Journal of Controlled Release 217 (2015) 273–283

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Solid lipid nanoparticle-based vectors intended for the treatment of

X-linked juvenile retinoschisis by gene therapy: In vivo approaches in
Rs1h-deficient mouse model
P.S. Apaolaza a, A. del Pozo-Rodríguez a, J. Torrecilla a, A. Rodríguez-Gascón a, J.M. Rodríguez b, U. Friedrich c,
B.H.F. Weber c, M.A. Solinís a,⁎
a
Pharmacokinetic, Nanotechnology and Gene Therapy Group (PharmaNanoGene), Faculty of Pharmacy, Centro de investigación Lascaray ikergunea,
University of the Basque Country UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain
b
Physiology Laboratory, Faculty of Pharmacy, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain
c
Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: X-linked juvenile retinoschisis (XLRS), which results from mutations in the gene RS1 that encodes the protein
Received 25 June 2015 retinoschisin, is a retinal degenerative disease affecting between 1/5000 and 1/25,000 people worldwide. Cur-
Received in revised form 16 September 2015 rently, there is no cure for this disease and the treatment is based on the application of low-vision aids. The
Accepted 18 September 2015
aim of the present work was the in vitro and in vivo evaluation of two different non-viral vectors based on
Available online 21 September 2015
solid lipid nanoparticles (SLNs), protamine and two anionic polysaccharides, hyaluronic acid (HA) or dextran
Keywords:
(DX), for the treatment of XLRS. First, the vectors containing a plasmid which encodes both the reporter green
Solid lipid nanoparticles fluorescent protein (GFP) and the therapeutic protein retinoschisin, under the control of CMV promoters, were
X-linked juvenile retinoschisis characterized in vitro. Then, the vectors were subretinally or intravitreally administrated to C57BL/6 wild type
Non-viral vector mice. One week later, GFP was detected in all treated mice and in all retinal layers except in the Outer Nuclear
Hyaluronic acid Layer (ONL) and the Inner Nuclear Layer (INL), regardless of the administration route and the vector employed.
Dextran Finally, two weeks after subretinal or intravitreal injection to Rs1h-deficient mice, GFP and retinoschisin expres-
Gene therapy sion was detected in all retinal layers, except in the ONL, which was maintained for at least two months after
subretinal administration. The structural analysis of the treated Rs1h-deficient eyes showed a partial recovery
of the retina related to the production of retinoschisin. This work shows for the first time a successful RS1 gene
transfer to Rs1h-deficient animals using non-viral nanocarriers, with promising results that point to non-viral
gene therapy as a feasible future therapeutic tool for retinal disorders.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
X-linked juvenile retinoschisis (XLRS) is a retinal degenerative dis-
ease caused by mutations in the RS1 gene, which encodes a secreted
protein named retinoschisin. This disease affects between 1/5000 and
1/25,000 worldwide [1] and is characterized by cystic cavities in the
inner and outer retina and deterioration in vision due to retinal disorga-
nization [2]. Common phenotypic features are the loss of central vision
acuity, schisis radiating from the fovea with the classical spoke-wheel
pattern and other peripheral abnormalities such as splitting of the
inner retinal layers, pigmented scars after involution of cysts, and a
remarkable decrease in the b-wave amplitude after electroretinogram
⁎ Corresponding author at: Pharmacy and Pharmaceutical Technology Laboratory,
Pharmacy Faculty, University of the Basque Country UPV/EHU, Paseo de la Universidad
7, 01006 Vitoria-Gasteiz, Spain.
E-mail address: marian.solinis@ehu.eus (M.A. Solinís).
analysis [3]. Currently, there is no cure for the schisis formation and
treatment is based on the application of low-vision aids.
The recessive monogenic origin makes this disease an excellent can-
didate for gene therapy treatment. Viral gene therapy for the treatment
of XLRS has shown promising results in animal models of the disease [4],
proving visual recovery for weeks and months after subretinal or intra-
vitreal administration [2,5–11]. However, the clinical application of viral
vectors is limited due to concerns over their immunogenicity and toxic-
ity [12,13]. In this sense, DNA delivery nanocarriers are a safer strategy
with advantages such as more favourable safety profile, lower produc-
tion cost, easy modification, better stability and key features related to
technical feasibility [14]. Among non-viral vectors, solid lipid nanoparti-
cles (SLNs) represent one of the most effective lipid-based colloidal
carriers [15,16], and for gene delivery to the posterior segment of the
eye, they present several advantages: can be easily administered in a
liquid form, are biocompatible, easy to produce at large scale, can be
autoclaved, sterilized [17] or lyophilized [18] and, they are stable in
biological fluids and during storage, for at least one week at room

