In vivo targeted delivery of nucleic acids and CRISPR genome editors enabled by GSH-responsive silica nanoparticles

Article abstract of DOI:10.1016/j.jconrel.2021.06.030

The rapid development of gene therapy and genome editing techniques brings up an urgent need to develop safe and efficient nanoplatforms for nucleic acids and CRISPR genome editors. Herein we report a stimulus-responsive silica nanoparticle (SNP) capable of encapsulating biomacromolecules in their active forms with a high loading content and loading efficiency as well as a well-controlled nanoparticle size (~50 nm). A disulfide crosslinker was integrated into the silica network, endowing SNP with glutathione (GSH)-responsive cargo release capability when internalized by target cells. An imidazole-containing component was incorporated into the SNP to enhance the endosomal escape capability. The SNP can deliver various cargos, including nucleic acids (e.g., DNA and mRNA) and CRISPR genome editors (e.g., Cas9/sgRNA ribonucleoprotein (RNP), and RNP with donor DNA) with excellent efficiency and biocompatibility. The SNP surface can be PEGylated and functionalized with different targeting ligands. In vivo studies showed that subretinally injected SNP conjugated with all-trans-retinoic acid (ATRA) and intravenously injected SNP conjugated with GalNAc can effectively deliver mRNA and RNP to murine retinal pigment epithelium (RPE) cells and liver cells, respectively, leading to efficient genome editing. Overall, the SNP is a promising nanoplatform for various applications including gene therapy and genome editing.

Full text of DOI:10.1016/j.jconrel.2021.06.030

Journal of Controlled Release 336 (2021) 296–309
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
In vivo targeted delivery of nucleic acids and CRISPR genome editors
enabled by GSH-responsive silica nanoparticles
,
,
,
,
,
Yuyuan Wanga b, Pawan K. Shahic d, Xiuxiu Wanga b, Ruosen Xieb e, Yi Zhaoa b, Min Wua,
Seth Rogea, Bikash R. Pattnaikc ,f, Shaoqin Gonga
,
d
,b,d,e,g,*
a Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA
b Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI 53715, USA
c Department of Pediatrics, University of Wisconsin-Madison, Madison, WI 53706, USA
d McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53705, USA
e Department of Materials Science and Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA
f Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA
g Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53715, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
The rapid development of gene therapy and genome editing techniques brings up an urgent need to develop safe
and efficient nanoplatforms for nucleic acids and CRISPR genome editors. Herein we report a stimulus-responsive
silica nanoparticle (SNP) capable of encapsulating biomacromolecules in their active forms with a high loading
content and loading efficiency as well as a well-controlled nanoparticle size (~50 nm). A disulfide crosslinker
was integrated into the silica network, endowing SNP with glutathione (GSH)-responsive cargo release capability
when internalized by target cells. An imidazole-containing component was incorporated into the SNP to enhance
the endosomal escape capability. The SNP can deliver various cargos, including nucleic acids (e.g., DNA and
mRNA) and CRISPR genome editors (e.g., Cas9/sgRNA ribonucleoprotein (RNP), and RNP with donor DNA) with
excellent efficiency and biocompatibility. The SNP surface can be PEGylated and functionalized with different
targeting ligands. In vivo studies showed that subretinally injected SNP conjugated with all-trans-retinoic acid
(ATRA) and intravenously injected SNP conjugated with GalNAc can effectively deliver mRNA and RNP to
murine retinal pigment epithelium (RPE) cells and liver cells, respectively, leading to efficient genome editing.
Overall, the SNP is a promising nanoplatform for various applications including gene therapy and genome
editing.
Silica nanoparticle
Gene delivery
CRISPR-Cas9 genome editing
1. Introduction
nucleic acid complexes such as RNP or RNP together with single-
stranded oligonucleotide DNA (i.e., RNP + ssODN) for genome editing
Safe and efficient delivery of biomacromolecules (e.g., nucleic acids
and CRISPR ribonucleoproteins (RNPs)) to target cells is of great
importance, yet it remains a challenge. Nucleic acids, including DNA
and mRNA, are widely used for gene therapy because of their relatively
rapid and safe protein production [1–3]. CRISPR-Cas9 RNPs can achieve
genome editing by introducing gene deletion, correction, and/or inser-
tion with high efficiency and specificity [4–6]. However, under physi-
ological conditions, naked nucleic acids and RNPs are prone to
enzymatic degradation. Moreover, the transfection/gene editing effi-
ciency is negligible due to the lack of cellular uptake and endosomal
escape capability [7–10]. In addition, efficient delivery of protein/
is hindered by its heterogenous charges and complicated structures
[10,11].
