Delivery of probiotics and enzymes in self-assemblies of lipids and biopolymers based on colloidal principles

Food is a complex soft matter, because various components, such as proteins, lipids, and carbohydrates, are self-assembled via non-covalent, colloidal interactions and form hierarchical structures at multiple length scales. Soft matter scientists have shown an increasing interest in understanding the general principles governing the food structure formation. During the last several decades, an increasing number of studies have shown that the maintenance of healthy gastrointestinal tract and its microbiome is essential for human health and wellbeing. The realization of the importance of the gastrointestinal microbiome has led to the development of probiotics, which are defined as living bacteria that confer a health benefit on the host. Probiotic bacteria and enzymes can be delivered to the intestinal system by formulating appropriate carriers and including these into food ingested by humans. Despite this simple statement, it involves many challenges in the field of soft matter science. This review aims to highlight how the key concepts in soft matter science can be used to design, characterize, and evaluate self-assembled formulations of probiotics and enzymes based on lipids and biopolymers. The topics covered in this review includes the emulsification of oil-water mixtures, the self-assembly of lipids and polymers at interfaces, the electrostatics and viscoelasticity of interfaces, and the wetting/adhesion of colloidal particles

Encapsulation of sugar beet phytoglobin BvPgb 1.2 and myoglobin in a lipid sponge phase system

Globins are usually associated with oxygen carriage in vertebrates. However, plants also contain similar heme-containing proteins, called phytoglobins (Pgbs). Unlike conventional hemoglobin, these proteins are often linked to nitric oxide metabolism, energy metabolism and redox maintenance under hypoxic and related abiotic and biotic stress conditions. Class I and II non-symbiotic Pgbs (nsPgbs) have different degrees of heme hexacoordination. This involves direct interaction of the distal histidine in the E-helix with the sixth coordination site of the central iron, resulting in increased stability, in contrast to the oxygen storage linked to pentacoordinated globins, such as myoglobin (Mb). Due to their robustness, nsPgbs have substantial potential for various biomedical applications, particularly for iron supplementation. In this study, a class I nsPgb from sugar beet (Beta vulgaris ssp. vulgaris) was encapsulated in a lipid sponge phase system for potential protein delivery purposes and compared to a similar system of Mb containing nanoparticles. Bulk phases and dispersions were made with two lipid compositions (30/45/25 diglycerol monooleate (DGMO)/Capmul GMO-50/sorbitan monooleate (P80) and 28/42/30 DGMO/GMO-50/P80, where the DGMO/GMO-50 ratio was kept constant at 40/60). In addition, buffer effects on protein loading and particle formation were investigated. High concentrations of BvPgb1.2 (60 mg/mL) showed higher aggregation tendencies than Mb but these appeared to be transient. This property could be coupled to the higher isoelectric point (pI) of the BvPgb1.2 (7.85, compared to 6.8 for Mb), which make it more sensitive to small pH changes. In addition, excess protein/leakage was observed with Mb from the nanoparticles when analysed with size exclusion chromatography. This work highlighted the encapsulation efficiency of these proteins, which might be directly linked to difference in iron coordination and therefore, reactivity and lipid peroxidation. The interactions between the bulk phases and dispersion of the hemeproteins are complex, more research is needed to proper elucidate these relations in more detail, in order to facilitate the encapsulation capacity for heme-containing proteins in similar lipid-based systems

A review of the biology of calcium phosphate sequestration with special reference to milk

In milk, a stable fluid is formed in which sequestered nanoclusters of calcium phosphate are substructures in casein micelles. As a result, calcium and phosphate concentrations in milk can be far in excess of their solubility. Variations of calcium, phosphate and casein concentrations in milks, both within and among species, are mainly due to the formation of the nanocluster complexes. Caseins evolved from tooth and bone proteins well before the evolution of lactation. It has therefore been suggested that the role of caseins in milk is an adaptation of an antecedent function in the control of some aspect of biomineralisation. There is new evidence that nanocluster-type complexes are also present in blood serum and, by implication, in many other closely related biofluids. Because such fluids are stable but nevertheless supersaturated with respect to the bone and tooth mineral hydroxyapatite, they allow soft and mineralised tissues to co-exist in the same organism with relative ease. An appreciable concentration of nanocluster complexes exists in fresh saliva. Such saliva may stabilise tooth mineral and help to repair demineralised lesions. In the extracellular matrix of bone, nanocluster complexes may be involved in directing the amorphous calcium phosphate to intrafibrillar spaces in collagen where they can mature into oriented apatite crystals. Thus, evidence is accumulating that calcium phosphate sequestration by phosphopeptides to form equilibrium complexes, first observed in milk, is more generally important in the control of physiological calcification

Structural studies of hydrated samples of amorphous calcium phosphate and phosphoprotein nanoclusters