http://dx.doi.org/10.1016/j.jconrel.2015.09.033
0168-3659/© 2015 Elsevier B.V. All rights reserved.
274 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283
temperature and 1 month at 4 °C [18]. Moreover, the features of SLNs
can be improved by the incorporation of different ligands, such as prot-
amine sulphate [19], cell penetration peptides [20], dextran (DX) [21],
chitosan [22] or hyaluronic acid (HA) [23], among others. These ligands
increase nucleic acid protection and cell internalization and/or modu-
late the trafficking inside the cells.
Our research group has demonstrated the ability of SLNs combined
with protamine and DX (DX-SLN) to transfect retinal cells in vivo after
intravitreal and subretinal administration to rats [21]. Later on, the
replacement of DX by HA (HA-SLN) turned out to be more effective to
transfect the retinal pigment epithelial cell line ARPE-19 [23]. The aim
of the present study was to evaluate two vectors based on SLN, prot-
amine, and HA or DX, in C57BL/6 wild type and Rs1h-deficient mice
for the treatment of XLRS. After in vitro characterization, the vectors
were injected by subretinal or intravitreal route to the mice. Gene
expression was evaluated over time, and structural analysis of the retinas
of the Rs1h-deficient mice was also performed.
2. Materials and methods
2.1. Plasmid pCAG-GFP_CMV-RS1 development
The expression unit containing the CMV promoter, the RS1 open
reading frame, and a PolyA adenylation site was extracted from a
retinoschisin expression plasmid generated before [24] by digestion
with SalI. It was inserted into the pCAG-GFP plasmid [25] via the SalI
restriction site of this plasmid. After digesting pCAG-GFP with SalI, the
CAG promoter dropped out due to two SalI restriction sites within the
pCAG-GFP plasmid. We made sure that after ligation of all required
elements (RS1 expression unit, CAG-promoter and plasmid backbone),
they were present in the correct quantity and orientation in the final
plasmid by Sanger sequencing of plasmid DNA. The pCAG-GFP_CMV-
RS1 plasmid, which encodes both Green Fluorescent Protein (GFP) and
retinoschisin, was analysed and amplified by the research group at the
Institute of Human Genetics, University of Regensburg (Germany).
2.2. Preparation of the DX-SLN and HA-SLN vectors
SLNs were prepared by a solvent emulsification-evaporation
technique previously described [26]. Following the protocol described
by our group [23], an aqueous solution of protamine (Sigma-Aldrich;
Madrid, Spain) (P), DNA and the corresponding polysaccharide,
DX (Mw of 3.26 kDa) (Sigma-Aldrich; Madrid, Spain) or HA (Mw of
150 kDa) (Sigma-Aldrich; Madrid, Spain), was added to SLNs in
order to form the final vectors DX-SLN or HA-SLN. The component
weight ratio (DX/HA:P:DNA:SLN) was 1:2:1:5 in the DX-SLN vector,
and 0.5:2:1:2 in the HA-SLN vector.
2.3. Characterization of the vectors
2.3.1. Size, superficial charge and electrophoretic gel study
Particle size and zeta potential of DX-P-DNA and HA-P-DNA
complexes, and of DX-SLN and HA-SLN vectors were measured on a
Zetasizer Nano series-Nano ZS (Malvern Instruments, Worcestershire,
UK) using previously described methods [23].
2.3.2. DNA binding, protection from DNase I and DNA release
The studies about the DNA binding capacity by the vectors, protection
from DNase I and DNA release from the vectors were performed by 0.7%
agarose gel electrophoresis containing Gel Red™ (Biotium; Hayward,
USA) as described in [23]. A final concentration of 0.03 μg DNA/μl was
exposed to 1 U DNase I/2.5 μg DNA and incubated at 37 °C for 30 min;
afterwards, 4% sodium dodecyl sulfate (SDS) solution was added to
the samples to a final concentration of 1% to release the DNA from the
SLNs. The encapsulation efficiency was confirmed by the PicoGreen®
assay, as previously described [26].
2.3.3. Transmission electron microscope (TEM)
Electron microscopy negative staining was performed for the visual-
ization of the vectors. For that purpose, 10 μl of the vector suspension
were adhered onto glow discharged carbon coated grids for 60 s. The
remaining liquid was removed by blotting on filter paper and stained
with 2% uranyl acetate for 60 s. Samples were visualized in a Philips
EM208S TEM. Digital images were acquired with an Olympus SIS purple
digital camera.
2.4. In vitro studies in cell cultures
2.4.1. Transfection studies
Transfection studies were carried out in cultured human retinal
pigment epithelial (ARPE-19) cells obtained from the American Type
Culture Collection (ATCC), following previously reported methods [23].
ARPE-19 cells were seeded on 12 well plates (6 × 104 cells/well) in
Dulbecco's Modified Eagle's Medium–Han's Nutrient Mixture F-12
(1:1) medium (D-MEM/F-12) purchased from Life Technologies
(GIBCO™; Madrid, Spain). Cells were incubated 24 h at 37 °C before
the addition of vectors corresponding to a single dose of 2.5 μg
DNA. Four hours later, the medium was replaced with 1 ml of fresh-
media. The evaluation of the transfection efficacy was performed 72 h
after addition of vectors. Transfectin® (Bio-Rad; Madrid, Spain) was
used as control. Briefly, plasmid and Transfectin® (ratio 1:1.5) were
mixed separately but in an equal total volume of 50 μl with OPTI-
MEM (GIBCO™; Madrid, Spain) at room temperature. Then, the solu-
tion of the Transfectin® was added to the plasmid solution and, incu-
bated 20 min. After 2 h the media was replaced. Free DNA and the
complexes DX-P-DNA (1:2:1) and HA-P-DNA (0.5:2:1) were also
evaluated.
2.4.2. Quantification of GFP and retinoschisin
Quantification of GFP was performed by a fluorometric assay [23]
and it was expressed as Relative Fluorescent Units (RFU) per milligram
of total amount of protein quantified by the Micro BCA™ Protein Assay
Kit (Thermo-Fisher Scientific Inc.; Madrid, Spain). The percentage of
transfected cells was measured by flow cytometry [23]. The levels of
secreted retinoschisin were measured by the Enzyme-linked Immuno-
sorbent Assay (ELISA) Kit (USCN LIFE Science Inc.) [23].
2.4.3. GFP and retinoschisin visualization by fluorescence microscopy
Retinoschisin was detected via immunocytochemistry as described
by Delgado et al., 2012 [21], using a secondary antibody AlexaFluor
647-conjugated goat anti-mouse IgG instead of Alexa Fluor 488-
conjugated goat anti-rabbit, and with minor modifications on the
nuclei staining step, using DAPI-fluoromount-G™ (SouthernBiotech;
Birmingham, USA) as mounting media. Images of both proteins GFP
(green) and retinoschisin (red) were captured with an Axio Observer
Inverted Microscope Z.1 with Apotome (Zeiss) using 20× magnification.
2.4.4. Cellular uptake of the vectors
For the evaluation of the uptake, SLNs were labelled with the fluores-
cent dye Nile Red (Sigma-Aldrich; Madrid, Spain) (λ = 590 nm) accord-
ing to a previously reported method [27]. ARPE-19 cells were treated
with the Nile Red labelled vectors as described in Section 2.4.1, and
2 h later the cellular uptake was evaluated by flow cytometry [23].
Images of the cells were also captured with an Axio Observer Inverted
Table 1
Size, polydispersity index (PDI) and zeta potential SLNs-based vectors bearing the plasmid
pCAG-GFP_CMV-RS1. Mean ± SD (n = 3).
Vector Size (nm) PDI Zeta potential (mV)
DX-SLN 218 ± 30 0.24 ± 0.01 43.9 ± 1.1
HA-SLN 235 ± 28 0.23 ± 0.01 31.4 ± 0.8⁎
⁎ p b 0.05 respect to DX-SLN.
 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283 275

