Long-term biophysical stability of nanodiamonds combined with lipid nanocarriers for non-viral gene delivery to the retina

Abstract

Nanodiamonds were combined with niosome, and resulting formulations were named as nanodiasomes, which were evaluated in terms of physicochemical features, cellular internalization, cell viability and transfection efficiency both in in vitro and in in vivo conditions. Such parameters were analyzed at 4 and 25 °C, and at 15 and 30 days after their elaboration. Nanodiasomes showed a particle size of 128 nm that was maintained over time inside the ±10% of deviation, unless after 30 days of storage at 25°C. Something similar occurred with the initial zeta potential value, 35.2 mV, being both formulations more stable at 4°C. The incorporation of nanodiamonds into niosomes resulted in a 4-fold increase of transfection efficiency that was maintained over time at 4 and 25°C. In vivo studies reported high transgene expression of nanodiasomes after subretinal and intravitreal administration in mice, when injected freshly prepared and after 30 days of storage at 4°C.

Full text

International Journal of Pharmaceutics 639 (2023) 122968
Available online 18 April 2023
0378-5173/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Long-term biophysical stability of nanodiamonds combined with lipid
nanocarriers for non-viral gene delivery to the retina
Nuseibah H. AL Qtaish
a
,
b
,
1
, Ilia Villate-Beitia
a
,
b
,
c
,
1
, Idoia Gallego
a
,
b
,
c
, Gema Martínez-
Navarrete
b
,
f
, Cristina Soto-S´
anchez
b
,
f
, Myriam Sainz-Ramos
a
,
b
,
c
, Tania B Lopez-Mendez
a
,
b
,
c
,
Alejandro J. Paredes
d
,
e
, Francisco Javier Chich´
on
g
, Noelia Zamarre˜
no
g
, Eduardo Fern´
andez
b
,
f
,
Gustavo Puras
a
,
b
,
c
,
*
, Jos´
e Luis Pedraz
a
,
b
,
c
,
*
a
NanoBioCel Research Group, Laboratory of Pharmacy and Pharmaceutical Technology. Faculty of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de
la Universidad, 7, 01006 Vitoria-Gasteiz, Spain
b
Networking Research Centre of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Institute of Health Carlos III, Paseo de la Universidad, 7, 01006 Vitoria-
Gasteiz, Spain
c
Bioaraba, NanoBioCel Research Group, 01009 Vitoria-Gasteiz, Spain
d
Research and Development Unit in Pharmaceutical Technology (UNITEFA), CONICET and Department of Pharmaceutical Sciences, Chemistry Sciences Faculty,
National University of C´
ordoba, Haya de la Torre y Medina Allende, X5000XHUA C´
ordoba, Argentina
e
School of Pharmacy, Queen’s University Belfast, Medical Biology Centre, 97, Lisburn Road, Belfast, BT9 7BL Northern Ireland, UK
f
Neuroprothesis and Neuroengineering Research Group, Institute of Bioengineering, Miguel Hern´
andez University, Avenida de la Universidad, 03202 Elche, Spain
g
CryoEM CSIC Facility. Centro Nacional de Biotecnología (CNB-CSIC). Structure of Macromolecules Department. Calle Darwin n◦3, 28049 Madrid, Spain
ARTICLE INFO
Keywords:
Nanodiamond
Niosome
Non-viral
Gene delivery
Stability
Retina
ABSTRACT
Nanodiamonds were combined with niosome, and resulting formulations were named as nanodiasomes, which
were evaluated in terms of physicochemical features, cellular internalization, cell viability and transfection ef-
ciency both in in vitro and in in vivo conditions. Such parameters were analyzed at 4 and 25
◦
C, and at 15 and 30
days after their elaboration. Nanodiasomes showed a particle size of 128 nm that was maintained over time
inside the ±10% of deviation, unless after 30 days of storage at 25 ◦C. Something similar occurred with the
initial zeta potential value, 35.2 mV, being both formulations more stable at 4 ◦C. The incorporation of nano-
diamonds into niosomes resulted in a 4-fold increase of transfection efciency that was maintained over time at 4
and 25 ◦C. In vivo studies reported high transgene expression of nanodiasomes after subretinal and intravitreal
administration in mice, when injected freshly prepared and after 30 days of storage at 4 ◦C.
1. Introduction
The pharmaceutical science has directed considerable efforts to-
wards discovering and developing safe and efcient vectors for gene
therapy purposes. While most studies focus on overcoming specic is-
sues related to conventional gene delivery platforms, such as unpre-
dictability, incompatibility with biological systems or low efciency,
few studies conduct an exhaustive assessment of the storage stability of
gene carriers, a critical quality to achieve both large-scale production
and clinical application (Suzuki et al., 2015).