Non-viral nanovectors, including lipid-based [12–16] nanoparticles
(NPs), polymer-based NPs [17–20] and functionalized inorganic NPs
[21–27] have been actively investigated for the delivery of bio-
macromolecules. However, current state-of-the-art non-viral nano-
vectors often suffer a number of limitations, including low payload
encapsulation content/efficiency, high cytotoxicity, insufficient in vivo
stability, and/or relatively large nanoparticle sizes (typically around
100 nm) [28–30]. To overcome these limitations, we came up with a set
of design criteria of a desirable biomolecule nanocarrier including: (1)
* Corresponding author at: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53715, USA.
E-mail address: shaoqingong@wisc.edu (S. Gong).
https://doi.org/10.1016/j.jconrel.2021.06.030
Received 3 March 2021; Received in revised form 1 June 2021; Accepted 21 June 2021
Available online 23 June 2021
0168-3659/© 2021 Elsevier B.V. All rights reserved.

Y. Wang et al.
Journal of Controlled Release 336 (2021) 296–309
high loading content and loading efficiency, while maintaining the
payload activity, (2) small NP size (e.g., hydrodynamic diameter < 50
nm), (3) versatile surface chemistry (e.g., ligand conjugation) to facili-
tate the payload delivery to target cells, (4) excellent biocompatibility,
(5) efficient endo/lysosomal escape capability, (6) rapid payload release
in the target cells, and (7) ease of handling, storage, and transport.
Silica-based delivery platforms have been extensively investigated
due to their unique characteristics, such as high stability, multi-
functionality, biocompatibility and versatility in chemistry [31,32]. The
most studied ones are mesoporous silica nanoparticles, which have been
developed for biomacromolecule delivery, but the payloads are mostly
exposed on the nanoparticle surface and subject to premature release or
degradation [33]. Considering most biomacromolecules have good sol-
ubility in an aqueous solution but not in organic solvents, we sought to
use a water-in-oil microemulsion method to synthesize a biodegradable
silica nanoparticle (SNP), which can encapsulate and protect the pay-
loads before reaching the target cells, but can also rapidly release the
payloads intracellularly through stimulus-triggered NP degradation.
Furthermore, when appropriate surfactants, oil phase, water phase and
nanoparticle formulations are chosen, various hydrophilic payloads can
be encapsulated into SNP with high loading content and efficiency via in
situ SNP formulation [34,35]. The size and polydispersity of the final
SNP can be controlled by emulsification conditions (e.g., water to oil
ratio and type/ratio of surfactant) [34,36], while functional moieties
can be integrated into the SNP by the addition of silica reactants, making
the SNP a versatile nanoplatform [35,37].
SNP treated mice was observed. Overall, this multifunctional SNP is an
efficient, biocompatible, and versatile nanoplatform for targeted de-
livery of a broad range of biomacromolecule cargos both in vitro and in
vivo.
2. Experimental section
2.1. Materials
Tetraethyl orthosilicate (TEOS), 1H-imidazole-4-carboxylic acid,
thionyl chloride (SOCl2), Traut’s reagent (2-iminothiolane), Triton X-
100, acetone, ethanol, glutathione (GSH), 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) and ammonia (30% in
water) were purchased from Fisher Scientific, USA. Hexanol, cyclo-
hexane, and (3-aminopropyl)triethoxysilane (APTES) were bought from
Tokyo Chemical Industry Co., Ltd., USA. Triethylamine (TEA) and
dimethyl sulfoxide (DMSO) were purchased from Alfa Aesar, USA. Bis[3-
(triethoxysilyl)propyl]-disulfide (BTPD) was purchased from Gelest,
Inc., USA. Methoxy-poly(ethylene glycol)-silane (mPEG-silane, Mn
5000), amine-poly(ethylene glycol)-silane (NH2-PEG-silane, Mn = 5000)
and maleimide-poly(ethylene glycol)-silane (Mal-PEG-silane, Mn
=
=
5000) were purchased from Biochempeg Scientific Inc., USA. All-trans-
retinoic acid (ATRA) and 4-aminophenyl 2-acetamido-2-deoxy-β-D-
glucopyranoside were purchased from Santa Cruz Biotechnology, USA.