Fig. 1. DNA binding, protection against DNase I and SDS-induced release from HA-SLN and DX-SLN vectors. Lane 1 = DNA ladder; lane 2 = free DNA; lane 3 = HA-SLN; lane 4 = DX-SLN;
lane 5 = free DNA treated with DNase I; lane 6 = HA-SLN treated with DNase I and SDS; lane 7 = DX-SLN treated with DNase I and SDS; lane 8 = HA-SLN treated with SDS; and lane 9 = DX-
SLN treated with SDS.

Microscope Z.1 with ApoTome (Zeiss) using 63× magnification. As con-
trol, cells treated with a solution of free Nile Red (total amount equal to
that in the cells treated with the labelled vectors) were also analysed.
2.4.5. Cell viability
The viability of the cells was evaluated by the CCK-8 assay (Sigma-
Aldrich; Madrid, Spain), as described by Torrecilla et al. [28]. 5 × 103
cells/well were cultured on a 96-well culture plate followed by transfec-
tion. Seventy-two hours post-transfection, CCK-8 reagent was added
and after 4 h of incubation at 37 °C, the absorbance at 450 nm was
measured.
2.5. In vivo studies
The animal experiments were carried out according to the European
Union regulations for the use of animals in research. The animal proto-
col was approved by the Animal Care and Use Committee of the Univer-
sity of Regensburg (The German Animal Welfare Act. § 8 Abs 1. Des
TierschutzgesetzesA). The method of generation and the phenotypic
characterization of the Rs1h-deficient mice used in this study have
been reported previously by Weber et al. [4]. C57BL/6 wild type and
deficient mice were housed under specific pathogen-free barrier condi-
tions at the Central Animal Facility of the University of Regensburg and
maintained under conditions established by the institution for their use,
in strict compliance with NIH guidelines. Mice were sacrificed by
cervical dislocation after inhalation of carbon dioxide. Animals were dis-
sected and their eyes were immediately stored at − 80 °C until their
subsequent analysis.
2.5.1. Intraocular administration
A single dose of the vectors (DX-SLN or HA-SLN) bearing the pCAG-
GFP_CMV-RS1 plasmid was injected into the left eye of the mice at post-
natal day 14. The right eye of each mouse was kept as non-treated
control. The injections were performed under general anaesthesia,
using a stereo microscope SZT 300 (VWR). The sclera was perforated
by a 27-gauge hypodermic needle. Immediately afterwards, a Hamilton
syringe (75 SN, 5 μl), with needle point style 3 and a 32-gauge needle
was inserted through the hole. Once the tip of the needle had reached
the desired location, 0.75 μl of a suspension of DX-SLN or HA-SLN
(0.15 μg of DNA) was injected either subretinally or into the vitreous
cavity (n = 3–5). After intraocular injections, the needle was held in
place for 10 s and withdrawn slowly. Animals with retinal bleeding or
lens injury after the injection procedure were excluded from the study.
2.5.2. Processing murine eyes for analysis
At the established times (1 week, 2 weeks or 2 months post-
administration) eyes were enucleated and immediately processed by
immersion-fixed in 4% paraformaldehyde in 0.1 M phosphate buffer,
pH 7.4, (PB) for 30 min. Afterwards, the eyes were washed several
times in 0.1 M PB. Before the freezing procedure, the eyes were
cryoprotected in 0.1 M PB containing 18% sucrose for 45 min. Thus,
they were embedded in OCT (Tissue Tek, Sakura Finetek) and fast
frozen immersed in dry ice. Eyes were cryosectioned; 14 μm-thick ver-
tical sections were prepared.
2.5.3. Evaluation of gene expression
2.5.3.1. Antibodies. Mouse polyclonal anti-retinoschisin, mouse poly-
clonal anti-Rhodopsin and mouse monoclonal anti-RPE65 were pur-
chased from Abcam (Cambridge, UK). Rabbit polyclonal anti-GFP,
Alexa 488-conjugated goat anti-rabbit, AlexaFluor 647-conjugated
goat anti-mouse and AlexaFluor 488-conjugated goat anti-mouse
were purchased from Life Technologies (Madrid, Spain).
2.5.3.2. Protein analysis in C57BL/6 wild type and Rs1h-deficient mice eyes.
A total of 6–9 sections by eye were analysed for GFP and retinoschisin
detection by immunolabelling, as described by Friedrich et al. [24]
modifying the nuclei staining step, using the mounting media