Nowadays, few gene therapy drugs have been marketed globally, and
most of these products are based on viral vectors (Al Qtaish et al., 2020;
Shahryari et al., 2019). However, because of specic issues associated to
viral gene carriers, including low DNA packing capacity, high costs and
complex production, non-viral vectors are gaining increasing interest
(Do et al., 2019; Ibraheem et al., 2014; Ginn et al., 2018). In addition to
overcoming these specic challenges, non-viral vectors offer high
versatility due to the wide variety of available nanomaterials that can be
used to produce gene delivery systems (Grijalvo et al., 2019; Riley and
Vermerris, 2017). Among these, niosomes have been reported in
repeated occasions as efcient vehicles for gene delivery to brain
(Mashal et al., 2018) and retina (Puras et al., 2015), among others.
* Corresponding authors at: NanoBioCel Research Group, Laboratory of Pharmacy and Pharmaceutical Technology. Faculty of Pharmacy, University of the Basque
Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain.
E-mail addresses: gustavo.puras@ehu.eus (G. Puras), joseluis.pedraz@ehu.es (J.L. Pedraz).
1
These authors contributed equally to this work.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
https://doi.org/10.1016/j.ijpharm.2023.122968
Received 9 December 2022; Received in revised form 6 March 2023; Accepted 14 April 2023

International Journal of Pharmaceutics 639 (2023) 122968
2
Niosomes are cationic lipid nanoparticles with a bilayer distribution
similar to liposomes, but, additionally, niosomes can also contain a
“helper” component and a non-ionic surfactant to obtain more stable
colloidal dispersions. All these mentioned components, provide nio-
somes superior chemical and storage stability than liposomes (Bartelds
et al., 2018; Ojeda et al., 2016). All the components of niosome for-
mulations inuence on their biocompatibility and transfection ef-
ciency. In particular, the characteristics of the “helper” component
inuence directly on relevant biological processes, such as the cellular
uptake and the subsequent intracellular disposition, which are critical
factors that determine successful gene delivery efciency (Ojeda et al.,
2016). Among the most studied “helper” components, lipid-based ones
such as lycopene, cholesterol, squalane, squalene and sphingolipids (Al
Qtaish et al., 2021; Mashal et al., 2017; Ojeda et al., 2016) have been the
most employed to date, but also non-lipid ones such as chloroquine are
gaining interest with encouraging results (Mashal et al., 2019).
Recently, nanodiamonds (NDs) have emerged as an interesting ma-
terial to elaborate non-viral vectors for gene delivery applications. The
high biocompatibility, low toxicity, along with their versatile surface
chemistry (Lim et al., 2016), which allows multiple combination forms
as “helper” components with other nanomaterials such as polymers or
lipids have captured the interest of scientics. NDs are allotropes of
carbon that contain a core diamond crystalline structure and present
unique physicochemical properties, such as almost spherical shape, low
size polydispersity and high specic area. Additionally, NDs can be
easily functionalized with many chemical compounds (Chauhan et al.,
2020). In previous research for gene therapy purposes, authors com-
bined NDs with hydrophilic cationic polymers such as polyethylenimine
800 (PEI 800) (Alhaddad et al., 2011); Chen et al., 2021; Zhang et al.,
2009) and polyallylamine hydrochloride (PAH) (Alhaddad et al., 2011),
or with cationic monomer such as lysine (Alwani et al., 2016) by elec-
trostatic interactions. On the other hand, covalent derivatization of NDs
has been performed with silane-NH
2
groups (Edgington et al., 2018;
Zhang et al., 2009) and polyamidoamine (PANAM) (Lim et al., 2017). In
other study, Bi et al designed and synthetized a complex structure of ND-
CONH(CH
2
)
2
NH-VDGR/survivin-siRNA with antitumoral effect (Bi
et al., 2016). Finally, our research group combined NDs with niosomes,
demonstrating their superiority in enhancing the transfection efciency
of these non-viral vectors (Al Qtaish et al., 2022). However, to the best of
our knowledge, the combination of NDs with niosomes to evaluate their
stability along with their retinal gene delivery efciency has not been
explored yet.