Nuclear localization signal (NLS)-tagged Streptococcus pyogenes Cas9
nuclease (sNLS-SpCas9-sNLS) was obtained from Aldevron, USA. In vitro
transcribed single guide RNAs (sgRNAs) and ssODNs were purchased
from Integrated DNA Technologies, Inc., USA.
Herein, we report the facile fabrication of a sub-50 nm, multifunc-
tional and stimuli-responsive SNP via a water-in-oil microemulsion
method. A disulfide bond-containing crosslinker was integrated into the
SNP network to achieve glutathione (GSH)-responsive degradation and
cargo release in the cytosol. Functional moieties including imidazole
capable of facilitating endosomal escape can be incorporated into SNP-
during the fabrication process. The surface of SNP can be further tuned
with charges from negative to positive, or conjugated with poly
(ethylene glycol) (PEG). PEG can shield the surface charge of the SNP,
enhance its in vivo stability and circulation time, and allow versatile
ligand conjugations onto the SNP, which is necessary for targeted de-
livery [38]. This multifunctional SNP can encapsulate various bio-
macromolecules (i.e., DNA, mRNA, Cas9 RNP and Cas9 RNP with single-
stranded oligonucleotide DNA (ssODN)) with superior loading content
and loading efficiency, independent of payload types or surface charges.
The gene transfection or editing efficiencies of the multifunctional SNP
for the delivery of nucleic acids (i.e., DNA and mRNA) and CRISPR gene
editors (e.g., Cas9 RNP and RNP + ssODN) were systematically
investigated.
2.2. Synthesis of N-(3-(triethoxysilyl)propyl)-1H-imidazole-4-
carboxamide (TESPIC)
A mixture of 1H-imidazole-4-carboxylic acid (250 mg, 1.9 mmol)
and SOCl2 (4 mL) was refluxed at 75 ◦C overnight. The reaction mixture
was then cooled down to room temperature (RT) and added into
anhydrous toluene (20 mL). The precipitate was collected by filtration
and vacuum-dried to yield the intermediate, 1H-imidazole-4‑carbonyl
chloride. The as-prepared 1H-imidazole-4‑carbonyl chloride was sus-
pended in anhydrous THF (5 mL), followed by the addition of triethyl-
amine (232 mg, 2.3 mmol) and APTES (420 mg, 1.9 mmol). The mixture
was stirred at RT overnight under a nitrogen atmosphere, and then
filtered. The solvent was removed by rotary evaporation to yield the
final product TESPIC. Since the silica reactants have the tendency to
undergo hydrolysis/polymerization during column purification, TESPIC
was synthesized and used without purification [37,45]. 1H NMR (400
MHz, DMSO-D6): δ 0.62 (dd, 2H, J = 14.6, 6.2 Hz), δ 1.12 (t, 9H, J = 7.0
Hz), δ 1.60 (dt, 2H, J = 15.9, 8.0 Hz), δ 2.70 (m, 2H), δ 3.83 (q, 6H, J =
6.0 Hz), δ 7.00 (s, 1H), δ 7.40 (s, 1H) ppm. 13C NMR (100 MHz, DMSO-
D6): δ 166, 137, 134, 128, 58, 43, 23, 18, and 7.6 ppm.
For in vivo study, we proved efficient delivery of mRNA and Cas9
RNP in the retinal pigment epithelium (RPE) using SNP in Ai14 trans-
genic mice. As RPE plays an important role in neuroretina survival and
visual function, the dysfunction of RPE will result in various eye diseases
[39,40]. To enhance the RPE-specific internalization, a targeting ligand,
all-trans-retinoic acid (ATRA) was conjugated on SNP-PEG (i.e., SNP-
PEG-ATRA). ATRA binds to the inter-photoreceptor retinoid-binding
protein that selectively transports all-trans-retinol to the RPE [41,42].