Fig. 2. TEM photographs of (A) free DNA; (B) P-DNA; (C) DX-P-DNA; and (D) HA-P-DNA. Scale bar: 200 nm.
276 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283

Fig. 3. TEM photographs of (A) HA-SLN; and (B) DX-SLN. White arrows: surround layer on the HA-SLN vector. Scale bar: 200 nm.

DAPI-Fluoromount-G®. Sections were examined under an Olympus
Fluoview FV500 confocal microscope, using sequential acquisition to
avoid overlapping of fluorescent emission spectra.
Sections were considered positive if the fluorescent protein was
detected in any of the retinal layers. By using a 20× magnification, pro-
tein expression was scored in every layer according to a scale of 0–3
(0: no fluorescence; 1: dotted fluorescence, 2: discontinuous, and 3:
continuous fluorescence in the entire layer). The mean value of all the
analysed sections was used as a result for the protein expression in
every layer. The sections of non-treated eyes underwent the same
procedure.
2.5.4. Structural analysis of the retinal tissue
The structure of the retinal sections was analysed by the Masson's
trichrome staining technique. The samples were histologically evaluat-
ed and scored, including the thickness of the whole retina and the
outer nuclear layer, the general organization of the layers, the loss of
photoreceptors (PR), and the presence of schisis between the retinal
layers. The layer organization, the PR loss, and the presence of schisis
in every sample were scored as + (low), ++ (medium), and +++
(high). Sections were examined with an optical microscope (Olympus
BX50). Every eye (n = 3) was assessed based on its own control eye.
2.6. Statistical analysis
Statistical analysis was performed with IBM® SPSS® Statistics 21
(IBM). Normal distribution of samples was assessed by the Shapiro–
Wilk test, and homogeneity of variance, by the Levene test. The results
were compared with ANOVA and student's t test or H Kruskal–Wallis,
whereby differences were considered statistically significant at p b 0.05.
3. Results
3.1. Characterization of DX-SLN and HA-SLN vectors
Table 1 shows the particle size, the polydispersity index and the zeta
potential of the vectors bearing the plasmid pCAG-GFP_CMV-RS1. No
significant difference in the mean particle size of the formulations
(p N 0.05) was detected. The polydispersity index (PDI) was around
0.2, indicating a homogeneous distribution of the particle size. The

Fig. 4. GFP and retinoschisin expression in ARPE-19 cells 72 h after treatment with Transfectin, free DNA, HA-P-DNA, DX-P-DNA, DX-SLN or HA-SLN vectors. (A) Amount of GFP (RFUs per
milligram of total protein); (B) percentage of GFP positive cells; and (C) concentration levels of secreted retinoschisin per milligram of total protein. Error bars represent SD (n = 3). *
p b 0.05 respect to Transfectin; # p b 0.05 respect to DX-SLN and HA-SLN; and ¥ p b 0.05 respect to DX-SLN. (D) Fluorescence microscopy images of intracellular expression of GFP
(green) and retinoschisin (red). The nuclei are observed in blue. Images are at 20× magnification. Scale bar: 1 μm.
 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283 277

Fig. 5. (A) Fluorescence microscopy images of ARPE-19 cells at 2 h after addition of free-Nile Red (NR) and NR labelled vectors. Images are at 63× magnification. Scale bar: 20 μm. (B) Flow
cytometry histograms of ARPE-19 cells 2 h after treatment with free-NR or NR-labelled vectors. Grey filled curve = untreated cells; dark blue curve = free NR; light blue curve = HA-SLN
and pink curve = DX-SLN.

size of the complexes without SLNs (DX-P-DNA and HA-P-DNA) ranged
from 134 to 167 nm (p N 0.05), with a PDI of 0.5. The zeta potential of the
HA-SLN vector (+31.4 mV) was significantly lower (p b 0.05) than that
of the DX-SLN vector (+43.9 mV); in both cases the superficial charge
was higher than that obtained with the DX-P-DNA (+22.3 ± 0.3) and
HA-P-DNA (+15.8 ± 0.5) complexes.
As observed in Fig. 1, the absence of band corresponding to free DNA
in lanes 3 and 4 indicates that DNA was completely bound to the
vectors. When the vectors were analysed by the PicoGreen® assay no
free DNA was detected, which confirms a binding efficiency of 100%.
Lanes 6 and 7 show the bands of the DNA released from the vectors
after the treatment first with DNase I and later with SDS, which indi-
cates the ability of the vectors to protect the nucleic acid. Finally, the
capacity of the vectors to release the DNA was confirmed after the treat-
ment of the vectors with SDS only. The absence of DNA in the loading
well of lanes 8 and 9 indicates that all DNA was released from the
vectors.
Fig. 2 shows TEM images of the plasmid alone (Fig. 2A), and the com-
plexes P-DNA (Fig. 2B), DX-P-DNA (Fig. 2C) and HA-P-DNA (Fig. 2D). It
can be seen that the DNA (in dark) is highly condensed when it is