In this work, we prepared and comparatively evaluated the trans-
fection efciency and long-term stability at different storage tempera-
tures of two niosome-based formulations that only differed on the use or
not of NDs as “helper” components. Formulations were based on cationic
lipid N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium chlo-
ride (DOTMA) and non-ionic surfactant polysorbate 20. NDs were added
as “helper” components to one of the two formulations. Resulting for-
mulations were named as niosomes and nanodiasomes depending on
their ND content and were incubated with pCMS-EGFP plasmid in order
to obtain nanocomplexes, named as nioplexes and nanodiaplexes,
respectively. Formulations were evaluated in terms of physicochemical
properties, including size distribution, supercial charge and poly-
dispersity index at different periods of time (0, 15 and 30 days) and
storage temperatures (4 ◦C and 25 ◦C). In addition, in vitro biological
studies were performed to evaluate the toxicity of the formulations
along with their cellular uptake and gene delivery efciency over time at
different storage temperatures in HEK-293 cells. Further assays were
carried out in primary retinal cells and in mice after both intravitreal
and subretinal administration of the formulations in order to determine
the effect of NDs as “helper” components on the gene delivery efciency
and long-term storage of the formulations.
2. Materials and methods
2.1. Preparation of nanodiasome and niosome formulations
For the preparation of the nanodiasome formulations, the water in
oil emulsion technique was used as previously described. Briey. 250
μ
L
of NDs (10 mg/ml in H
2
O, Sigma-Aldrich Madrid, Spain, product ID:
900180) were ultrasonicated for 30 min and mixed with an aqueous
phase composed of 2 mL of 0.5% polysorbate 20 (Sigma-Aldrich Madrid,
Spain) plus 1.75 mL of MilliQ water. On the other hand, 5 mg of DOTMA
(Avanti Polar Lipids, Inc., Alabama, USA) were accurately weighted and
diluted in 1 mL of the organic solvent dichloromethane (DCM) (Panreac,
Barcelona). This oil phase was incorporated into the aqueous phase and
sonicated for 30 s at 50 W (Branson Sonier 250, Danbury). The
emulsion was maintained under magnetic stirring for 2 h at room tem-
perature (RT) until evaporation of DCM to obtain the nanodiasome
formulation. The preparation of the niosome formulation followed the
same procedure, but the aqueous phase did not contain NDs. Fig. 1
summarizes the main components and their disposition in both
formulations.
2.2. Preparation of the nanocomplexes
Nanocomplexes were obtained by incubating both niosomes and
nanodiasomes with the previously propagated pEGFP plasmid, as
described elsewhere (Ojeda et al., 2016), to obtain complexes (nioplexes
and nanodiaplexes, respectively) at 5/1 cationic lipid/DNA ratio (w/w).
2.3. Physicochemical studies
Niosomes, nanodiasomes, and their corresponding complexes were
physicochemically characterized by means of mean particle size, dis-
persity index (ᴆ) and zeta potential, following previously reported
methodology (Mashal et al., 2017).
Microscopy studies were carried out to determine the morphology
and the disposition of NDs in the nanodiasomes, by cryo-electron
tomogram, as previously described (Al Qtaish et al., 2022).
2.4. Biophysical stability studies of formulations
Stability studies were performed with all formulations by means of
physicochemical characterization and biological performance. For that
purpose, particle size, dispersity, zeta potential, cell viability and
transfection were evaluated at 0, 15 and 30 days with stored formula-
tions at 4 ◦C and 25 ◦C. Cellular uptake, transfection in primary retinal
cell cultures, along with in vivo retinal assays were performed with
freshly prepared formulations and with formulations stored at 4 ◦C for
30 days.
2.5. Transfection studies
To perform transfection experiments, human embryonic kidney 293
cell line (HEK-293; ATCC® CRL1573
TM
) was cultured and maintained as
previously described (Ojeda et al., 2016). For this, HEK-293 cells were
seeded at 20 ×10
4
cells per well in 24 well plates and incubated to reach
70% of conuence the next day. After discarding the medium from the
wells, cells were transfected using OptiMEM (Gibco, San Diego, CA,
USA) transfection medium, for 4 h with nioplexes and nanodiaplexes,
from freshly prepared and stored for 15 and 30 days at 4 ◦C and 25 ◦C
formulations, at the cationic lipid/DNA mass ratio 5/1, as previously
reported (Al Qtaish et al., 2022). Positive control of transfection con-
sisted in cells transfected with Lipofectamine 2000™ (Invitrogen,
Carlsbad, CA, USA), while negative control were non-treated cells but in
OptiMEM for 4 h. Each condition was carried out in triplicate.
N.H. AL Qtaish et al.

International Journal of Pharmaceutics 639 (2023) 122968
3
2.6. EGFP expression and cell viability assays
The efciency of the transfection process was assessed both quali-
tative and quantitatively 48 h after the transfection assay. Qualitative
determination of EGFP signal was performed using an inverted uo-
rescence microscope (Eclipse TE2000-S, Nikon). Quantitative studies of
plasmid expression, cell viability and mean uorescence intensity (MFI)
were carried out by ow cytometry using a FACSCalibur system (Becton
Dickinson Bioscience, San Jose, USA), as reported previously (Al Qtaish
et al., 2022).
