RESUMO
Enzyme replacement therapy (ERT) has been used to treat a few of the many existing diseases which are originated from the lack of, or low enzymatic activity. Exogenous enzymes are administered to contend with the enzymatic activity deficiency. Enzymatic nanoreactors based on the enzyme encapsulation inside of virus-like particles (VLPs) appear as an interesting alternative for ERT. VLPs are excellent delivery vehicles for therapeutic enzymes as they are biodegradable, uniformly organized, and porous nanostructures that transport and could protect the biocatalyst from the external environment without much affecting the bioactivity. Consequently, significant efforts have been made in the production processes of virus-based enzymatic nanoreactors and their functionalization, which are critically reviewed. The use of virus-based enzymatic nanoreactors for the treatment of lysosomal storage diseases such as Gaucher, Fabry, and Pompe diseases, as well as potential therapies for galactosemia, and Hurler and Hunter syndromes are discussed.
Assuntos
Doenças por Armazenamento dos Lisossomos , Nanopartículas , Humanos , Terapia de Reposição de Enzimas , Doenças por Armazenamento dos Lisossomos/tratamento farmacológicoRESUMO
Gaucher disease is a genetic disorder and the most common lysosomal disease caused by the deficiency of enzyme ß-glucocerebrosidase (GCase). Although enzyme replacement therapy (ERT) is successfully applied using mannose-exposed conjugated glucocerebrosidase, the lower stability of the enzyme in blood demands periodic intravenous administration that adds to the high cost of treatment. In this work, the enzyme ß-glucocerebrosidase was encapsulated inside virus-like nanoparticles (VLPs) from brome mosaic virus (BMV), and their surface was functionalized with mannose groups for targeting to macrophages. The VLP nanoreactors showed significant GCase catalytic activity. Moreover, the Michaelis-Menten constants for the free GCase enzyme (KM =0.29â mM) and the functionalized nanoreactors (KM =0.32â mM) were similar even after chemical modification. Importantly, the stability of enzymes under physiological conditions (pHâ 7.4, 37 °C) was enhanced by ≈11-fold after encapsulation; this is beneficial for obtaining a higher blood circulation half-life, which may decrease the cost of therapy by reducing the requirement of multiple intravenous injections. Finally, the mannose receptor targeted enzymatic nanoreactors showed enhanced internalization into macrophage cells. Thus, the catalytic activity and cell targeting suggest the potential of these nanoreactors in ERT of Gaucher's disease.
Assuntos
Doença de Gaucher , Terapia de Reposição de Enzimas , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Glucosilceramidase/genética , Humanos , Manose , NanotecnologiaRESUMO
Different types of gold nanoparticles have been synthesized that show great potential in medical applications such as medical imaging, bio-analytical sensing and photothermal cancer therapy. However, their stability, polydispersity and biocompatibility are major issues of concern. For example, the synthesis of gold nanorods, obtained through the elongated micelle process, produce them with a high positive surface charge that is cytotoxic, while gold nanoshells are unstable and break down in a few weeks due to the Ostwald ripening process. In this work, we report the self-assembly of the capsid protein (CP) of cowpea chlorotic mottle virus (CCMV) around spherical gold nanoparticles, gold nanorods and gold nanoshells to form virus-like particles (VLPs). All gold nanoparticles were synthesized or treated to give them a negative surface charge, so they can interact with the positive N-terminus of the CP leading to the formation of the VLPs. To induce the protein self-assembly around the negative gold nanoparticles, we use different pH and ionic strength conditions determined from a CP phase diagram. The encapsidation with the viral CP will provide the nanoparticles better biocompatibility, stability, monodispersity and a new biological substrate on which can be introduced ligands toward specific cells, broadening the possibilities for medical applications.
Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/química , Ouro/química , Nanopartículas Metálicas/química , Nanoconchas/química , Vírion/metabolismo , LigantesRESUMO
Virus-like particles (VLPs) are being used for therapeutic developments such as vaccines and drug nanocarriers. Among these, plant virus capsids are gaining interest for the formation of VLPs because they can be safely handled and are noncytotoxic. A paradigm in virology, however, is that plant viruses cannot transfect and deliver directly their genetic material or other cargos into mammalian cells. In this work, we prepared VLPs with the CCMV capsid and the mRNA-EGFP as a cargo and reporter gene. We show, for the first time, that these plant virus-based VLPs are capable of directly transfecting different eukaryotic cell lines, without the aid of any transfecting adjuvant, and delivering their nucleic acid for translation as observed by the presence of fluorescent protein. Our results show that the CCMV capsid is a good noncytotoxic container for genome delivery into mammalian cells.