Fig. 6. CLSM images of retinas 1 week after subretinal (A–C) or intravitreal administration (D–F) to C57BL/6 wild type mice. A, D: non-treated eyes. B, C, E, F: GFP (green) and Rhodopsin
(red). Cell nuclei (INL, ONL and GC) are observed in blue. Images were captured at 20× magnification (Zoom 2×). Scale bar: 100 μm.
278 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283

Table 2 3.3. Cellular uptake

Levels of GFP in retinal sections of C57BL/6 wild type mice 1 week after subretinal or intra-
vitreal administration of vectors.
Flow cytometry histograms shown in Fig. 5B reveal an effective cel-
Subretinal administration Intravitreal administration lular internalization of both formulations. The higher displacement of
Retinal layer
DX-SLN HA-SLN DX-SLN HA-SLN the histogram corresponding to the DX-SLN vector indicates that this
RPE 0.7 0.9 0.5 0.6 formulation was internalized to a higher extent. As can be observed
PR 0.9 0.7 0.6 1.0 both in Fig. 5A and 5B, Nile Red is only uptaken when loaded into SLNs.
ONL 0.0 0.0 0.0 0.0
OPL 0.2 0.5 0.5 0.8
INL 0.0 0.0 0.0 0.0
3.4. In vivo evaluation
IPL 0.4 0.7 0.7 0.4
GC 0.1 1.2 1.1 0.9 3.4.1. Analysis of C57BL/6 wild type retinas
Positive mice 4/4 4/4 4/4 5/5 One week after administration, the GFP expression in the eye sec-
Positive sections (%) 90 91 97 90 tions was evaluated by immunohistochemistry. No green fluorescence
Nine sections per eye were evaluated. Light grey: dotted fluorescence; and dark grey: was detected in the samples from untreated mice. Evidence of toxicity
discontinuous fluorescence. Data represent mean values of all the sections analysed. GC: was not detected in the animals, irrespective of the formulation or the
Ganglion Cells Layer; IPL: Inner Plexiform Layer; INL: Inner Nuclear Layer; OPL: Outer administration route. Fig. 6 (and supplementary material) shows the
Plexiform Layer; ONL: Outer Nuclear Layer; PR: Photoreceptors; and RPE: Retinal Pigment
GFP fluorescence location of sectioned retinas treated with HA-SLN
Epithelium.
and DX-SLN vectors after subretinal and intravitreal injection, and
Table 2 shows the relative GFP expression levels in the different retinal
complexed with P, with P and DX or with P and HA. Fig. 3 features TEM layers.
pictures of DX-SLN and HA-SLN vectors. On the HA-P-SLN vector sur- GFP was detected in all treated mice, with positive sections ranging
face, a well-formed spherical corona was observed (white arrow). from 90 to 97%. After subretinal administration, GFP was detected in all
retinal layers except for the ONL and INL. Both vectors induced high
expression of GFP in PR and in RPE, and the HA-SLN vector also induced
3.2. Transfection and cell viability in ARPE-19 cells a higher expression of GFP in GC compared to DX-SLN vector.
After intravitreal injection GFP was detected in all retinal layers
In ARPE-19 cells, the HA-SLN vector induced a higher production of except for the ONL and INL. The highest expression with the DX-SLN
GFP (7216.87 vs 4954.54 RFU/mg, p b 0.05.) and a higher proportion vector was detected in the GC, and a modest expression was found in
of transfected cells (16.22 vs 11.43% GFP positive cells, p b 0.05) than the other layers. In contrast, after the administration of the HA-SLN vec-
the DX-SLN vector (Fig. 4A and 4B), and both vectors resulted more tor, the highest expression of GFP was found in the GC and in the PR.
effective than the commercial agent Transfectin®. The percentage of (For interpretation of the references to colour in this figure legend, the
transfected cells after treatment with free DNA or with the DX-P-DNA reader is referred to the web version of this article.)
and HA-P-DNA complexes was lower than 1%, and the amount of GFP
was lower than 150 RFU/mg.. 3.4.2. Analysis of Rs1h-deficient retinas
When transfection was measured in terms of retinoschisin produced GFP and retinoschisin expression levels in the different retinal layers
and secreted to the culture medium (Fig. 4C), a higher level was also are collected in Tables 3 and 4. Fig. 7 shows GFP and retinoschisin loca-
detected after the treatment of the cells with HA-SLN than with DX- tion in the sectioned retinas two weeks and two months after subretinal
SLN (11.16 vs 7.05 ng/ml per mg of total protein, p b 0.05). Intracellular administration and, two weeks after intravitreal administration of the
retinoschisin and GFP were analysed by immunocytochemistry. In Fig. vectors to Rs1h-deficient mice. The expression of both proteins was
4D, both proteins can be seen in the cells treated with either HA-SLN detected in all treated mice. No fluorescence was detected in the sam-
or DX-SLN vectors, in which a higher expression of retinoschisin and ples from untreated mice, and evidence of toxicity was not detected in
GFP was detected with the HA-SLN vector. the animals, irrespective of the formulation or the administration route.
Cell viability of treated ARPE-19 cells measured at 72 h with regard Two weeks after subretinal and intravitreal administration GFP and
to the non-treated cells was 103.8 ± 7.4% and 98.9 ± 6.7% for HA-SLN retinoschisin were detected in all retinal layers, except for the ONL,
and DX-SLN vectors, respectively. although the highest levels were observed in PR by subretinal injection.
By this route, high levels of retinoschisin were also detected in RPE, OPL,
INL and GC layers. By intravitreal route, the highest levels of GFP were
detected in GC and PR, whereas noteworthy levels of retinoschisin
were detected in the RPE, INL and GC layers.
Table 3 Two months after subretinal administration of the formulations,
Levels of GFP and retinoschisin in retinal sections of Rs1h-deficient mice 2 weeks after
retinoschisin and GFP were barely detected in the outer retina, with
subretinal or intravitreal administration of the vectors.
higher expression in GC, where values were comparable to the two-
Subretinal administration Intravitreal administration week samples. Moreover, the retinoschisin expression observed in the
GFP Retinoschisin GFP Retinoschisin
Retinal layer INL was also significant although PR hardly expressed both proteins.
DX- HA- HA- DX- HA- HA-
DX-SLN DX-SLN
SLN SLN SLN SLN SLN SLN
RPE 0.3 0.6 1.0 1.5 0.3 0.2 1.1 1.2 3.4.3. Structural analysis of the retinal tissue
PR 1.6 1.8 1.4 1.7 1.0 0.8 0.3 0.7
ONL 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Fig. 8 depicts histological sections from the Rs1h-deficient retinas of
OPL 0.2 0.9 1.3 1.6 0.3 0.3 0.6 0.6 mice treated with the HA-SLN and the DX-SLN vectors, and their own
INL 0.7 0.2 1.1 1.1 0.3 0.1 1.1 1.3 control eye. A histological section from an untreated wild type mouse
IPL 0.1 0.0 0.6 0.1 0.9 0.4 0.4 0.2
GC 1.3 0.8 1.3 1.3 1.4 1.1 1.3 1.5 has also been included as control. The analysis of all the retinal samples
Positive mice 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 is compiled in Table 5.
Positive
sections (%) 100 100 100 100 100 83.3 100 100 Two weeks after the treatment with the vectors, subretinally or
intravitreally, a lower PR loss, a decrease in gaps between bipolar cells
Six sections per eye were evaluated. Light grey: dotted fluorescence; dark grey: discontin-
uous fluorescence. Data represent mean values of all the sections analysed. GC: Ganglion
and the plexiform layers, and an improvement of the retinal layers orga-
Cells Layer; IPL: Inner Plexiform Layer; INL: Inner Nuclear Layer; OPL: Outer Plexiform nization were detected in all treated eyes, which indicates recovery of
Layer; ONL: Outer Nuclear Layer; PR: Photoreceptors; RPE: Retinal Pigment Epithelium. the retina. Two months after treatment with either HA-SLN or DX-
 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283 279