2.7. Cellular uptake
To analyse the cellular internalization process of nioplexes and
nanodiaplexes, using freshly prepared and stored for 30 days at 4 ◦C
formulations, niosomes and nanodiasomes were condensed with FITC-
labelled pEGFP plasmid. Fluorescence microscopy and ow cytometry
equipment were used to elaborate cellular uptake process in a qualita-
tive and quantitative way, respectively (Al Qtaish et al., 2022).
2.8. Animals and anesthetics
Procedures were performed following the RD 53/2013 Spanish and
2010/63/EU European Union regulations, as well as the Association for
Research in Vision and Ophthalmology (ARVO), once obtained the
approval of the Miguel Hernandez University Standing Committee for
Animal Use in the Laboratory.
2.9. Transfection studies in rat primary central nervous system cell
cultures and immunocytochemistry assays
E17-E18 rat embryos (Sprague Dawley) were employed for the
extraction of primary central nervous system (CNS) cells, from the brain
cortex and retinal tissue. Cells were removed and cultured onto pre-
coated glass coverslips in 24 well plates.
Cortical and retinal cells were transfected with nanodiaplexes from
freshly prepared and 30 days stored nanodiasomes. Lipofectamine™
2000 (ThermoFisher Scientic) was used as a positive control. Trans-
fections experiments were repeated three times for each condition and
GFP expression was analyzed at 96 h after transfection.
Cell xation was carried out with 4% paraformaldehyde for 25 min
and permeabilized using 0.5% Triton X-100 during 5 min. After blocking
with a solution of 10% BSA (v/v) in PBS for 1 h at RT, cells were
incubated with primary antibody chicken anti-EGFP (ThermoFisher
Scientic) overnight at 4 ◦C. Secondary antibody Alexa Fluor 555 goat
anti-chicken IgG (ThermoFisher Scientic) and Hoechst 33,342 (Sigma-
Aldrich, Spain) were applied for 1 h at 4 ◦C. Coverslips were analyzed
by a Zeiss AxioObserver Z1 (Carl Zeiss) microscope equipped with an
ApoTome system and Leica TCS SPE spectral confocal microscope (Leica
Microsystems GmbH, Wetzlar, Germany).
2.10. Intravitreal and subretinal administration of formulations
In vivo transfections of nanodiaplexes were carried in C57BL/6J mice
with freshly prepared (n =10) and 30 days stored nanodiasomes (n =
10). Animals were anesthetized, and intravitreal (n =5) or subretinal (n
=5) injections were administered under microscope (Zeiss OPMI® pico;
Carl Zeiss Meditec GmbH, Jena, Germany) using a Hamilton micro-
syringe with a blunt 34-gauge needle (Hamilton Co., Reno, NV). The
nanodiaplexes solution injected was 0.5
μ
L which contained 100 ng of
EGFP plasmid. As negative controls, the untreated right eyes were used.
EGFP expression was analyzed qualitatively one week after the in-
jection of complexes from freshly or 30 days stored nanodiasomes in
frozen sections of the retina, as previously described (Mashal et al.,
2017). Cryosections were incubated with the primary antibodies
chicken anti-EGFP (ThermoFisher Scientic) and rabbit anti-Iba1
(Abcam) overnight at 4 ◦C. Secondary antibodies Alexa Fluor 488
donkey anti-rabbit and Alexa Fluor 555 goat anti-chicken (both Ther-
moFisher Scientic) were applied for 1 h at 4 ◦C. Nuclei were stained
with Hoechst 33,342 (Thermo Fisher Scientic). The samples were
analyzed and photographed using a Leica TCS SPE spectral confocal
microscope (Leica Microsystems GmbH, Wetzlar, Germany).
2.11. Statistical analysis
Data were analyzed using SPSS 15.0 software. Normality and ho-
mogeneity of variances were evaluated with the Shapiro-Wilk test and
the Levene test, respectively. Students t test or ANOVA followed by post-
Fig. 1. Overview of formulations and their components.
N.H. AL Qtaish et al.

International Journal of Pharmaceutics 639 (2023) 122968
4
hoc HSD Tukey test were employed under parametric conditions. On the
contrary, Kruskal-Wallis test and/or Mann-Whitney U test were used
under non-parametric conditions. In all cases, P value ≤0.05 was
considered statistically signicant. Data were represented as mean ±
standard deviation (SD).
3. Results
3.1. Physicochemical characterization of formulations
In general, formulations containing NDs presented higher mean
particle size values than their counterparts in all conditions (Fig. 2A). At
day 0, freshly prepared nanodiasome formulations showed a mean
particle size of 128.7 ±4.2 nm, which maintained stable over time and
was signicantly increased (P <0.05) only after 30 days of storage at
25 ◦C. Regarding the niosome formulation, the mean particle size at day
0 was 90.5 ±10.3 nm and presented signicant oscillations (P <0.05) at
day 15 of storage at 25 ◦C and at day 30 of storage at 4 ◦C.
