro test methods for evaluating high molecular weight polyethylene oxide polymer induced hemolytic and thrombotic potential.

To combat opioid abuse, the U.S. Food and Drug Administration (FDA) released a comprehensive action plan to address opioid addiction, abuse, and overdose that included increasing the prevalence of abuse-deterrent formulations (ADFs) in opioid tablets. Polyethylene oxide (PEO) has been widely used as an excipient to deter abuse via nasal insufflation. However, changes in abuse patterns have led to unexpected shifts in abuse from the nasal route to intravenous injection. Case reports identify adverse effects similar to thrombotic thrombocytopenic purpura (TTP) syndrome following the intravenous (IV) abuse of opioids containing PEO excipient. Increased risk of IV opioid ADF abuse compared to clinical benefit of the drug led to the removal of one opioid product from the market in 2017. Because many generic drugs containing PEO are still in development, there is interest in assessing safety consistent with generic drug regulation and unintended uses. Currently, there are no guidelines or in vitro assessment tools to characterize the safety of PEO excipients taken via intravenous injection. To create a more robust excipient safety evaluation tool and to study the mechanistic basis of HMW PEO-induced TMA, a dynamic in vitro test system involving blood flow through a needle model has been developed.

Development of Nanofibers with Embedded Liposomes Containing an Immunomodulatory Drug Using Green Electrospinning.

Conventional treatments for chronic wounds are often ineffective, thus new therapeutic approaches are needed, such as the delivery of immunomodulatory drugs that can reduce inflammation, restore immune cell function, and facilitate tissue regeneration. A potential drug for such an approach is simvastatin, which has major drawbacks including poor solubility and chemical instability. With the aim of developing a dressing for wound healing, simvastatin and an antioxidant were incorporated into alginate/poly(ethylene oxide) nanofibers by green electrospinning without the use of organic solvents, thanks to their prior encapsulation into liposomes. The composite liposome-nanofiber formulations exhibited fibrillar morphology (160-312 nm) and unprecedentedly high phospholipid and drug content (76%). Transmission electron microscopy revealed dried liposomes as bright ellipsoidal spots homogeneously distributed over the nanofibers. After nanofiber hydration, the liposomes reconstituted in two size populations (~140 and ~435 nm), as revealed by cutting-edge MADLS® analysis. Lastly, in vitro assays demonstrated that composite liposome-nanofiber formulations are superior to liposomal formulations due to a better safety profile in keratinocytes and peripheral blood mononuclear cells. Furthermore, both formulations exhibited similarly advantageous immunomodulatory effects, measured as decreased inflammation in vitro. A synergistic combination of the two nanodelivery systems shows promise for the development of efficient dressings for chronic wound treatment.

Safety of a Novel Listeria monocytogenes-Based Vaccine Vector Expressing NcSAG1 (Neospora caninum Surface Antigen 1).

Listeria monocytogenes (LM) has been proposed as vaccine vector in various cancers and infectious diseases since LM induces a strong immune response. In this study, we developed a novel and safe LM-based vaccine vector platform, by engineering a triple attenuated mutant (Lm3Dx) (ΔactA, ΔinlA, ΔinlB) of the wild-type LM strain JF5203 (CC 1, phylogenetic lineage I). We demonstrated the strong attenuation of Lm3Dx while maintaining its capacity to selectively infect antigen-presenting cells (APCs) in vitro. Furthermore, as proof of concept, we introduced the immunodominant Neospora caninum (Nc) surface antigen NcSAG1 into Lm3Dx. The NcSAG1 protein was expressed by Lm3Dx_SAG1 during cellular infection. To demonstrate safety of Lm3Dx_SAG1 in vivo, we vaccinated BALB/C mice by intramuscular injection. Following vaccination, mice did not suffer any adverse effects and only sporadically shed bacteria at very low levels in the feces (<100 CFU/g). Additionally, bacterial load in internal organs was very low to absent at day 1.5 and 4 following the 1st vaccination and at 2 and 4 weeks after the second boost, independently of the physiological status of the mice. Additionally, vaccination of mice prior and during pregnancy did not interfere with pregnancy outcome. However, Lm3Dx_SAG1 was shed into the milk when inoculated during lactation, although it did not cause any clinical adverse effects in either dams or pups. Also, we have indications that the vector persists more days in the injected muscle of lactating mice. Therefore, impact of physiological status on vector dynamics in the host and mechanisms of milk shedding requires further investigation. In conclusion, we provide strong evidence that Lm3Dx is a safe vaccine vector in non-lactating animals. Additionally, we provide first indications that mice vaccinated with Lm3Dx_SAG1 develop a strong and Th1-biased immune response against the Lm3Dx-expressed neospora antigen. These results encourage to further investigate the efficiency of Lm3Dx_SAG1 to prevent and treat clinical neosporosis.

