Implementation of mRNA-Lipid Nanoparticle Technology in Atlantic Salmon ().

Background: This study was conducted to investigate whether mRNA vaccine technology could be adapted for the ectothermic vertebrate Atlantic salmon (). Lipid nanoparticle (LNP) technology has been developed and optimized for mRNA vaccines in mammals, stabilizing mRNA and facilitating its delivery into cells. However, its utility at the temperatures and specific biological environments present in ectotherms remains unclear.

Quality assessment of LNP-RNA therapeutics with orthogonal analytical techniques.

The availability of analytical methods for the characterization of lipid nanoparticles (LNPs) for in-vivo intracellular delivery of nucleic acids is critical for the fast development of innovative RNA therapies. In this study, analytical protocols to measure (i) chemical composition, (ii) drug loading, (iii) particle size, concentration, and stability as well as (iv) structure and morphology were evaluated and compared based on a comprehensive characterization strategy linking key physical and chemical properties to in-vitro efficacy and toxicity. Furthermore, the measurement protocols were assessed either by testing the reproducibility and robustness of the same technique in different laboratories, or by a correlative approach, comparing measurement results of the same attribute with orthogonal techniques.

Polyethylene glycol (PEG) as a broad applicability marker for LC-MS/MS-based biodistribution analysis of nanomedicines.

Polyethylene glycol (PEG) conjugation (PEGylation) is a well-established strategy to improve the pharmacokinetic and biocompatibility properties of a wide variety of nanomedicines and therapeutic peptides and proteins. This broad use makes PEG an attractive 'allround' candidate marker for the biodistribution of such PEGylated compounds. This paper presents the development of a novel strategy for PEG quantification in biological matrices.

Cryo-XPS for Surface Characterization of Nanomedicines.

Nanoparticles used for medical applications commonly possess coatings or surface functionalities intended to provide specific behavior , for example, the use of PEG to provide stealth properties. Direct, quantitative measurement of the surface chemistry and composition of such systems in a hydrated environment has thus far not been demonstrated, yet such measurements are of great importance for the development of nanomedicine systems. Here we demonstrate the first use of cryo-XPS for the measurement of two PEG-functionalized nanomedicines: a polymeric drug delivery system and a lipid nanoparticle mRNA carrier.

A state of the art in analytical quality-by-design and perspectives in characterization of nano-enabled medicinal products.

Quality-by-Design (QbD) guidance is a risk-based and proactive approach to drug development proposed in the early 2000s and now widely used in the pharmaceutical field in compliance with the ICH Q8-Q11 guidelines. Analytical Quality by Design (AQbD), introduced in 2010, is the adaptation of the QbD paradigm for the development of analytical methods. AQbD aims at optimizing the accuracy and robustness of analysis results by identifying and controlling critical analytical variables and method parameters over the entire protocol, including biological sample preparation, measurement technology and statistical analysis.

Physiologically based pharmacokinetic modeling of intravenously administered nanoformulated substances.

The use of nanobiomaterials (NBMs) is becoming increasingly popular in the field of medicine. To improve the understanding on the biodistribution of NBMs, the present study aimed to implement and parametrize a physiologically based pharmacokinetic (PBPK) model. This model was used to describe the biodistribution of two NBMs after intravenous administration in rats, namely, poly(alkyl cyanoacrylate) (PACA) loaded with cabazitaxel (PACA-Cbz), and LipImage™ 815.

A Decision Support System for preclinical assessment of nanomaterials in medical products: the REFINE DSS.

The application of nanomaterials in medicine has led to novel pharmaceuticals and medical devices that have demonstrated a strong potential for increasing the efficacy/performance and safety of therapeutic and diagnostic procedures to address a wide range of diseases. However, the successful translation of these technologies from their inception (proof-of-concept) to clinical practice has been challenged by substantial gaps in the scientific and technical capacity of R&D companies, especially SMEs, to keep up with the ever-evolving regulatory expectations in the emerging area of nanomedicine. To address these challenges, the EU Horizon 2020 project REFINE has developed a Decision Support System (DSS) to support developers of nanotechnology-enabled health products in bringing their products to the clinic.

A comparative biodistribution study of polymeric and lipid-based nanoparticles.

Biodistribution of nanoencapsulated bioactive compounds is primarily determined by the size, shape, chemical composition and surface properties of the encapsulating nanoparticle, and, thus, less dependent on the physicochemical properties of the active pharmaceutical ingredient encapsulated. In the current work, we aimed to investigate the impact of formulation type on biodistribution profile for two clinically relevant nanoformulations. We performed a comparative study of biodistribution in healthy rats at several dose levels and durations up to 14-day post-injection.

Delivery of oligonucleotide-based therapeutics: challenges and opportunities.

Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues.

Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval.

Therapeutic Effect of Cabazitaxel and Blood-Brain Barrier opening in a Patient-Derived Glioblastoma Model.

Treatment of glioblastoma and other diseases in the brain is especially challenging due to the blood-brain barrier, which effectively protects the brain parenchyma. In this study we show for the first time that cabazitaxel, a semi-synthetic derivative of docetaxel can cross the blood-brain barrier and give a significant therapeutic effect in a patient-derived orthotopic model of glioblastoma. We show that the drug crosses the blood-brain barrier more effectively in the tumor than in the healthy brain due to reduced expression of p-glycoprotein efflux pumps in the vasculature of the tumor.