The measurement of zeta potential of nanodiasomes and niosomes at
different days and temperatures of storage revealed more oscillations in
the case of niosome formulations than their counterparts (Fig. 2B). The
mean zeta potential value of freshly prepared nanodiasomes at day
0 was 35.2 ±0.3 mV and presented a statistically relevant increase (P <
0.05) after 30 days of storage at both 4 ◦C and 25 ◦C. On the other hand,
niosome formulations showed a mean zeta potential value of 20.2 ±2.5
mV at day 0, which signicantly increased after 15 days of storage at
25 ◦C (P <0.05) and decreased after 30 days of storage at 4 ◦C (P <0.05)
and 25 ◦C (P <0.01).
Dispersity values of nanodiasomes were lower than niosomes and
remained stable with little oscillations at all conditions tested, while
niosome formulations showed higher values and more variations,
especially after being stored during 15 days at 25 ◦C (Fig. 2C).
Nanodiasomes under electron cryo-tomography microscopy
(Fig. 2D) showed a spherical shape, with the NDs integrated in the lipid
layer (Fig. 2D, white arrow).
3.2. Gene delivery efciency and toxicity of nioplexes and nanodiaplexes
The comparative evaluation of cell viability and gene delivery ef-
ciency in cells between nanodiaplexes and nioplexes, prepared with
fresh formulations or with formulations stored for 15 and 30 days at
different temperatures, showed that nanodiaplexes were better tolerated
by cells and achieved signicantly higher transfection rates at all con-
ditions. Data were normalized to Lipofectamine 2000
TM
which reported
39.5 ±12.2% of EGFP expression in live cells, and 79.7 ±14.6% cell
viability in HEK-293 cells (data not shown). The mean percentage of live
cells exposed to freshly prepared nanodiaplexes was 90.79 ±2.5%,
while this value was signicantly lower (P <0.001) for nioplexes which
presented a mean percentage of live cells of 78.8 ±5.8% (Fig. 3A, lines).
These values remained relatively stable over time and different storage
temperatures, with little oscillations but no statistically relevant differ-
ences compared to the values of day 0 in both formulations.
Regarding transfection efciency, the percentage of EGFP expressing
live cells exposed to freshly prepared nanodiaplexes and nioplexes were,
respectively, 89.8 ±3.4% and 23.3 ±1.1% (Fig. 3A, bars). These values
remained stable for both formulations over time and storage conditions,
always maintaining signicantly higher transfection percentages in cells
treated with nanodiaplexes than with nioplexes (P <0.001). In addition,
the MFI data (Fig. 3B) corroborated the advantage of nanodiaplexes over
nioplexes, with signicantly higher MFI values obtained in cells exposed
to nanodiaplexes prepared with nanodiasome formulations at all days
and storage conditions tested (P <0.001). Fig. 3C and D show repre-
sentative uorescence microscopy images of HEK-239 transfected cells
with both formulations at day 0 and after 30 days of storage at 4 ◦C,
respectively.
Fig. 2. Physicochemical characterization and stability of formulations at different days and storage temperature. A. Mean particle size. B. Zeta potential. C. Dis-
persity. Each value shows the mean ±SD of 3 readings. Blue and orange stripes represent ±10% deviation respect to nanodiasomes and niosomes parameters at day
0, respectively. D. Cryo-electron tomogram slice of a nanodiasome; asterisk indicates the aqueous phase; white arrow indicates the lipid layer of the nanodiasome
with nanodiamonds integrated in the lipid structure; black arrow indicates higher densities of the tomogram (more electron-dense material), which correspond to
gold nanoparticles added to the sample for tilt series alignment. Scale bar: 100 nm. (For interpretation of the references to colour in this gure legend, the reader is
referred to the web version of this article.)
N.H. AL Qtaish et al.

International Journal of Pharmaceutics 639 (2023) 122968
5
3.3. Cellular uptake of nioplexes and nanodiaplexes
The analysis of cellular uptake in HEK-293 cells 4 h after exposure to
complexes prepared with fresh and 30 days at 4 ◦C stored nanodiasomes
and niosomes, revealed signicantly higher (P <0.05) cell internaliza-
tion percentages for ND based formulations, at both conditions (Fig. 4A).
These cell uptake percentages remained stable over time for both for-
mulations, with statistically relevant differences. Such values were
normalized to Lipofectamine 2000
TM
which reported 43.5 ±2.7% of
FITC-pEGFP positive cells 4 h after transfection (data not shown).
