Polyphosphoester-stabilized cubosomes encapsulating a Ru(II) complex for the photodynamic treatment of lung adenocarcinoma.

The clinical translation of photosensitizers based on ruthenium(II) polypyridyl complexes (RPCs) in photodynamic therapy of cancer faces several challenges. To address these limitations, we conducted an investigation to assess the potential of a cubosome formulation stabilized in water against coalescence utilizing a polyphosphoester analog of Pluronic F127 as a stabilizer and loaded with newly synthesized RPC-based photosensitizer [Ru(dppn)2(bpy-morph)](PF6)2 (bpy-morph = 2,2'-bipyridine-4,4'-diylbis(morpholinomethanone)), PS-Ru. The photophysical characterization of PS-Ru revealed its robust capacity to induce the formation of singlet oxygen (1O2). Furthermore, the physicochemical analysis of the PS-Ru-loaded cubosomes dispersion demonstrated that the encapsulation of the photosensitizer within the nanoparticles did not disrupt the three-dimensional arrangement of the lipid bilayer. The biological tests showed that PS-Ru-loaded cubosomes exhibited significant phototoxic activity when exposed to the light source, in stark contrast to empty cubosomes and to the same formulation without irradiation. This promising outcome suggests the potential of the formulation in overcoming the drawbacks associated with the clinical use of RPCs in photodynamic therapy for anticancer treatments.

On the interactions between RNA and titrateable lipid layers: implications for RNA delivery with lipid nanoparticles.

Characterising the interaction between cationic ionisable lipids (CIL) and nucleic acids (NAs) is key to understanding the process of RNA lipid nanoparticle (LNP) formation and release of NAs from LNPs. Here, we have used different surface techniques to reveal the effect of pH and NA type on the interaction with a model system of DOPC and the CIL DLin-MC3-DMA (MC3). At only 5% MC3, differences in the structure and dynamics of the lipid layer were observed. Both pH and %MC3 were shown to affect the absorption behaviour of erythropoietin mRNA, polyadenylic acid (polyA) and polyuridylic acid (polyU). The adsorbed amount of all studied NAs was found to increase with decreasing pH and increasing %MC3 but with different effects on the lipid layer, which could be linked to the NA secondary structure. For polyA at pH 6, adsorption to the surface of the layer was observed, whereas for other conditions and NAs, penetration of the NA into the layer resulted in the formation of a multilayer structure. By comparison to simulations excluding the secondary structure, differences in adsorption behaviours between polyA and polyU could be observed, indicating that the NA's secondary structure also affected the MC3-NA interactions.

The density of anionic lipids modulates the adsorption of α-Synuclein onto lipid membranes.

α-Synuclein is an intrinsically disordered presynaptic protein associated with Parkinson's disease. The physiological role of α-Synuclein is not fully understood, but the protein is known to interact with lipid membranes. We here study how membrane charge affects the adsorption of α-Synuclein to (i) supported lipid bilayers and (ii) small unilamellar vesicles with varying amounts of anionic lipids. The results showed that α-Synuclein adsorbs onto membranes containing ≥5% anionic phosphatidylserine (DOPS) lipids, but not to membranes containing ≤1% DOPS. The density of adsorbed α-Synuclein increased steadily with the DOPS content up to 20% DOPS, after which it leveled off. The vesicles were saturated with α-Synuclein at a 3-5 times higher protein density compared to the supported bilayers, which suggests that a more deformable membrane binds more α-Synuclein. Altogether, the results show that both membrane charge density and flexibility influence the association of α-Synuclein to lipid membranes.

Single-vesicle intensity and colocalization fluorescence microscopy to study lipid vesicle fusion, fission, and lipid exchange.