Table 4 detected after the administration of both vectors by the two admin-
Levels of GFP and retinoschisin in retinal sections of Rs1h-deficient mice 2 months after istration routes.
subretinal administration of vectors.

GFP Retinoschisin 4. Discussion

Retinal layer
DX-SLNHA -SLN DX-SLN HA-SLN
The SLN-based vectors evaluated in this study include protamine, a
RPE 0.0 0.3 0.5 0.3
cationic peptide which condenses DNA, presents nuclear localization
PR 0.2 0.3 0.3 0.0 signals, improves the transcription, and enhances the transfection effi-
ONL 0.0 0.0 0.0 0.0 ciency of SLNs in retinal cells [19,29]. A polysaccharide was also includ-
OPL 0.0 0.1 0.1 0.3 ed, specifically DX or HA, with beneficial properties contributing to
INL 0.1 0.6 0.8 0.9 improve nucleic acid delivery and widely used for pharmaceutical and
IPL 0.2 0.3 0.3 0.2
biomedical applications [30]. Both polyanions present low cytotoxicity,
are easily subject to chemical modification, and have stealth properties.
GC 1.2 1.1 0.8 1.1
HA also shows mucoadhesive properties, is non-immunogenic and pre-
Positive mice 2/2 2/2 2/2 2/2
sents targeting moieties [31].
Positive sections (%) 100 100 100 100 Electrostatic interactions between the components of the formula-
Six sections per eye for each protein were evaluated. Light grey: dotted fluorescence; and tion play a major role in the vector formation. The DNA is fully con-
dark grey: discontinuous fluorescence. Data represent mean values of all the sections densed by protamine [19] and the features of the polysaccharide
analysed. GC: Ganglion Cells Layer; IPL: Inner Plexiform Layer; INL: Inner Nuclear Layer; determine the interactions between the components of the formulation,
OPL: Outer Plexiform Layer; ONL: Outer Nuclear Layer; PR: Photoreceptors; RPE: Retinal
conditioning the final structure and the physicochemical characteristics
Pigment Epithelium.
of the vectors. Indeed, the corona surrounding the surface of the HA-SLN
vector, observed in TEM images (Fig. 3), could be due to HA segment
SLN vectors, a retinal recovery was also observed, although less loops around the particle [32]. Moreover, HA-SLN presented a lower
noticeable. zeta potential than DX-SLN, although the electrophoresis on agarose
The box plots presented in Fig. 9 display the retinal thickness of gels showed that both formulations protected and released the plasmid
the eyes from untreated wild type mice, untreated Rs1h-deficient mice properly (Fig. 1). In vitro studies showed that DX-P-DNA and HA-P-DNA
and Rs1h-deficient mice two weeks and two months after the treatment were not able to transfect ARPE-19 cells, highlighting the importance of
with the vectors. Although high variability in the retina organization the SLNs on the transfection efficacy (Fig. 4A and 4B). The HA-SLN
of the Rs1h-deficient mice was observed, a partial recovery was vector proved to be more effective, even though it was internalized in