Fig. 4B shows representative images of cellular uptake in HEK-293 cells
exposed to both formulations at days 0 and 30.
3.4. Gene delivery efciency of nanodiaplexes in rat primary cell cultures
The transfection assay of nanodiaplexes in rat primary retinal cells
with freshly prepared (Fig. 5A) and 30 days stored nanodiasomes at 4 ◦C
(Fig. 5B) showed similar EGFP expression, indicating that the trans-
fection efciency of that formulation maintained stable over a month.
Additionally, the transfection efciency of fresh and 30 days stored
nanodiasomes was also evaluated in another CNS cell type, specically
in rat primary neuronal cell culture, which clearly corroborated the high
gene delivery capacity, by means of EGFP expression, of stored formu-
lations over a month and even 3 months (Supplementary Fig. S1).
Fig. 3. Gene delivery efciency and toxicity of formulations in HEK-293 cells 48 h after transfection with nanodiaplexes and nioplexes at 5/1 cationic lipid/DNA
ratio (w/w) over time at 4˚C and 25˚C. A. Normalized percentages of EGFP positive live cells (bars) and cell viability (dots) obtained by ow cytometry. B. Mean
uorescence intensity values obtained by ow cytometry. Each value represents the mean ±SD of 3 measurements. C-D. Merged images showing EGFP signal in
HEK-293 transfected cells with both complexes at 5/1 lipid/DNA ratio (w/w) at day 0 (C) and after 30 days of storage at 4
◦C (D). Scale bars: 200
μ
m. *** P <0.001;
** P <0.01 for nanodiaplexes vs nioplexes, no negative signicant differences in term of live cells (%) for nioplexes between day 0 and the rest of days and
temperatures; # P <0.05 for nioplexes between day 0 and the rest of days and temperatures; $ P <0.05 for nanodiaplexes at day 30 compared with the rest of days
and temperatures.
Fig. 4. Cellular uptake in HEK-293 cells 4 h after exposure to nanodiasomes and niosomes at 5/1 lipid/DNA ratio (w/w) at day 0 and after 30 days of formulations
storage at 4˚C. A. Normalized percentages of FITC-pEGFP positive live cells after the exposure to these complexes. Each value represents the mean ±SD of 3
measurements. B. Confocal microscopy images. Cell nuclei were colored in blue (DAPI); F-actin in red (Phalloidin); nanodiaplexes and nioplexes in green (FITC).
Scale bars: 50
μ
m.* P <0.05 for nanodiaplexes vs nioplexes. (For interpretation of the references to colour in this gure legend, the reader is referred to the web
version of this article.)
N.H. AL Qtaish et al.

International Journal of Pharmaceutics 639 (2023) 122968
6
3.5. In vivo transfection efciency of nanodiaplexes
Nanodiaplexes, prepared with fresh and stored nanodiasomes, were
administered to the mouse eye through intravitreal (Fig. 6A and C) and
subretinal injections (Fig. 6B and D), and uorescence signal was
detected in different retinal cell layers after one week. Both subretinally
and intravitreally administered nanodiaplexes showed that EGFP
expression colocalized mainly with microglial marker Iba-1 and was also
located in the ganglion cell layer (GCL), as well as in the inner nuclear
layer (INL) with some diffused uorescence signal in the outer nuclear
layer (ONL), and even the retinal pigment epithelial cell layer (RPE)
after subretinal injections (Fig. 6B and D). Results also showed that the
intensity of the uorescence signal was comparable in both transfections
with freshly prepared and 30 days stored formulations. Additionally,
mouse retinal cells tolerated well the exposition to nanodiaplexes, in
terms of cell viability, considering the results reported in the qualitative
analysis.
4. Discussion
The high versatility of non-viral vectors relies on the large variety of
available nanomaterials and preparation methods that can be employed.
Among the wide plethora, NDs have been recognized as powerful tools
to increase the transfection efciency of many non-viral vector systems
due to their unique physicochemical properties, including versatile
surface chemistry and ease of functionalization, together with their high
biocompatibility and low toxicity (Al Qtaish et al., 2022). In addition,
NDs show a favourable particle distribution, being almost spherical in
shape. Interestingly, they can also be easily functionalized with many
chemical compounds, show a high surface area-to-volume ratio, and
their production process can be easily scalable (Krüger et al., 2006; Liu
et al., 2007). As NDs present low stability in suspension, their combi-
nation with niosome formulations could be necessary to provide
enhanced stability, which is necessary for gene delivery applications.
