Liposomal formulations for treating lysosomal storage disorders.

Lysosomal storage disorders (LSD) are a group of rare life-threatening diseases caused by a lysosomal dysfunction, usually due to the lack of a single enzyme required for the metabolism of macromolecules, which leads to a lysosomal accumulation of specific substrates, resulting in severe disease manifestations and early death. There is currently no definitive cure for LSD, and despite the approval of certain therapies, their effectiveness is limited. Therefore, an appropriate nanocarrier could help improve the efficacy of some of these therapies. Liposomes show excellent properties as drug carriers, because they can entrap active therapeutic compounds offering protection, biocompatibility, and selectivity. Here, we discuss the potential of liposomes for LSD treatment and conduct a detailed analysis of promising liposomal formulations still in the preclinical development stage from various perspectives, including treatment strategy, manufacturing, characterization, and future directions for implementing liposomal formulations for LSD.

DELOS Nanovesicles-Based Hydrogels: An Advanced Formulation for Topical Use.

Topical delivery has received great attention due to its localized drug delivery, its patient compliance, and its low risk for side effects. Recent developments have focused on studying new drug delivery systems as a strategy for addressing the challenges of current topical treatments. Here we describe the advances on an innovative drug delivery platform called DELOS nanovesicles for topical drug delivery. Previously, the production of DELOS nanovesicles demonstrated potentiality for the topical treatment of complex wounds, achieving well-tolerated liquid dispersions by this route. Here, research efforts have been focused on designing these nanocarriers with the best skin tolerability to be applied even to damaged skin, and on exploring the feasibility of adapting the colloidal dispersions to a more suitable dosage form for topical application. Accordingly, these drug delivery systems have been efficiently evolved to a hydrogel using MethocelTM K4M, presenting proper stability and rheological properties. Further, the integrity of these nanocarriers when being gellified has been confirmed by cryo-transmission electron microscopy and by Förster resonance energy transfer analysis with fluorescent-labeled DELOS nanovesicles, which is a crucial characterization not widely reported in the literature. Additionally, in vitro experiments have shown that recombinant human Epidermal Growth Factor (rhEGF) protein integrated into gellified DELOS nanovesicles exhibits an enhanced bioactivity compared to the liquid form. Therefore, these studies suggest that such a drug delivery system is maintained unaltered when hydrogellified, becoming the DELOS nanovesicles-based hydrogels, an advanced formulation for topical use.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Application of Quality by Design to the robust preparation of a liposomal GLA formulation by DELOS-susp method.

Fabry disease is a lysosomal storage disease arising from a deficiency of the enzyme α-galactosidase A (GLA). The enzyme deficiency results in an accumulation of glycolipids, which over time, leads to cardiovascular, cerebrovascular, and renal disease, ultimately leading to death in the fourth or fifth decade of life. Currently, lysosomal storage disorders are treated by enzyme replacement therapy (ERT) through the direct administration of the missing enzyme to the patients. In view of their advantages as drug delivery systems, liposomes are increasingly being researched and utilized in the pharmaceutical, food and cosmetic industries, but one of the main barriers to market is their scalability. Depressurization of an Expanded Liquid Organic Solution into aqueous solution (DELOS-susp) is a compressed fluid-based method that allows the reproducible and scalable production of nanovesicular systems with remarkable physicochemical characteristics, in terms of homogeneity, morphology, and particle size. The objective of this work was to optimize and reach a suitable formulation for in vivo preclinical studies by implementing a Quality by Design (QbD) approach, a methodology recommended by the FDA and the EMA to develop robust drug manufacturing and control methods, to the preparation of α-galactosidase-loaded nanoliposomes (nanoGLA) for the treatment of Fabry disease. Through a risk analysis and a Design of Experiments (DoE), we obtained the Design Space in which GLA concentration and lipid concentration were found as critical parameters for achieving a stable nanoformulation. This Design Space allowed the optimization of the process to produce a nanoformulation suitable for in vivo preclinical testing.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

Highly Stable and Red-Emitting Nanovesicles Incorporating Lipophilic Diketopyrrolopyrroles for Cell Imaging.