Interactions of lipid vesicles play important roles in a large variety of functions and dysfunctions in the human body. Vital for several biochemical functions is the interaction between monomeric proteins and lipid membranes, and the induced phenomena such as fusion between vesicles and cell membranes, lipid exchange between the membranes, or vesicle fission. Identification of single events and their frequency of occurrence would provide valuable information about protein-lipid interactions in both healthy and degenerative pathways. In this work, we present a single-vesicle intensity and colocalization fluorescence microscopy assay with a custom-written MATLAB analysis program. The assay can be used to study lipid exchange as well as vesicle fusion and fission between two vesicle populations labeled with different fluorescent dyes. Vesicles from the two populations are first mixed and docked to a glass surface. The sample is then simultaneously imaged using two separate wavelength channels monitoring intensity changes and colocalization of vesicles from the two populations. The monomeric pre-synaptic protein α-synuclein (α-syn) and small unilamellar vesicles consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DOPS), and monosialotetrahexosylganglioside (GM1) were used as a model system to evaluate the method. From our analysis, neither α-syn induced fusion nor lipid exchange was observed for vesicles consisting of DOPC:DOPS (7:3). However, including 10% GM1 in the vesicles resulted in a 91% increase of the number of vesicles within 10 min, combined with a 57% decrease in the average fluorescence intensity per vesicle, indicating that approximately half of the vesicles underwent fission. The method facilitates the study of lipid vesicle fusion, fission, and lipid exchange under controlled conditions. It also allows these events to be studied for systems with more complex composition including exosomes and lipid-based drug carriers, to enable a better understanding of their physicochemical properties.

Hybrid Theranostic Cubosomes for Efficient NIR-Induced Photodynamic Therapy.

In recent years, lipid bicontinuous cubic liquid-crystalline nanoparticles known as cubosomes have been under investigation because of their favorable properties as drug nanocarriers useful for anticancer treatments. Herein, we present organic/inorganic hybrid, theranostic cubosomes stabilized in water with a shell of alternate layers of chitosan, single strand DNA (model genetic material for potential gene therapy), and folic acid-chitosan conjugate (the outmost layer), coencapsulating up-converting Er3+ and Yb3+ codoped NaYF4 nanoparticles and daunorubicin. The latter acts as a chemotherapeutic drug of photosensitizing activity, while up-converting nanoparticles serve as energy harvester and diagnostic agent. Cellular uptake and NIR-induced photodynamic therapy were evaluated in vitro against human skin melanoma (MeWo) and ovarian (SKOV-3) cancer cells. Results evidenced the preferential uptake of the theranostic cubosomes in SKOV-3 cells in comparison to uptake in MeWo cells, and this effect was enhanced by the folic acid functionalization of the cubosomes surface. Nanocarriers coloaded with the hybrid fluorophores exhibited a superior NIR-induced photodynamic activity, also confirmed by the improved mitochondrial activity and the most affecting f-actin fibers of cytoskeleton. Similar results, but with higher photocytotoxicity, were detected when folic acid-functionalized cubosomes were incubated with SKOV-3 cells. Taken on the whole, these results prove these hybrid cubosomes are good candidates for the photodynamic treatment of tumor lesions.

In Vitro Evaluation of Nanoerythrosome Cytotoxicity and Uptake in Pancreatic Endothelial Cells: Implications for ß-Cell Imaging Applications.

Biomolecule-targeted imaging represents one of the most difficult challenges in medicine. Nanoerythrosomes (NERs) are nanovesicles obtained after lysis of red blood cells, and they are promising tools for drug delivery and imaging. In this work, a formulation based on NERs functionalized with 7-amino-3-methylcoumarin via cross-linking was tested on rat INS-1E and mouse MIN6 ß-cells and endothelial MSI cell lines. First, the morphology, size, ζ-potentials, and spectroscopic properties of the aggregates were investigated, highlighting that the functionalization did not significantly affect the nanoparticles' physicochemical features. In vitro, the nanoparticles did not significantly affect the proliferation and function of INS-1E and MIN6 ß-cells at different concentrations. Only at the highest concentration tested on the MSI cell line, the formulation inhibited proliferation. Furthermore, NER aggregates were not internalized in both INS-1E and MIN6 cell lines, while a diffuse fluorescence was noticed in the cytosol of the MSI cell line at the highest concentrations. These findings proved that NER formulations might represent a new nanotool for ß-cell imaging as a part of a strategy aimed to prevent any intracellular accumulation, thus reducing/avoiding side effects.

Lipid vesicular gels for topical administration of antioxidants.