Cabazitaxel-loaded Poly(2-ethylbutyl cyanoacrylate) nanoparticles improve treatment efficacy in a patient derived breast cancer xenograft.

The effect of poly(2-ethyl-butyl cyanoacrylate) nanoparticles containing the cytotoxic drug cabazitaxel was studied in three breast cancer cell lines and one basal-like patient-derived xenograft model grown in the mammary fat pad of immunodeficient mice. Nanoparticle-encapsulated cabazitaxel had a much better efficacy than similar concentrations of free drug in the basal-like patient-derived xenograft and resulted in complete remission of 6 out of 8 tumors, whereas free drug gave complete remission only with 2 out of 9 tumors. To investigate the different efficacies obtained with nanoparticle-encapsulated versus free cabazitaxel, mass spectrometry quantification of cabazitaxel was performed in mice plasma and selected tissue samples.

Analytical ultracentrifugation for analysis of doxorubicin loaded liposomes.

Analytical ultracentrifugation (AUC) is a powerful tool for the study of particle size distributions and interactions with high accuracy and resolution. In this work, we show how the analysis of sedimentation velocity data from the AUC can be used to characterize nanocarrier drug delivery systems used in nanomedicine. Nanocarrier size distribution and the ratio of free versus nanoparticle-encapsulated drug in a commercially available liposomal doxorubicin formulation are determined using interference and absorbance based AUC measurements and compared with results generated with conventional techniques.

Mapping global effects of the anti-sigma factor MucA in Pseudomonas fluorescens SBW25 through genome-scale metabolic modeling.

Background: Alginate is an industrially important polysaccharide, currently produced commercially by harvesting of marine brown sea-weeds. The polymer is also synthesized as an exo-polysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter, and these organisms may represent an alternative alginate source in the future. The current work describes an attempt to rationally develop a biological system tuned for very high levels of alginate production, based on a fundamental understanding of the system through metabolic modeling supported by transcriptomics studies and carefully controlled fermentations.

Initiation of polyene macrolide biosynthesis: interplay between polyketide synthase domains and modules as revealed via domain swapping, mutagenesis, and heterologous complementation.

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT(0) in the nystatin PKS loading module NysA with the propionate-specific AT(1) from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT(0) domain by site-directed mutagenesis were accomplished.

Violacein-producing Collimonas sp. from the sea surface microlayer of costal waters in Trøndelag, Norway.

A new strain belonging to the genus Collimonas was isolated from the sea surface microlayer off the coast of Trøndelag, Norway. The bacterium, designated Collimonas CT, produced an antibacterial compound active against Micrococcus luteus. Subsequent studies using LC-MS identified this antibacterial compound as violacein, known to be produced by several marine-derived bacteria.

Improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Streptomyces noursei.

Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei. In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model.

Analysis of the mycosamine biosynthesis and attachment genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455.

The polyene macrolide antibiotic nystatin produced by Streptomyces noursei contains a deoxyaminosugar mycosamine moiety attached to the C-19 carbon of the macrolactone ring through the beta-glycosidic bond. The nystatin biosynthetic gene cluster contains three genes, nysDI, nysDII, and nysDIII, encoding enzymes with presumed roles in mycosamine biosynthesis and attachment as glycosyltransferase, aminotransferase, and GDP-mannose dehydratase, respectively. In the present study, the functions of these three genes were analyzed.

Probing the structure-function relationship of polyene macrolides: engineered biosynthesis of soluble nystatin analogues.

Although polyene macrolides are efficient antifungal agents with fungicidal mode of action, their use in medical practice is problematic due to their low solubility and significant human toxicity. In an attempt to address the solubility problem, we have obtained two analogues of nystatin with hydroxy groups at positions C31 and C33 through manipulation of the nystatin polyketide synthase in the producing organism Streptomyces noursei. Structures of the analogues were confirmed by nuclear magnetic resonance (NMR), and their solubility was found to be more than 2000 times higher than that of nystatin.

Effect of glucose limitation and specific mutations in the module 5 enoyl reductase domains in the nystatin and amphotericin polyketide synthases on polyene macrolide biosynthesis.

Enoyl reductase (ER) domains in module 5 of nystatin and amphotericin polyketide synthase (PKS) are responsible for reduction of the C28-C29 unsaturated bond on the nascent polyketide chain during biosynthesis of both macrolides, resulting in production of tetraenes nystatin A(1) and amphotericin A, respectively. Data obtained in fermentations under glucose limitation conditions demonstrated that the efficiency of the ER5 domain can be influenced by carbon source availability in the amphotericin producer Streptomyces nodosus, but not in the nystatin producer Streptomyces noursei. Two S.

Nystatin biosynthesis and transport: nysH and nysG genes encoding a putative ABC transporter system in Streptomyces noursei ATCC 11455 are required for efficient conversion of 10-deoxynystatin to nystatin.

The genes nysH and nysG, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the nysH and nysG deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue.

Chemical diversity of polyene macrolides produced by Streptomyces noursei ATCC 11455 and recombinant strain ERD44 with genetically altered polyketide synthase NysC.

The gram-positive bacterium Streptomyces noursei ATCC 11455 produces a complex mixture of polyene macrolides generally termed nystatins. Although the structures for nystatins A(1) and A(3) have been reported, the identities of other components of the nystatin complex remain obscure. Analyses of the culture extract from the S.