PEGylated cationic nanoemulsions can efficiently bind and transfect pIDUA in a mucopolysaccharidosis type I murine model
Graphical abstract
Introduction
Mucopolysaccharidosis type I (MPS I, OMIM No 607014) is an autosomal recessive lysosomal disorder characterized by a deficiency of alpha-l-iduronidase (IDUA, EC 3.2.1.76) which results in the lysosomal storage of partially degraded heparan sulphate and dermatan sulphate glycosaminoglycans (GAGs). The clinical manifestations of this disease include stiff joints, coarse facial features, visual impairment, deafness, cardiac valve disease, and intellectual disability [1]. Although treatments such as enzyme replacement therapy (ERT) and haematopoietic stem cell transplantation (HSCT) are often prescribed to patients with MPS I, their effects are limited by the fact that intravenous ERT does not cross the blood–brain barrier, and, therefore, does not address the neurological complications of the disease, while HSCT is only effective when initiated early in life, and is associated with high morbidity and mortality. Despite some advances, these treatments still have several limitations [2], [3].
Non-viral gene therapy can be considered as an interesting treatment option for patients with MPS I, given that non-viral vectors are safer and easier to produce than viral ones [4], [5], [6]. The design of non-viral vectors, based on cationic polymers (such as polyethylenimine) and phospholipids/cationic lipids (such as DOPE and DOTAP), is among the most widespread strategies for circumventing the poor cell uptake and instability of nucleic acids against enzymatic degradation. A well-documented literature has showed an improved transfection efficiency of polyplexes and lipoplexes, mainly in vitro [7], [8]. Despite extensive research in the field, limitations in gene therapy need to be overcome to design an optimal system that is able to protect and carry the nucleic acid to the site of action for a sufficient period of time.
In recent years, considerable efforts have been made to refine the properties of the cationic nanoemulsions/pDNA complexes used in gene therapy, especially since studies revealed interesting results regarding transfection efficiency after administration by different routes [9], [10]. Nanoemulsions are composed of a liquid lipid oil core stabilized by a binary mixture of phospholipids and a cationic lipid. We have recently described PEGylated nanoemulsions composed of medium-chain triglycerides, dioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-trimethylammonium propane (DOTAP), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) as a carrier for a plasmid containing the alpha-l-iduronidase (IDUA) gene [11]. Transfection efficiency was evaluated in an animal model of MPS I developed by Neufeld et al., which has proved to be a remarkable tool for the study of MPS I pathogenesis and treatment options [12], [13], [14], since it was created by the disruption of exon 6 of the mouse IDUA gene, resulting in mice with a severe form of the disease [14]. Preliminary results showed that our pIDUA/PEGylated nanoemulsion complexes were able to increase IDUA activity after intravenous administration in MPS I mice when compared with non-treated MPS I animals [11].
Therefore, the goal of the present study was to follow up on this finding by investigating the effect of pIDUA loading processes obtained by adsorption or encapsulation of preformed pIDUA-cationic lipid complexes in nanoemulsions on transfection efficiency in the MPS I murine model. Based on previous findings regarding the preparation of reconstituted chylomicron remnant particles [15], [16] we hypothesized that pIDUA would behave differently when encapsulated in oil droplets of PEGylated nanoemulsions than it would in a single adsorption at the o/w interface of nanoemulsions. The size, charge, structural organization, and decomplexation of pIDUA from formulations were also investigated.
Section snippets
Construction of pIDUA
The plasmid used in this study was constructed as described by Camassola et al. [17]. Briefly, a HindIII/BamHI fragment containing the total span of the human IDUA cDNA in pH 2.1 was provided to us by Dr Elizabeth Neufeld, and subcloned in the mammalian expression vector pREP9 (Invitrogen, Carlsbad, USA). The plasmid was cultured in transformed Escherichia coli DH5α-T1R (Invitrogen, Carlsbad, USA) and isolated using MaxiPrep High Purity columns (Invitrogen, Carlsbad, USA) according to the
Physicochemical properties of the formulations
The physicochemical properties of the formulations are listed in Table 1. As the table shows, mean droplet size of nanoemulsions before complexation was close to 200 nm for both NE and NEP. However, mean diameter tended to increase after complexation, except for the PEGylated complexes containing the encapsulated pIDUA at a charge ratio of + 4/− (NEP/pIDUAE). The polydispersity index was lower than 0.15 in all formulations.
The NE formulation showed a positive ζ-potential of about + 50 mV, which
Discussion
The present report described the loading of a plasmid coding for alpha-l-iduronidase (pIDUA) onto cationic nanoemulsions either by its adsorption on blank formulations (NE/pIDUAA and NEP/pIDUAA) or by encapsulation of preformed pIDUA–DOTAP complex into the inner phase of nanoemulsions (NE/pIDUAE and NEP/pIDUAE). Our main goal was to investigate the effect of these loading procedures on the physicochemical properties of the complexes and on transfection efficiency in a MPS I murine model.
Two
Conclusion
The present study investigated the use of non-viral vectors in gene therapy for the multisystemic disease MPS I. PEGylation had a strong effect in maintaining colloidal stability, especially in the presence of proteins, so that PEGylated formulations were selected for in vivo studies. The overall results of EDXD experiments showed a lamellar structure with the insertion of DSPE-PEG in the lipid organization of blank formulations. In the presence of pIDUA, Bragg reflections corresponding to a
Abbreviation and nomenclature
- DOPE
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
- DOTAP
1,2-dioleoyl-sn-glycero-3-trimethylammonium propane
- DSPE-PEG
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]
- EDXD
energy-dispersive X-ray technique
- ERT
enzyme replacement therapy
- GAGs
glycosaminoglycans
- HSCT
haematopoietic stem cell transplantation
- IDUA
alpha-l-iduronidase
- MCT
medium chain triglycerides
- MPS I
mucopolysaccharidosis type I
- NE
blank nanoemulsions
- NEP
PEGylated blank nanoemulsions
- NE/pIDUAA
Complexes prepared by
Acknowledgments
This study was supported by the National Council for Scientific and Technological Development (grant number 478426/2011-9) and by the Research Incentive Fund of the HCPA (FIPE/HCPA). M.F. wishes to thank the Brazilian Federal Agency for the Support and Evaluation of Graduate Education for her scholarship. F.B. wishes to thank the Foundation for Research Support of the State of Rio Grande do Sul (DOCFIX/FAPERGS) for her postdoctoral grant. The authors wish to thank the Brazilian Synchrotron
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2018, International Journal of PharmaceuticsCitation Excerpt :Among nonviral delivery systems are cationic nanoemulsions, consisting of a liquid–liquid dispersion of two immiscible liquids (oil and water) stabilized by surfactants (Bouchemal et al., 2004; Liu and Yu, 2010). Recently, we have demonstrated that the administration of pegylated cationic nanoemulsions associated with a plasmid donor of IDUA gene (pIDUA) increased gene expression and functional enzyme production in human fibroblasts in vitro (Schuh et al., 2018) and in vivo in MPS I mice after intravenous delivery (Fraga et al., 2017, Fraga et al., 2015b,c), with high cell viability and in vivo tolerability. However, tissues which are difficult to reach, such as bone, brain, and joint show no improvements with intravascular administration, as IDUA is not able to reach sanctuary tissues (Baldo et al., 2014; Schuh et al., 2016).