Diketopyrrolopyrroles (DPPs) have recently attracted much interest as very bright and photostable red-emitting molecules. However, their tendency to form nonfluorescent aggregates in water through the aggregation-caused quenching (ACQ) effect is a major issue that limits their application under the microscope. Herein, two DPP molecules have been incorporated into the membrane of highly stable and water-soluble quatsomes (QS; nanovesicles composed of surfactants and sterols), which allow their nanostructuration in water and, at the same time, limits the ACQ effect. The obtained fluorescent organic nanoparticles showed superior structural homogeneity, along with long-term colloidal and optical stability. A thorough one- (1P) and two-photon (2P) fluorescence characterization revealed the promising photophysical features of these fluorescent nanovesicles, which showed a high 1P and 2P brightness. Finally, the fluorescent QSs were used for the in vitro bioimaging of Saos-2 osteosarcoma cell lines; this demonstrates their potential as nanomaterials for bioimaging applications.

Pressure-Responsive, Surfactant-Free CO2-Based Nanostructured Fluids.

Microemulsions are extensively used in advanced material and chemical processing. However, considerable amounts of surfactant are needed for their formulation, which is a drawback due to both economic and ecological reasons. Here, we describe the nanostructuration of recently discovered surfactant-free, carbon dioxide (CO2)-based microemulsion-like systems in a water/organic-solvent/CO2 pressurized ternary mixture. "Water-rich" nanodomains embedded into a "water-depleted" matrix have been observed and characterized by the combination of Raman spectroscopy, molecular dynamics simulations, and small-angle neutron scattering. These single-phase fluids show a reversible, pressure-responsive nanostructuration; the "water-rich" nanodomains at a given pressure can be instantaneously degraded/expanded by increasing/decreasing the pressure, resulting in a reversible, rapid, and homogeneous mixing/demixing of their content. This pressure-triggered responsiveness, together with other inherent features of these fluids, such as the absence of any contaminant in the ternary mixture (e.g., surfactant), their spontaneous formation, and their solvation capability (enabling the dissolution of both hydrophobic and hydrophilic molecules), make them appealing complex fluid systems to be used in molecular material processing and in chemical engineering.

α-Galactosidase-A Loaded-Nanoliposomes with Enhanced Enzymatic Activity and Intracellular Penetration.

Lysosomal storage disorders (LSD) are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of macromolecules, such as lipids, glycoproteins, and mucopolysaccharides. For instance, the lack of α-galactosidase A (GLA) activity in Fabry disease patients causes the accumulation of glycosphingolipids in the vasculature leading to multiple organ pathology. Enzyme replacement therapy, which is the most common treatment of LSD, exhibits several drawbacks mainly related to the instability and low efficacy of the exogenously administered therapeutic enzyme. In this work, the unprecedented increased enzymatic activity and intracellular penetration achieved by the association of a human recombinant GLA to nanoliposomes functionalized with Arginine-Glycine-Aspartic acid (RGD) peptides is reported. Moreover, these new GLA loaded nanoliposomes lead to a higher efficacy in the reduction of the GLA substrate named globotriasylceramide in a cellular model of Fabry disease, than that achieved by the same concentration of the free enzyme. The preparation of these new liposomal formulations by DELOS-SUSP, based on the depressurization of a CO2 -expanded liquid organic solution, shows the great potential of this CO2 -based methodology for the one-step production of protein-nanoliposome conjugates as bioactive nanomaterials with therapeutic interest.

Surfactant-free CO2-based microemulsion-like systems.

The presence of water-rich and water-lean nanodomains in a transparent, pressurized "water-acetone-CO2" mixture was revealed by Raman spectroscopy. This nano-structured liquid can be classified as a surfactant-free microemulsion-like system and has the capacity to dissolve hydrophobic compounds, such as ibuprofen, in the presence of large amounts of water. This finding opens new opportunities in the fields of confined reactions and material templating.