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Aggresomes are protein aggregates found in mammalian cells when the intracellular protein degradation machinery is over-titered. Despite that they abound in cells producing recombinant proteins of biomedical and biotechnological interest, the physiological roles of these protein clusters and the functional status of the embedded proteins remain basically unexplored. In this work, we have determined for the first time that, like in bacterial inclusion bodies, deposition of recombinant proteins into aggresomes does not imply functional inactivation. As a model, human α-galactosidase A (GLA) has been expressed in mammalian cells as enzymatically active, mechanically stable aggresomes showing higher thermal stability than the soluble GLA version. Since aggresomes are easily produced and purified, we propose these particles as novel functional biomaterials with potential as carrier-free, self-immobilized catalyzers in biotechnology and biomedicine. [ABSTRACT FROM AUTHOR]

Abstract: The human α-galactosidase A (EC 3.2.1.22, GLA), a lysosomal enzyme with important biotechnological and biomedical applications, has been successfully immobilized for the first time onto different versions of micro-sized magnetic particles by means of alternative coupling chemistries (covalent and metal affinity adsorption). The immobilized enzyme shows higher specific activity than its soluble counterpart and its enhanced stability as well as the magnetic-controlled positioning and reusability provided by coupling make these new bioconjugates excellent platforms for the presentation of highly active and pure versions of human GLA for both in vitro catalysis and therapeutic applications. [Copyright &y& Elsevier]

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme. [ABSTRACT FROM AUTHOR]

The central nervous system represents a major target tissue for therapeutic approach of numerous lysosomal storage disorders. Fabry disease arises from the lack or dysfunction of the lysosomal alpha-galactosidase A (GLA) enzyme, resulting in substrate accumulation and multisystemic clinical manifestations. Current enzyme replacement therapies (ERTs) face limited effectiveness due to poor enzyme biodistribution in target tissues and inability to reach the brain. We present an innovative drug delivery strategy centered on a peptide-targeted nanoliposomal formulation, designated as nanoGLA, engineered to selectively deliver a recombinant human GLA (rhGLA) to target tissues. In a Fabry mouse model, nanoGLA demonstrated improved efficacy, inducing a notable reduction in Gb3 deposits in contrast to non-nanoformulated GLA, even in the brain, highlighting the potential of the nano-GLA to address both systemic and cerebrovascular manifestations of Fabry disease. The EMA has granted the Orphan Drug Designation to this product, underscoring the potential clinical superiority of nanoGLA over authorized ERTs and encouraging to advance it toward clinical translation. [ABSTRACT FROM AUTHOR]

• Robust preparation of liposomal formulation by DELOS-susp method. • Implementation of Quality by Design methodology to liposomes preparation. • Influence of critical parameters on quality was studied through DoE analysis. • Design Space was obtained for GLA-loaded liposomes formulation. [Display omitted] 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. [ABSTRACT FROM AUTHOR]

The capability of HeLa cells to internalize large spherical microparticles has been evaluated by using inorganic, magnetic microparticles of 1 and 2.8 µm of diameter. In both absence but especially under the action of a magnet, both types of particles were uptaken, in absence of cytotoxicity, by a significant percentage of cells, in a non-endosomal process clearly favored by the magnetic field. The engulfed particles efficiently drive inside the cells chemically associated proteins such as GFP and human alpha-galactosidase A, without any apparent loss of protein functionalities. While 1 µm particles are completely engulfed, at least a fraction of 2.8 µm particles remain embedded into the cell membrane, with only a fraction of their surface in cytoplasmic contact. The detected tolerance to endosomal-independent cell penetration of microscale objects is not then restricted to organic, soft materials (such as bacterial inclusion bodies) as previously described, but it is a more general phenomenon also applicable to inorganic materials. In this scenario, the use of magnetic particles in combination with external magnetic fields can represent a significant improvement in the internalization efficiency of such agents optimized as drug carriers. This fact offers a wide potential in the design and engineering of novel particulate vehicles for therapeutic, diagnostic and theragnostic applications. [Display omitted] • Magnetic microparticles are uptaken by cells, a process favored by magnetic field. • Small particles are engulfed, while bigger ones remain embedded in cell membrane. • Magnetic microparticles efficiently drive into cells active proteins. • Tolerance to cell uptake is a general phenomenon applicable to inorganic particles. • Magnetic particles are potential nanovehicles as drug delivery systems. [ABSTRACT FROM AUTHOR]