Synthesis and characterization of modified magnetic nanoparticles as theranostic agents: in vitro safety assessment in healthy cells.

Over the past few decades nanotechnology has paved its way into cancer treatment procedures with the use of nanoparticles (NPs) for contrast media and therapeutic agents. Iron based NPs are the most investigated since they can be used for drug delivery, imaging and when magnetically activate employed as local heat sources in cancer hyperthermia. In this work, was performed synthesis, characterization and biological evaluation of different types of iron oxide nanoparticles (mNPs'), as promising material for tumor hyperthermia. The surface of mNPs' has modified with inorganic stabilizing agents to particularly improve characteristics such as their magnetic properties, colloidal stability and biocompatibility. The successful coating of mNPs' was confirmed by morphological and structural characterization by transmission electron microscopy (TEM) and Fourier-Transform Infra-Red spectroscopy (FT-IR), while their hydrodynamic diameter was studied by using Dynamic light scattering (DLS). X-ray Diffraction (XRD) proved that the crystallite phase of mNPs' is the same with the pattern of magnetite. Superparamagnetic behavior and mNPs' response under the application of alternating magnetic field (AMF) were also thoroughly investigated and showed good heating efficiency in magnetic hyperthermia experiments. The contrast ability in magnetic resonance imaging (MRI) is also discussed indicating that mNPs are negative MRI contrast types. Nonetheless the effects of mNPs on cell viability was performed by MTT on human keratinocytes, human embryonic kidney cells, endothelial cells and by hemolytic assay on erythrocytes. In healthy keratinocytes wound healing assay in different time intervals was performed, assessing both the cell migration and wound closure. Endothelial cells have also been studied in functional activity performing capillary morphogenesis. In vitro studies showed that mNPs are safely taken by the healthy cells and do not interfere with the biological processes such as cell migration and motility.

Screening, verification, and analysis of biomarkers for drug-induced cardiac toxicity in vitro based on RTCA coupled with PCR Array technology.

Cardiotoxicity is one of the most serious side effects of new drugs. Early detection of the drug induced cardiotoxicity based on the biomarkers provides an important preventative strategy for detecting potential cardiotoxicity of candidate drugs. In this study, we aim to identify the predictive genomics biomarkers for drug-induced cardiac toxicity based on the RTCA coupled with PCR Array technology in primary cells. Three prototypical cardiotoxic compounds (doxorubicin, isoproterenol, ouabain) with different mechanisms were firstly real-time monitored to diagnose the cytotoxicity by using the RTCA, while the functional alterations of cardiomyocytes were also monitored by analyzing the beating frequency of cardiomyocytes. Then cardiac specific toxicity gene expression changes were studied by using the technology of PCR Array, which can detect the changes of 84 cardiac functions related genes. Rps6kb1 was identified to be the common cardiac biomarkers by using multivariate statistical and integration analyses. The biomarker was further verified by selecting other drugs with or without cardiotoxicity, and the results showed that the gene exhibited specific changes in cardiac toxicity. Moreover, IPA was applied to combine relevant pathways of Rps6kb1, and identify the main types of cardiac toxicity. These results would further enrich the evaluating strategy of drug-induced cardiotoxicity in vitro, and Rps6kb1 could be used as the specific biomarker of cardiotoxcity during safety assessment of the novel drug candidates.