The application of a formulation on the skin represents an effective way to deliver bio-active molecules for therapeutical purposes. Moreover, the outermost skin layer, the stratum corneum, can be overcome by employing chemical permeation enhancers and edge activators as components. Several lipids can be considered as permeation enhancers, such as the ubiquitous monoolein, one of the most used building blocks for the preparation of lipid liquid crystalline nanoparticles which are applied as drug carriers for nanomedicine applications. Recent papers highlighted how bile salts can affect the phase behavior of monoolein to obtain drug carriers suitable for topical administration, given their role as edge activators into the formulation. Herein, the encapsulation of natural antioxidants (caffeic acid and ferulic acid) into lipid vesicular gels (LVGs) made by monoolein and sodium taurocholate (TC) in water was studied to produce formulations suitable for topical application. TC induces a bicontinuous cubic to multilamellar phase transition for monoolein in water at the given concentrations, and by increasing its content into the formulations, unilamellar LVGs are formed. The encapsulation of the two antioxidants did not affect significantly the structure of the gels. The oscillating rheological studies showed that ferulic acid has a structuring effect on the lipid matrix, in comparison with the empty dispersion and the one containing caffeic acid. These gels were then tested in vitro on new-born pig skin to evaluate their efficacy as drug carriers for topical administration, showing that caffeic acid is mostly retained in the gel whereas ferulic acid is released at a higher degree. The data herein reported provide some further information on the effect of bile salts on the lipid self-assembly to evaluate useful compositions for topical administration of natural antioxidants.

Cooperativity of α-Synuclein Binding to Lipid Membranes.

Cooperative binding is a key feature of metabolic pathways, signaling, and transport processes. It provides tight regulation over a narrow concentration interval of a ligand, thus enabling switching to be triggered by small concentration variations. The data presented in this work reveal strong positive cooperativity of α-synuclein binding to phospholipid membranes. Fluorescence cross-correlation spectroscopy, confocal microscopy, and cryo-TEM results show that in excess of vesicles α-synuclein does not distribute randomly but binds only to a fraction of all available vesicles. Furthermore, α-synuclein binding to a supported lipid bilayer observed with total internal reflection fluorescence microscopy displays a much steeper dependence of bound protein on total protein concentration than expected for independent binding. The same phenomenon was observed in the case of α-synuclein binding to unilamellar vesicles of sizes in the nm and µm range as well as to flat supported lipid bilayers, ruling out that nonuniform binding of the protein is governed by differences in membrane curvature. Positive cooperativity of α-synuclein binding to lipid membranes means that the affinity of the protein to a membrane is higher where there is already protein bound compared to a bare membrane. The phenomenon described in this work may have implications for α-synuclein function in synaptic transmission and other membrane remodeling events.

Bicontinuous cubic liquid crystalline phase nanoparticles stabilized by softwood hemicellulose.

The colloidal stability of lipid based cubosomes, aqueous dispersion of inverse bicontinuous cubic phase, can be significantly increased by a stabilizer. The most commonly used stabilizers are non-ionic tri-block copolymers, poloxamers, which adsorb at the lipid-water interface and hence sterically stabilize the dispersion. One of the challenges with these synthetic polymers is the effect on the internal structure of the cubosomes and the potential toxicity when these nanoparticles are applied as nanomedicine platforms. The natural polysaccharide, softwood hemicellulose, has been proved to be an excellent stabilizer for oil-in-water emulsions, partially due to the presence of hydrophobic lignin in the extract which to some extent is associated to hemicellulose. Herein, we reported for the first time cubosomes stabilized by two types of softwood hemicelluloses, where one is extracted through thermomechanical pulping (TMP, low lignin content) and the other obtained from sodium-based sulfite liquor (SSL, high lignin content). The effect of the two hemicellulose samples on the colloidal stability and structure of monoolein-based cubosomes have been investigated via DLS, SAXS, AFM and cryo-TEM. The data obtained suggest that both types of the hemicelluloses stabilize monoolein (GMO) based cubosomes in water without significantly affecting their size, morphology and inner structure. SSL-extracted hemicellulose yields the most stable cubosomes, likely due to the higher content of lignin in comparison to TMP-stabilized ones. In addition, the stability of these particles was tested under physiological conditions relevant to possible application as drug carriers.

Bioimaging Applications of Non-Lamellar Liquid Crystalline Nanoparticles.