Fig. 7. CLSM images of retinas 2 weeks and 2 months after subretinal or 2 weeks after intravitreal administration to Rs1h-deficient mice. A, F, K: non-treated eyes. B, D, G, I, L and N: GFP
(green) and rhodopsin (red). C, E, H, J, M and O: retinoschisin (green) and rhodopsin (red). Cell nuclei (INL, ONL and GC) are observed in blue. Images were captured at 20× magnification
(zoom 1.5×). Scale bar: 200 μm.
280 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283

a lesser extent than DX-SLN (Fig. 5). In this sense, the effect of HA on the
DNA condensation favours the intracellular loosening of the plasmid,
and thus, the transfection process [23], contributing to the higher effica-
cy of this vector.
The beneficial treatment for XLRS by gene replacement therapy may
not be necessarily restricted to the expression of retinoschisin in a spe-
cific cellular target, considering that it is a secreted protein. Accordingly,
we included in the non-viral vectors a construct containing both GFP
and retinoschisin cDNAs under the control of ubiquitous CAG and
CMV promoters. These promoters are widely used due to their ability
to induce robust protein expression in a wide variety of cell types [33].
By following the expression of the two genes, we could, on the one
hand, identify the retinal layers transfected by evaluating GFP fluo-
rescence, and, on the other hand, the distribution of the produced
retinoschisin.
One week after subretinal or intravitreal administration to C57BL/6
wild type mice (Fig. 6 and Table 2), vectors successfully protected and
delivered the plasmid, and were able to induce the expression of GFP
in several retinal layers, including PR, the main target cells to treat
XLRS. DX-SLN formulation transfected more efficiently the layers closest

Fig. 8. Microscopic images of Rs1h-deficient mouse retinas stained by Masson's trichrome technique. Green: connective tissue; dull green: muscle; dark blue: nuclei; and pink: cytoplasm
and muscle fibres. A: non-treated wild type mouse retina; C, E, G, I, K and M non-treated Rs1h-deficient mice retinas; B: 2 weeks after subretinal administration of HA-SLN; D: 2 weeks after
subretinal administration of DX-SLN; F: 2 weeks after intravitreal administration of HA-SLN; H: 2 weeks after intravitreal administration of DX-SLN; J: 2 months after subretinal admin-
istration of HA-SLN; and L: 2 months after subretinal administration of DX-SLN. Images were captured at 20× magnification. Scale bar: 200 μm.
 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283 281

Table 5
Evaluation of retinal morphology features in Rs1h-deficient mice after subretinal or intravitreal administration of vectors.

Sample Layers organization PR loss Schisis (gaps) Retinal thickness (μm) ONL thickness (μm)

No administration WT +++ – – 197.5 ± 5.0 42.5 ± 5.0

Subretinal administration (2 weeks) Rs1h-KO + ++ ++ 135.0 ± 18.7a 20.0 ± 0.0a
NT
Rs1h-KO ++ + + 146.7 ± 20.8a 26.7 ± 5.8a,b
HA-SLN
Rs1h-KO ++ + + 166.7 ± 45.1b 30.0 ± 0.0a,b
DX-SLN
Intravitreal administration (2 weeks) Rs1h-KO + ++ ++ 127.5 ± 21.0a 23.8 ± 5.8a
NT
Rs1h-KO ++ + + 187.5 ± 27.5b 35.0 ± 5.8b
HA-SLN
Rs1h-KO ++ + + 152.5 ± 31.0a 30.0 ± 0.0a
DX-SLN
Subretinal administration (2 months) Rs1h-KO + + +++ 140.8 ± 16.4a 27.5 ± 5.0a
NT
Rs1h-KO + + ++ 183.3 ± 32.2b 36.7 ± 11.6
HA-SLN
Rs1h-KO + + + 160.0 ± 10.0a 33.3 ± 5.8
DX-SLN

PR: Photoreceptor; ONL: Outer Nuclear Layer; WT: wild type mouse; NT: non-treated mouse; and Rs1h-KO: Rs1h-deficient mouse. NT eyes values are obtained from all the non-treated
eyes of each group. Qualitative values were given in order to evaluate the effect as: –: no effect; +: low; ++: medium; and +++: high. Quantitative results are expressed as mean ±
standard deviation (n ≥ 3).
a
p b 0.05 respect to WT.
b
p b 0.05 respect to NT.

