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.

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.

MKC-Quatsomes: a stable nanovesicle platform for bio-imaging and drug-delivery applications.

Quatsomes are outstanding new lipid-based nanovesicles that are highly homogeneous and stable in different media for years, but the composition must be carefully chosen to avoid any potentially toxic side effects in in vivo applications. To this end, we have developed and studied a novel type of Quatsomes composed of cholesterol and myristalkonium chloride (MKC), the latter being extensively used as antimicrobial preservative in many ophthalmic and parenteral formulations on the EU and USA market. We have synthesized these novel MKC-Quatsomes in different media that are suitable for parenteral administration, and confirmed their stability in these media for 18 months, as well as the stability in human serum for 24 hours. Biodistribution assays were performed after intravenous injection of fluorescently labeled MKC-Quatsomes in live mice bearing xenografted colorectal tumors, showing nanovesicle accumulation in tumors, liver, spleen, and kidneys. No histological alteration or toxicity was observed in any of these organs.

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.

Improved and safe transcorneal delivery of flurbiprofen by NLC and NLC-based hydrogels.

Flurbiprofen (FB)-loaded nanostructured lipid carriers (NLCs) based on Compritol®888 ATO (C888; FB-C888NLC) were developed for anti-inflammatory ocular therapy. NLCs prepared by high-pressure homogenization technique following a factorial design had low particle size (<199 nm), high entrapment efficiency (∼90%), and long-term physical stability. Previously optimized NLCs based on stearic acid (SA; FB-SANLC) were prepared for comparison studies. Both formulations were dispersed in freshly prepared carbomer hydrogel (HG) to check the suitability of semisolid-based NLC HGs to enhance the corneal residence time. FB-C888NLC remained in the nanometric range, whereas FB-SANLC suffered an increase in particle size up to 5 µm after incorporation. Consequently, modifications in the crystalline lattice structure were observed for FB-SANLC-enriched HG (HG_FB-SANLC) by X-ray diffractometry. Both HG formulations showed plastic and low or no thixotropic properties, making them suitable for ocular application while maintaining its predominant elastic component as an indicator of good physicochemical stability. Formulations depicted sustained FB release. Ex vivo permeation analysis in isolated rabbit cornea revealed enhanced transcorneal drug permeation from the systems. In vivo ocular tolerance was confirmed by the Draize test. Therefore, NLC are promising and effective systems for ocular delivery of FB.

Feasibility of lipid nanoparticles for ocular delivery of anti-inflammatory drugs.

Due to the multiple barriers imposed by the eye against the penetration of drugs, the ocular delivery and targeting are considered difficult to achieve. A major challenge in ocular drug therapy is to improve the poor bioavailability of topically applied ophthalmic drugs by overcoming the severe constraints imposed by the eye on drug absorption. One of the promising strategies nowadays is the use of colloidal carrier systems characterized by a submicron-meter size. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) represent promising alternatives to conventional and very popular ocular carrier systems, such as the nanoemulsions, liposomes, and polymeric nanoparticles. Nevertheless, taking into account the characteristics of the eye, morphometrical properties of the colloidal systems (e.g., average particle size and polydispersion) may represent a limiting factor for topical application without induced corneal irritation, being responsible for the selected system. This review article focuses on the application of lipid nanoparticles (SLN, NLC) as carriers for both non-steroidal and steroidal anti-inflammatory drugs for the treatment of ocular inflammatory disorders. Major benefits, as well as shortcomings, of ocular inflammation conditions are described, in particular upon management of inflammation induced by ocular surgery (e.g., cataracts, refractive surgery). Particular emphasis is given to the clinical choices currently available, while examining the most recent drugs that have been approved.