Self-assembling processes of amphiphilic lipids in water give rise to complex architectures known as lyotropic liquid crystalline (LLC) phases. Particularly, bicontinuous cubic and hexagonal LLC phases can be dispersed in water forming colloidal nanoparticles respectively known as cubosomes and hexosomes. These non-lamellar LLC dispersions are of particular interest for pharmaceutical and biomedical applications as they are potentially non-toxic, chemically stable, and biocompatible, also allowing encapsulation of large amounts of drugs. Furthermore, conjugation of specific moieties enables their targeting, increasing therapeutic efficacies and reducing side effects by avoiding exposure of healthy tissues. In addition, as they can be easy loaded or functionalized with both hydrophobic and hydrophilic imaging probes, cubosomes and hexosomes can be used for the engineering of multifunctional/theranostic nanoplatforms. This review outlines recent advances in the applications of cubosomes and hexosomes for in vitro and in vivo bioimaging.

Tuning lipid structure by bile salts: Hexosomes for topical administration of catechin.

The delivery of bio-active molecules through the skin is challenging given the complex structure of its outer layer, the stratum corneum. Here we explore the possibility to encapsulate natural compounds into nanocarriers containing permeation enhancers that can affect the fluidity of the stratum corneum lipids. This approach is expected to facilitate dermal or transdermal release. For this purpose, the application of bile salts, which are natural surfactants involved in vivo in lipid digestion, was exploited. Bile salts were added to lipid liquid crystalline nanoparticles (NPs) made of monoolein for antioxidant topical delivery. Monoolein self-assembly behaviour in water was affected by the presence of bile salts molecules, giving a transition from a bicontinuous cubic to unilamellar vesicles dispersion. By adding oleic acid (OA), the change of curvature in the system led to a reverse hexagonal phase. The morphology, structure and size of the nanocarriers was investigated before the nanoparticles were loaded with catechin, a natural antioxidant occurring in plants and food. The encapsulation did not affect significantly the formulation phase behaviour. The formulation loaded with bile salts and catechin was thereafter tested in vitro on the skin from new-born pig. The results for two different lipid formulations without bile salts were compared under the same experimental conditions and with the same antioxidant. The formulation with bile salts showed the best performance, allowing a superior permeation of catechin in the different skin layers in comparison with formulations without bile salt.

Surface-modified nanoerythrosomes for potential optical imaging diagnostics.

Nanoerythrosomes (NERs), vesicle-like nanoparticles derived from red blood cells, represent a new and interesting vector for therapeutic molecules and imaging probes, mainly thanks to their high stability and excellent biocompatibility. Aiming to present a proof-of-concept of the use of NERs as diagnostic tools for in vitro/in vivo imaging purposes, we report here several functionalization routes to decorate the surfaces of NERs derived from bovine blood with two different fluorophores: 7-amino-4-methylcumarin and dibenzocyclooctinecyanine5.5. Notably, the fluorophores were cross-linked to the NERs surface with glutaraldehyde and, in the case of dibenzocyclooctinecyanine5.5, also using a click-chemistry route, termed strain-promoted azide-alkyne cycloaddition. The physicochemical characterization highlighted the high stability of the NERs derivatives in physiological conditions. Furthermore, the loading efficiency of the fluorophores on the NERs surface was evaluated using both UV-Vis spectroscopy and fluorescence microscopy.

Cubosomes stabilized by a polyphosphoester-analog of Pluronic F127 with reduced cytotoxicity.

Lyotropic liquid crystalline nanoparticles with bicontinuous cubic internal nanostructure, known as cubosomes, have been proposed as nanocarriers in various medical applications. However, as these nanoparticles show a certain degree of cytotoxicity, particularly against erythrocytes, their application in systemic administrations is limited to date. Intending to produce a more biocompatible formulation, we prepared cubosomes for the first time stabilized with a biodegradable polyphosphoester-analog of the commonly used Pluronic F127. The ABA-triblock copolymer poly(methyl ethylene phosphate)-block-poly(propylene oxide)-block-poly(methyl ethylene phosphate) (PMEP-b-PPO-b-PMEP) was prepared by organocatalyzed ring-opening polymerization of MEP. The cytotoxic features of the resulting formulation were investigated against two different cell lines (HEK-293 and HUVEC) and human red blood cells. The response of the complement system was also evaluated. Results proved the poly(phosphoester)-based formulation was significantly less toxic than that prepared using Pluronic F127 with respect to all the tested cell lines and, more importantly, hemolysis assay and complement system activation tests demonstrated its very high hemocompatibility. The potentially biodegradable poly(phosphoester)-based cubosomes represent a new and versatile platform for preparation of functional and smart nanocarriers.