to the site of injection, whereas the HA-SLN vector, regardless of the
administration route, diffused more easily across the extracellular
matrix, and transfected efficiently both the outer and inner layers of
the retina. The higher capacity of HA-SLN to diffuse compared to DX-
SLN may be related to differences in the surface characteristics of both
vectors. It is important to note that the anionic nature of the vitreous
humour plays a key role in the charged particle distribution, limiting
the diffusion of cationic nanoparticles in the posterior segment of the
eye [34]. The transfection profile obtained with our positive charged
vectors after intravitreous injection reveals that the incorporation of
DX or HA polysaccharides to the formulations confers to the vectors
stealth properties that likely favour their diffusion through the vitreous
and also across the retina [35], facilitating the transfection of retinal
layers distant from the injection site.
Rs1h-deficient mice are considered an adequate model for XLRS dis-
ease; they exhibit many of the features found in XLRS patients, including
cystic cavities, disorganization of the PR-bipolar synapse, reduction in b-
wave of the electroretinogram, and progressive loss in rod and cone PR
cells [36,37]. Considering that PR cell death in the Rs1h-deficient mice is
triggered by apoptotic events initiating around the post-natal day 14
[38,39], DX-SLN and HA-SLN vectors were administered to mice aged
14 days. Although intravitreal injections may be the optimal route of
administration due to the fragile retina in XLRS, subretinal administra-
tion may be more effective to transfect PR [40]. Two weeks after the
administration (Fig. 7A–J and Table 3), the vectors led to transfection
of different retinal layers, from GC to PR and RPE cells, although unlike
in wild type mice, the location of GFP did not show differences
depending on the vector administered. The disorganization of the
retinal layers in the mice affected by the disease seems to result in a
less strong barrier to the diffusion of the vectors. The location of the
retinoschisin expressed after administration of the two vectors was dif-
ferent from that of GFP; it is important to take into account that the
presence of retinoschisin in a certain layer does not necessarily mean
that it was produced there, since this protein is secreted from the cells
where it is produced. In terms of biodistribution of retinoschisin, there
were no difference depending on the administration route, but in PR,
the level of this protein was higher when the vectors were injected by
subretinal route than after intravitreal administration. For this reason,
Rs1h-deficient mice were also evaluated two months after subretinal
administration and, GFP and retinoschisin were detected mainly in GC
and INL with both vectors (Fig. 7K–O; Table 4).
The structural analysis (Figs. 8, 9, Table 5) of the retinas showed a
partial recovery of the tissue with the two vectors by both administra-
tion routes two weeks after administration, related to the production

Fig. 9. Box plots of retinal thickness analysis 2 weeks after subretinal (A) and intravitreal (B) administrations, and (C) 2 months after subretinal administration of vectors to Rs1h-deficient
mice. Treated retinas were compared to retinas of wild type mice and to non-treated retinas of Rs1h-deficient mice. a: p b 0.05 respect to WT; and b: p b 0.05 respect to NT.
282 P.S. Apaolaza et al. / Journal of Controlled Release 217 (2015) 273–283
of the retinoschisin. This recovery was also maintained, although less
noticeably, two months after subretinal injection. It is known that the
delivery of the normal RS1 gene to defective retinal cells which do not
express retinoschisin can promote long-term rescue of retinal structure
and function and greatly slow PR degeneration [36]. Byrne et al. [2]
have recently observed a greatest long-term rescue in XLRS when
retinoschisin is produced in the PR, but they also mentioned that an
additional benefit from ubiquitous expression may be obtained during
retinal development, when retinoschisin is expressed strongly in a
wave of expression in other cell types. It should also be considered
that the unspecific production of retinoschisin achieved in the present
study, could be a good therapeutic option in advanced disease, when
the loss of PR is very pronounced.
Previous studies with viral vectors in Rs1h-deficient mice [6,8,36]
have shown significant improvement in the structure and function of
the retina related to the retinoschisin expression in the treated eyes.
Considering the promising results obtained in the present study in
terms of retinoschisin expression and retinal recovery, an improvement
of the visual function is expected, although additional studies should be
carried out in order to evaluate the long-term effect of these new
vectors and confirm the improvement of the visual function.
5. Conclusions
The non-viral SLN-based formulations evaluated in the present work
efficiently transfected different layers of the retina after subretinal or
intravitreal administration, providing retinoschisin levels that partially
restored the retinal structure in a Rs1h-deficient model.
To the best of our knowledge, this work shows for the first time the
administration of non-viral vectors to Rs1h-deficient animals, obtaining
promising results that point to non-viral gene therapy for retinal disor-
ders as a future perspective and feasible safer alternative to viral vectors,
which could contribute to the clinical approach of this kind of diseases.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jconrel.2015.09.033.
Author disclosure statement
Conflict of Interest statement. Nothing to declare.
Acknowledgements
This work was supported by the Basque Government's Department
of Education, Universities and Investigation (IT-341-10) and by the
Spanish Ministry of Economy and Competitiveness (SAF2010-19862).
We thank the University of Basque Country (UPV/EHU) for the
grant awarded to Paola S. Apaolaza (PIF09/2009/PIF09122) and the
grants from the Deutsche Forschungsgemeinschaft (DFG) to B.H.F.W.
(WE1259/12-4). Thanks to Kerstin Rückl for valuable and excellent
technical assistance. Technical and human support for TEM, flow
cytometry and CLSM provided by SGIker (UPV/EHU, MINECO, GV/EJ,
ERDF and ESF) is gratefully acknowledged as well.
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