Therefore, in this work we combined NDs with a niosome formulation,
based on a cationic lipid and non-ionic surfactant, obtaining a nal
formulation named nanodiasome in order to assess over time at several
storage temperatures the stability of nanodiasomes compared with
niosomes devoid of NDs. To evaluate the stability of the formulations,
relevant physicochemical parameters that affect to the transfection
process, along with biocompatibility and transfection efciency studies
were performed in in vitro and in vivo conditions.
The physicochemical characteristics constitute key parameters that
determine the biological behaviour of the formulations, including their
cellular internalization process, gene delivery efciency and biocom-
patibility. In the present work, nanodiasomes showed a slightly higher
mean particle size than niosomes at day 0, probably due to the incor-
poration of NDs as additional elements, which might have affected to the
packing of the formulation. The lower dispersity values observed with
nanodiasomes, indicated a more homogeneous particle size distribution
for that formulation compared to the niosome formulation. Both for-
mulations presented statistically relevant oscillations in their physico-
chemical parameters, especially after 30 days being stored at 25 ◦C,
suggesting that these parameters are better preserved if formulations are
kept at 4 ◦C rather than at higher temperatures. Hence, in general terms,
it can be said that nanodiasomes are physicochemically more stable over
time than niosomes. Therefore, NDs integration in the lipid structure of
niosomes is involved in supplying higher stability to the formulation,
probably providing more rigidity, by affecting the arrangement of the
lipid membrane and modifying the rheological and packing behaviour of
the formulation (Sainz-Ramos et al., 2021).
After the evaluation of physicochemical properties, biological in vitro
transfection studies were performed in HEK-293 cells. We found that
transfected cells with nanodiaplexes presented higher cell viability
values than the ones transfected with nioplexes, which suggests that the
formulations based on nanodiasomes are better tolerated by these cells.
These results are in accordance with the previously reported high
biocompatibility and low toxicity of NDs (Krüger et al., 2006; Liu et al.,
2007; Zhang et al., 2009). In addition, the gene delivery efciency of
nanodiaplexes was approximately 4-fold superior than the one of nio-
plexes, and this difference was maintained over time. The MFI values
refer to the quantity of the expressed GFP protein, and also indicated a
higher transfection capacity of nanodiaplexes over nioplexes. In this
sense, it is noteworthy that the number of DNA copies per cell decreased
progressively for nioplexes from day 15, while nanodiaplexes did not
suffer any alteration in this parameter until day 30. Taken all together,
this could suggest that the combination of NDs with niosomes enhances
the stability of the formulation, achieving more consistent and suc-
cessful transfection results over time. To better understand the differ-
ences observed between both formulations, we studied their cellular
uptake at 0 and 30 days after being stored at 4 ◦C. We found statistically
relevant differences in the percentage of cellular uptake between both
Fig. 5. EGFP signal in primary culture of rat retinal transfected cells with freshly prepared (A) and 30 days stored at 4
◦C (B) nanodiasomes at 5/1 lipid/DNA ratio
(w/w). Scale bar: 40 µm. Blue: Hoechst 33,342 (cell nuclei); Green: EGFP. Scale bars: 40
μ
m. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of this article.)
N.H. AL Qtaish et al.

International Journal of Pharmaceutics 639 (2023) 122968
7
formulations, obtaining almost 100% normalized values of cellular up-
take with nanodiasomes and around 70% with niosomes at both storage
conditions, which could support the idea that NDs increase the rigidity
of the niosome formulation, enhancing the cellular entry (Manzanares
and Cena. 2020). This higher cellular uptake could in part explain the
differences in transfection efciency between the two formulations, but
further aspects need also to be taken into account. Traditionally, cellular
endocytosis of non-viral vectors is mediated through the endosomal
pathway, which eventually leads to endosomal vesicles with an acidic
environment and digestive enzymes (Agirre et al., 2015). In these vesi-
cles, the DNA risks of being degraded before reaching the nucleus.
Therefore, DNA endosomal escape becomes a key step in order to ach-
ieve successful gene delivery. In this sense, it has been reported that NDs
are able to escape the endosome connement by rupturing the vesicle
membrane shortly after their cellular uptake (Chu et al., 2015), which
would also contribute to justify the higher gene delivery efciency of
nanodiaplexes compared to nioplexes counterparts. In addition, further
transfection assays in CNS cells, both retinal and neuronal primary cells,
conrmed effective transgene expression after transfection with both
freshly prepared and 30 days stored nanodiaplexes.
Therefore, based on these results, we performed an in vivo assay in
order to determine the gene delivery efciency of nanodiaplexes from
fresh and 30 days stored nanodiasomes formulation in mouse retina.
