Characterisation of locust bean gum with asymmetric flow field-flow fractionation (AF4) and light scattering.

We present a detailed characterisation of locust bean gum (LBG), an industrially significant galactomannan, utilising asymmetric flow field-flow fractionation (AF4) and light scattering. Molecular weight and size determination of galactomannans is complicated by their tendency to aggregate, even in dilute solutions; AF4 allows us to confirm the presence of aggregates, separate these from well-dispersed polymer, and characterise both fractions. For the dispersed polymer, we find Mw=9.2×105 g mol-1 and Rg,z=82.1 nm; the distribution follows Flory scaling (Rg∼Mν) with ν∼ 0.63, indicating good solvent conditions. The aggregate fraction exhibited radii of up to 1000 nm and masses of up to 3×1010 g mol-1. Furthermore, we demonstrate how both fractions are influenced by changes to filtration procedure and solvent conditions. Notably, a 200 nm nylon membrane effectively removes the aggregated fraction; we present a concentration-dependent investigation of solutions following this protocol, using static and dynamic light scattering, which reveals additional weak aggregation in these unfractionated samples. Overall, we demonstrate that AF4 is highly suited to LBG characterisation, providing structural information for both well-dispersed and aggregated fractions, and expect the methods employed to apply similarly to other galactomannans and associating polymer systems.

Derivatized chitosan-oil-in-water nanocapsules for trans-cinnamaldehyde delivery.

trans-Cinnamaldehyde, known for its bacterial anti-quorum sensing activity when applied at sublethal concentrations, has gained traction given its potential use against multidrug resistant bacteria. In this work, trans-cinnamaldehyde-loaded oil-in-water nanocapsules coated with chitosan, N,N,N-trimethyl chitosan chloride, N-(2-(N,N,N-trimethylammoniumyl)acetyl) chitosan chloride or N-(6-(N,N,N-trimethylammoniumyl)hexanoyl)chitosan chloride were obtained. All the formulated nanocapsules showed a Z-average hydrodynamic diameter ~ 160 nm and ζ-potential higher than +40 mV. N,N,N-trimethyl chitosan-coated oil-in-water nanocapsules showed the greatest trans-cinnamaldehyde association efficiency (99.3 ± 7.6) % and total payload release (88.6 ± 22.5) %, while N-(6-(N,N,N-trimethylammoniumyl)hexanoyl)chitosan chloride chitosan-coated oil-in-water nanocapsules were the only formulations stable in phosphate buffer saline PBS (pH 7.4) upon incubation at 37 °C for 24 h. Future work should address the stability of the developed nanocapsules in culture media and their biological performance.

Aptamer-Target-Gold Nanoparticle Conjugates for the Quantification of Fumonisin B1.

Fumonisin B1 (FB1), a mycotoxin classified as group 2B hazard, is of high importance due to its abundance and occurrence in varied crops. Conventional methods for detection are sensitive and selective; however, they also convey disadvantages such as long assay times, expensive equipment and instrumentation, complex procedures, sample pretreatment and unfeasibility for on-site analysis. Therefore, there is a need for quick, simple and affordable quantification methods. On that note, aptamers (ssDNA) are a good alternative for designing specific and sensitive biosensing techniques. In this work, the assessment of the performance of two aptamers (40 and 96 nt) on the colorimetric quantification of FB1 was determined by conducting an aptamer-target incubation step, followed by the addition of gold nanoparticles (AuNPs) and NaCl. Although MgCl2 and Tris-HCl were, respectively, essential for aptamer 96 and 40 nt, the latter was not specific for FB1. Alternatively, the formation of Aptamer (96 nt)-FB1-AuNP conjugates in MgCl2 exhibited stabilization to NaCl-induced aggregation at increasing FB1 concentrations. The application of asymmetric flow field-flow fractionation (AF4) allowed their size separation and characterization by a multidetection system (UV-VIS, MALS and DLS online), with a reduction in the limit of detection from 0.002 µg/mL to 56 fg/mL.