Multifunctional cubic liquid crystalline nanoparticles for chemo- and photodynamic synergistic cancer therapy.

With the aim of engineering multifunctional nanoparticles useful for cancer therapy, a diketopyrrolopyrrole-porphyrin based photosensitizer was here conjugated to a block copolymer (Pluronic F108), and used to stabilize in water lipidic cubic liquid crystalline nanoparticles (cubosomes), also loaded with the antineoplastic agent docetaxel. The physicochemical characterization by SAXS, DLS, and cryo-TEM demonstrated that the formulation consisted of cubosomes, about 150 nm in size, possessing a bicontinuous cubic structure (space group Pn3m). The cellular imaging experiments proved that these nanoparticles localized in lysosomes and mitochondria, while cytotoxicity tests evidenced a slight but significant synergistic effect which, after irradiation, increased the toxicity induced by docetaxel alone, allowing further reduction of cell viability.

Branched alkyldimethylamine oxide surfactants: An effective strategy for the design of high concentration/low viscosity surfactant formulations.

HYPOTHESIS: The rational design of branched-tail surfactants is a suitable strategy to obtain low-viscosity surfactant-rich isotropic aqueous mixtures with negligible effects on biodegradability. This opens a way to the design of concentrated ("water-free") surfactant formulations, highly attractive for their ecological and economic benefits. EXPERIMENTS: The aggregation behaviour of N,N-dimethyl-2-propylheptan-1-amine oxide (C10DAO-branched) in aqueous mixtures is investigated across the entire composition range by polarized optical microscopy, small angle X-ray and neutron scattering, electron paramagnetic resonance, and pulse-gradient stimulated echo nuclear magnetic resonance. The humidity scanning quartz crystal microbalance with dissipation monitoring technique is validated as a tool for the fast screening of surfactants phase behaviour. Furthermore, the shear viscosities and viscoelastic moduli of the systems are determined by rheological measurements. FINDINGS: With respect to the linear isomer, C10DAO-branched presents a much lower tendency to form lyotropic liquid crystalline phases. Except for a narrow composition and temperature range in which a lamellar structure is observed, C10DAO-branched aqueous mixtures are isotropic liquids whose microstructure changes, with increasing concentration, from micellar solutions to unstructured dispersions of hydrated surfactant molecules. Low-viscosity was found for all these mixtures, including the most concentrated ones. Thus, the introduction of a single short side-chain in the tail is demonstrated to be an effective approach to increase the active concentration in surfactant formulations.

3-hydroxycoumarin loaded vesicles for recombinant human tyrosinase inhibition in topical applications.

Tyrosinase is one of the key enzymes in mammalian melanin biosynthesis. Decreasing tyrosinase activity has been targeted for the prevention of conditions related to the hyperpigmentation of the skin, such as melasma and age spots. This paper is devoted to the engineering of vesicle formulations loaded with 3-hydroxycoumarin for topical pharmaceutical applications. At first, it was demonstrated the strong inhibiting ability of 3-hydroxycoumarin against recombinant human tyrosinase. Then, such a drug was effectively encapsulated within liquid or gel-like vesicle formulations, both based on monoolein and lauroylcholine chloride. In vitro skin penetration and permeation studies proved these formulations efficiently overcome the barrier represented by the stratum corneum, delivering 3-hydroxycoumarin to the deeper skin layers. The effect of applying for different times the liquid and the gel formulation was also evaluated. Results revealed that application of the gel formulation for 2 h favored the drug accumulation into the skin with low transdermal delivery, thus indicating this combination of administration time and formulation as ideal to locally inhibit tyrosinase activity with minimal systemic absorption. Moreover, when incubated with B16F10 melanoma cells, the liquid vesicle formulations did not show cytotoxic activity.