There are abundant examples of nanoclusters and inorganic microcrystals in biology. Their study under physiologically relevant conditions remains challenging due to their heterogeneity, instability, and the requirements of sample preparation. Advantages of using neutron diffraction and contrast matching to characterize biomaterials are highlighted in this article. We have applied these and complementary techniques to search for nanocrystals within clusters of calcium phosphate sequestered by bovine phosphopeptides, derived from osteopontin or casein. The neutron diffraction patterns show broad features that could be consistent with hexagonal hydroxyapatite crystallites smaller than 18.9 Å. Such nanocrystallites are, however, undetected by the complementary X-ray and FTIR data, collected on the same samples. The absence of a distinct diffraction pattern from the nanoclusters supports the generally accepted amorphous calcium phosphate structure of the mineral core

RNA and DNA interactions with zwitterionic and charged lipid membranes — A DSC and QCM-D study

AbstractThe aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na+) or divalent (Ca2+) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC–DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC–DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA–phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine

Structural insights on ionizable Dlin-MC3-DMA lipids in DOPC layers by combining accurate atomistic force fields, molecular dynamics simulations and neutron reflectivity

Ionizable lipids such as the promising Dlin-MC3-DMA (MC3) are essential for the successful design of lipid nanoparticles (LNPs) as drug delivery agents. Combining molecular dynamics simulations with experimental data, such as neutron reflectivity experiments and other scattering techniques, is essential to provide insights into the internal structure of LNPs, which is not fully understood to date. However, the accuracy of the simulations relies on the choice of force field parameters and high-quality experimental data is indispensable to verify the parametrization. For MC3, different parameterizations in combination with the CHARMM and the Slipids force fields have recently emerged. Here, we complement the existing efforts by providing parameters for cationic and neutral MC3 compatible with the AMBER Lipid17 force field. Subsequently, we carefully assess the accuracy of the different force fields by providing a direct comparison to neutron reflectivity experiments of mixed lipid bilayers consisting of MC3 and DOPC at different pHs. At low pH (cationic MC3) and at high pH (neutral MC3) the newly developed MC3 parameters in combination with AMBER Lipid17 for DOPC give good agreement with the experiments. Overall, the agreement is similar compared to the Park-Im parameters for MC3 in combination with the CHARMM36 force field for DOPC. The Ermilova-Swenson MC3 parameters in combination with the Slipids force field underestimate the bilayer thickness. While the distribution of cationic MC3 is very similar, the different force fields for neutral MC3 reveal distinct differences ranging from strong accumulation in the membrane center (current MC3/AMBER Lipid17 DOPC), over mild accumulation (Park-Im MC3/CHARMM36 DOPC) to surface accumulation (Ermilova-Swenson MC3/Slipids DOPC). These pronounced differences highlight the importance of accurate force field parameters and their experimental validation

Structural biology of calcium phosphate nanoclusters sequestered by phosphoproteins

Biofluids that contain stable calcium phosphate nanoclusters sequestered by phosphopeptides make it possible for soft and hard tissues to co-exist in the same organism with relative ease. The stability diagram of a solution of nanocluster complexes shows how the minimum concentration of phosphopeptide needed for stability increases with pH. In the stable region, amorphous calcium phosphate cannot precipitate. Nevertheless, if the solution is brought into contact with hydroxyapatite, the crystalline phase will grow at the expense of the nanocluster complexes. The physico-chemical principles governing the formation, composition, size, structure, and stability of the complexes are described. Examples are given of complexes formed by casein, osteopontin, and recombinant phosphopeptides. Application of these principles and properties to blood serum, milk, urine, and resting saliva is described to show that under physiological conditions they are in the stable region of their stability diagram and so cannot cause soft tissue calcification. Stimulated saliva, however, is in the metastable region, consistent with its role in tooth remineralization. Destabilization of biofluids, with consequential ill-effects, can occur when there is a failure of homeostasis, such as an increase in pH without a balancing increase in the concentration of sequestering phosphopeptides

Cation and buffer specific effects on the DNA-lipid interaction

Knowledge of DNA - lipid layer interactions is key for the development of biosensors, synthetic nanopores, scaffolds, and gene-delivery systems. These interactions are strongly affected by the ionic composition of the solvent. We have combined quartz crystal microbalance (QCM) and ellipsometry measurements to reveal how pH, buffers and alkali metal chloride salts affect the interaction of DNA with lipid bilayers (DOTAP/DOPC 30:70 in moles). We found that the thickness of the DNA layer adsorbed onto the lipid bilayer decreased in the order citrate > phosphate > Tris > HEPES. The effect of cations on the thickness of the DNA layer decreased in the order (K+ > Na+ > Cs+ ∼ Li+). Rationalization of the experimental results requires that adsorption, due to cation specific charge screening, is driven by the simultaneous action of two mechanisms namely, the law of matching water affinities for kosmotropes (Li+) and ion dispersion forces for chaotropes (Cs+). The outcome of these two opposing mechanisms is a “bell-shaped” specific cations sequence. Moreover, a superimposed buffer specificity, which goes beyond the simple effect of pH regulation, further modulated cation specificity. In summary, DNA-lipid bilayer interactions are maximized if citrate buffer (50 mM, pH 7.4) and KCl (100 mM) are used