Capsaicin-Loaded Chitosan Nanocapsules for wtCFTR-mRNA Delivery to a Cystic Fibrosis Cell Line.

Cystic fibrosis (CF), a lethal hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene coding for an epithelial chloride channel, is characterized by an imbalanced homeostasis of ion and water transports in secretory epithelia. As the disease is single-gene based, transcript therapy using therapeutic mRNA is a promising concept of treatment in order to correct many aspects of the fatal pathology on a cellular level. Hence, we developed chitosan nanocapsules surface-loaded with wtCFTR-mRNA to restore CFTR function. Furthermore, we loaded the nanocapsules with capsaicin, aiming to enhance the overall efficiency of transcript therapy by reducing sodium hyperabsorption by the epithelial sodium channel (ENaC). Dynamic light scattering with non-invasive back scattering (DLS-NIBS) revealed nanocapsules with an average hydrodynamic diameter of ~200 nm and a Zeta potential of ~+60 mV. The results of DLS-NIBS measurements were confirmed by asymmetric flow field-flow fractionation (AF4) with multidetection, while transmission electron microscopy (TEM) images confirmed the spherical morphology and size range. After stability measurements showed that the nanocapsules were highly stable in cell culture transfection medium, and cytotoxicity was ruled out, transfection experiments were performed with the CF cell line CFBE41o-. Finally, transepithelial measurements with a new state-of-the-art Ussing chamber confirmed successfully restored CFTR function in transfected cells. This study demonstrates that CS nanocapsules as a natural and non-toxic delivery system for mRNA to target cells could effectively replace risky vectors for gene delivery. The nanocapsules are not only suitable as a transcript therapy for treatment of CF, but open aspiring possibilities for safe gene delivery in general.

Characterisation of the Interaction among Oil-In-Water Nanocapsules and Mucin.

Mucins are glycoproteins present in all mucosal surfaces and in secretions such as saliva. Mucins are involved in the mucoadhesion of nanodevices carrying bioactive molecules to their target sites in vivo. Oil-in-water nanocapsules (NCs) have been synthesised for carrying N,N'-(di-m-methylphenyl)urea (DMTU), a quorum-sensing inhibitor, to the oral cavity. DMTU-loaded NCs constitute an alternative for the treatment of plaque (bacterial biofilm). In this work, the stability of the NCs after their interaction with mucin is analysed. Mucin type III from Sigma-Aldrich has been used as the mucin model. Mucin and NCs were characterised by the multi-detection asymmetrical flow field-flow fractionation technique (AF4). Dynamic light scattering (DLS) and ζ-potential analyses were carried out to characterise the interaction between mucin and NCs. According to the results, loading DMTU changes the conformation of the NC. It was also found that the synergistic interaction between mucin and NCs was favoured within a specific range of the mucin:NC ratio within the first 24 h. Studies on the release of DMTU in vitro and the microbial activity of such NCs are ongoing in our lab.

Interaction Between Chitosan and Mucin: Fundamentals and Applications.

The term chitosan (CS) refers to a family of aminopolysaccharides derived from chitin. Among other properties, CS is nontoxic, mucoadhesive and can be used for load and transport drugs. Given these and other physicochemical and biological properties, CS is an optimal biopolymer for the development of transmucosal drug delivery systems, as well as for the treatment of pathologies related to mucosal dysfunctions. Mucins are glycoprotein macromolecules that are the major components of mucus overlaying epithelia. CS interacts with mucin and adsorbs on and changes the rheology of mucus. However, CS and mucins denote families of polymers/macromolecules with highly variable chemical structure, properties, and behavior. To date, their interactions at the molecular level have not been completely unraveled. Also, the properties of complexes composed of CS and mucin vary as a function of the sources and preparation of the polymers. As a consequence, the mucoadhesion and drug delivery properties of such complexes vary as well. The breadth of this review is on the molecular interactions between CS and mucin. In particular, in vitro and ex vivo characterization methods to investigate both the interactions at play during the formation of CS-mucin complexes, and the advances on the use of CS for transmucosal drug delivery are addressed.