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. 2019 Oct 11;11(10):522.
doi: 10.3390/pharmaceutics11100522.

Enzyme-Loaded Gel Core Nanostructured Lipid Carriers to Improve Treatment of Lysosomal Storage Diseases: Formulation and In Vitro Cellular Studies of Elosulfase Alfa-Loaded Systems

J Víctor Álvarez  1   2   3 , Carolina Herrero Filgueira  4   5 , Alexandre de la Fuente González  6 , Cristóbal Colón Mejeras  7 , Andrés Beiras Iglesias  8 , Shunji Tomatsu  9 , José Blanco Méndez  10 , Asteria Luzardo Álvarez  11 , María Luz Couce  12 , Francisco J Otero Espinar  13
Affiliations

Enzyme-Loaded Gel Core Nanostructured Lipid Carriers to Improve Treatment of Lysosomal Storage Diseases: Formulation and In Vitro Cellular Studies of Elosulfase Alfa-Loaded Systems

J Víctor Álvarez et al. Pharmaceutics. .

Abstract

Mucopolysaccharidosis IVA (Morquio A) is a rare inherited metabolic disease caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS). Until now, treatments employed included hematopoietic stem cell transplantation and enzyme replacement therapy (ERT); the latter being the most commonly used to treat mucopolysaccharidoses, but with serious disadvantages due to rapid degradation and clearance. The purpose of this study was to develop and evaluate the potential of nanostructured lipid carriers (NLCs) by encapsulating elosulfase alfa and preserving its enzyme activity, leading to enhancement of its biological effect in chondrocyte cells. A pegylated elosulfase alfa-loaded NLC was characterized in terms of size, ζ potential, structural lipid composition (DSC and XRD), morphology (TEM microscopy), and stability in human plasma. The final formulation was freeze-dried by selecting the appropriate cryoprotective agent. Viability assays confirmed that NLCs were non-cytotoxic to human fibroblasts. Imaging techniques (confocal and TEM) were used to assess the cellular uptake of NLCs loaded with elosulfase alfa. This study provides evidence that the encapsulated drug exhibits enzyme activity inside the cells. Overall, this study provides a new approach regarding NLCs as a promising delivery system for the encapsulation of elosulfase alfa or other enzymes and the preservation of its activity and stability to be used in enzymatic replacement therapy (ERT).

Keywords: elosulfase alfa; enzyme activity; in vitro cell studies; lysosomal storage diseases; nanostructured lipid carrier (NLC).

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Conflict of interest statement

Víctor Álvarez, Asteria Luzardo, Cristóbal Colón, Francisco J. Otero-Espinar, and María L. Couce declare the patent application, PCT/EP2019/068629. This patent application covers the underlying concept of immobilized ERT on nanostructured lipid systems to treat MPS IVA, as described in the manuscript. Carolina Herreo and Alexandre Fuente Gonzalez from the company Nasasbiothech declares that their role in the work was to support the enzyme determination and that the company had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Effect of the percentage of sucrose and enzyme loading on NLC size.
Figure 2
Figure 2
Representative TEM microphotographs of an NLC fixed with phosphor-tungstic acid (top) and sectioned with a microtome after lipid staining with osmium tetroxide and uranyl acetate (bottom).
Figure 3
Figure 3
Activity of elosulfase alfa immobilized in the NLC before and after the complete disintegration of particles.
Figure 4
Figure 4
Effect of plasma on elosulfase alfa-NLC stability (size and ζ potential).
Figure 5
Figure 5
Microscopy confocal images of TC28a2 chondrocyte cells (green) incubated with enzyme-NLC (red) at 37 °C. (a) 200 mg/mL, (b) 100 mg/mL, (c) 50 mg/mL, and (d) 25 mg/mL.
Figure 6
Figure 6
Microscopy confocal images of TC28a2 chondrocyte cells (green) incubated with 50 mg/mL of enzyme-NLC (red) at 4 °C. Left: 30 min; right: 1 h.
Figure 7
Figure 7
TEM images illustrating elosulfase alfa-loaded NLC internalized into TC28a2 chondrocytes during co-incubation.
Figure 8
Figure 8
TEM images of the TC28a2 chondrocytes incubated with enzyme-loaded NLCs. The formation of large phagosomes after NLC incubation was observed. Additionally, details regarding the rest of the lipid components after NLC digestion were ascertained.
Figure 9
Figure 9
Electrophoretic mobility shift assay of the extracts of cells after co-incubation with elosulfase alfa-NLC. Line 1—molecular weight standards; line 2—elosulfase alfa; Line 3—untreated chondrocytes; Line 4—chondrocytes with elosulfase alfa-NLC.
Figure 10
Figure 10
N-acetylgalactosamine 6-sulphatase (GALNS) enzyme activity in untreated chondrocytes and chondrocytes incubated with elosulfase alpha-NLC, quantified by MALDI-TOF analysis.
Figure 11
Figure 11
TEM images of pathological fibroblasts from mucopolysaccharoidosis IVA (MPS IVA) patients.
Figure 12
Figure 12
TEM images of pathological fibroblasts incubated with elosulfase alfa-NLC for 3 h.
Figure 13
Figure 13
GALNS activity (left graph) and reduction of the glycosaminoglycan (GAG) lysosomal deposits of pathological fibroblasts before and after incubation in a suspension of elosulfase alfa-NLC.
Figure 14
Figure 14
Cell viability of human primary fibroblasts incubated with elosulfase alpha-NLC for 24 h.
Figure 15
Figure 15
Confocal microscopy images of mice organs 24 h after intravenous administration of elosulfase alpha-NLC stained with DiD. From left to right: Confocal images at 10×, 20×, 40×, and 63×.
Figure 15
Figure 15
Confocal microscopy images of mice organs 24 h after intravenous administration of elosulfase alpha-NLC stained with DiD. From left to right: Confocal images at 10×, 20×, 40×, and 63×.
Figure 16
Figure 16
TEM images of the brain in mice 24 h after administration of elosulfase alpha-NLC passing blood-brain barrier (BBB) (three left images), in the interior of the astrocytes (fourth image), and neurons (fifth image).
Figure 17
Figure 17
TEM images of (left to right) fibroblasts, vascular endothelia between muscle fibers, spleen macrophages, renal tubular cells, and hepatocytes of mice 24 h after administration of elosulfase alpha-NLC.
See this image and copyright information in PMC

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