Formulations were injected through intravitreal and subretinal routes,
which are widely used into the clinic for the treatment of genetically
based retinal disorders (Conley and Naash. 2010). In most cases, after
intravitreal injection, ganglion cell layer of the retina shows high
transgene expression levels (Farjo et al., 2006), which, for instance,
could be interesting for the treatment of glaucoma, a highly prevalent
inherited retinal disorder that causes blindness (Almasieh et al., 2012;
Kachi et al., 2005). On the other hand, the more invasive subretinal
route is useful for transfecting the outer layer of the retina (Almasieh
et al., 2012; Kachi et al., 2005), which would be interesting to face
retinal diseases related to mutations at the photoreceptors and the
retinal pigment epithelium level, such as Leber’s congenital amaurosis,
Stagardt’s disease or retinitis pigmentosa (Lipinski et al., 2013). In the
present study, EGFP signal was found mainly in microglial cells. Such
expression was also located in both, the inner and outer layers of the
retina both after intravitreal and subretinal injection of nanodiaplexes,
which suggest that this formulation is able to efciently diffuse along the
different retinal layers achieving high transgene expression at different
levels, which would be relevant from the therapeutic point of view. In
Fig. 6. In vivo assays showing EGFP signal in mouse retina after intravitreal (IV) (A-C) and subretinal (SR) (B-D) administration of freshly prepared (A-B) and 30 days
stored (C-D) nanodiasomes vectoring EGFP plasmid at 5/1 lipid/DNA ratio (w/w). Blue: Hoechst 33,342 (cell nuclei); Green: EGFP; Red: Iba-1. OS: outer segments;
ONL: outer nuclear layer; INL; inner nuclear layer; GCL: ganglion cell layer. Scale bar: 20
μ
m. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of this article.)
N.H. AL Qtaish et al.

International Journal of Pharmaceutics 639 (2023) 122968
8
addition, results revealed high EGFP expression in vivo after the
administration of 30 days stored formulation, indicating that the storage
of the formulation at 4 ◦C for 30 days does not affect its transfection
efciency.
5. Conclusions
Taken together, the main conclusions of the present work are that (i)
nanodiasomes present higher mean particle size, lower dispersity and
higher zeta potential values than niosomes, (ii) nanodiasomes preserve
more constant their physicochemical parameters over time than nio-
somes and both formulations prefer low temperatures for storage, (iii)
nanodiaplexes present an around 4-fold superior transfection efciency
than nioplexes, in terms of percentage of live transfected cells, although
both maintain their transfection efciency over time, (iv) nanodiaplexes
are more efciently uptaken by HEK-293 cells than nioplexes, (v) high
gene delivery efciency of nanodiaplexes is maintained over time in rat
central nervous primary cell cultures and (vi) also in vivo after subretinal
and intravitreal injection of nanodiaplexes in mouse retina.
CRediT authorship contribution statement
Nuseibah H. AL Qtaish: Investigation, Methodology, Visualization,
Writing – original draft. Ilia Villate-Beitia: Formal analysis, Visuali-
zation, Writing – original draft. Idoia Gallego: Formal analysis, Visu-
alization, Writing – review & editing. Gema Martínez-Navarrete:
Investigation, Visualization, Writing – review & editing. Cristina Soto-
S´
anchez: Investigation, Visualization, Writing – review & editing.
Myriam Sainz-Ramos: Investigation, Writing – review & editing. Tania
B Lopez-Mendez: . Alejandro J. Paredes: . Francisco Javier Chich´
on:
Formal analysis, Data curation. Noelia Zamarre˜
no: Methodology.
Eduardo Fern´
andez: Supervision, Writing – review & editing. Gustavo
Puras: Conceptualization, Supervision, Project administration, Writing
– review & editing. Jos´
e Luis Pedraz: Conceptualization, Supervision,
Project administration, Writing – review & editing, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Data availability
The data that has been used is condential.
Acknowledgements
This work was supported by the Basque Country Government
(Consolidated Groups, IT1448-22 and by CIBER -Consorcio Centro de
Investigaci´
on Biom´
edica en Red- CB06/01/1028, Instituto de Salud
Carlos III, Ministerio de Ciencia e Innovaci´
on. Authors wish to thank:
ICTS “NANBIOSIS”, specically the Drug Formulation Unit (U10) of the
CIBER in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)
for the intellectual and technical assistance. Authors also thank SGIker
(UPV/EHU) for technical and human support. Authors acknowledge
Rocio Arranz, access to the cryoEM CSIC facility in the context of the
CRIOMECORR project (ESFRI-2019-01-CSIC-16). I.V.B. thanks the
University of the Basque Country (UPV/EHU) for the granted post-
doctoral fellowship (call for the Specialization of Doctor Researcher
Personnel of the UPV/EHU, grant reference: ESPDOC19/47). M.S.R.
thanks the University of the Basque Country (UPV/EHU) for the granted
pre-doctoral fellowship (PIF17/79).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2023.122968.
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