SNP-PEG-ATRA can efficiently deliver both mRNA and RNP into RPE in
transgenic Ai14 mice, leading to high genome editing.
2.3. Preparation of GalNAc-PEG-silane
A mixture of 4-Aminophenyl 2-Acetamido-2-deoxy-β-D-glucopyr-
anoside (37.5 mg, 0.12 mmol) and Traut’s reagent (2-iminothiolane,
13.8 mg, 0.1 mmol) was dissolved in anhydrous DMSO (2 mL) and
stirred at RT for 24 h. Thereafter, a portion of Mal-PEG-silane (500 mg,
0.1 mmol) dissolved in 10 mL anhydrous DMSO was added to the above
solution. After 24 h, the reaction was terminated by precipitation into
diethyl ether (100 mL). The precipitate was washed twice with diethyl
ether, and vacuum dried to obtain the product GalNAc-PEG-silane
without further purification. 1H NMR (400 MHz, DMSO-D6) spectrum
of GalNAc-PEG-silane was shown in Fig. S1.
SNP can also effectively deliver mRNA and genome editors in adult
Ai14 mice through systemic administration routes. Without any tar-
geting moieties, SNP-PEG showed a preferential delivery to liver. To
further enhance the liver targeting capability, a targeting ligand, N-
acetylgalactosamine (GalNAc) was conjugated onto SNP-PEG (i.e., SNP-
PEG-GalNAc), as GalNAc is known for its ability to bind with higher
selectivity to the asialoglycoprotein receptors (ASGPRs) overexpressed
on hepatocytes [43,44]. SNP-PEG-GalNAc showed a 2-fold higher
mRNA and RNP delivery efficiency in liver than non-targeted SNP-PEG
according to the tdTomato expression levels induced by genome editing.
Additionally, no significant change in the blood biochemical profile of
297

Y. Wang et al.
Journal of Controlled Release 336 (2021) 296–309
2.4. Preparation of the GSH-responsive silica nanoparticles (SNPs)
detection angle with a sample concentration at 0.1 mg/mL. The
morphology of DNA-loaded SNP-PEG was characterized by transmission
electron microscopy (TEM, Tecnai 12, Thermo Fisher, USA).
SNPs were synthesized by a water-in-oil microemulsion method.
Triton X-100 (1.8 mL) and hexanol (1.8 mL) were dissolved in cyclo-
To calculate the loading content and loading efficiency of the pay-
hexane (7.4 mL) to form the oil phase. Separately, 30
μ
L of an aqueous
loads in the SNPs, SNPs were re-suspended in water (1 mg/mL, 40
μ
L)
solution containing the desired payload (e.g., DNA, mRNA, RNP or RNP
+ ssODN) (5 mg/mL) were mixed with TEOS (3.1 L, 14 mol), BTPD (6
mol) for imidazole incorporation
and incubated with 0.1 M GSH aqueous solution (pH 7.4, 160 μL) with a
μ
μ
pH of 7.4 for 1 h to allow for complete release of the payload. The RNP
loading contents and loading efficiencies were measured via a bicin-
choninic acid assay (BCA assay, Thermo Fisher, USA). DNA and mRNA
loading contents and loading efficiencies were quantified using a
μ
L, 13 μmol) and TESPIC (1 mg (i.e., 3 μ
at a 10% molar ratio, or 2 mg for 20% molar ratio). After shaking, this
mixture was added to the oil phase (1.1 mL), and then the water-in-oil
microemulsion was formed by vortex for 1 min. Under stirring (1500
NanoDrop One (Thermo Fisher, USA) by measuring OD260
.
rpm), an aliquot of 30% aqueous ammonia solution (5 μL) was added
and the water-in-oil microemulsion was under stirring at RT for 4 h to
obtain unmodified SNP with negative surface charge. To prepare posi-
tively charged SNP (SNP-NH2), the as-prepared SNP was modified with
amine groups by the addition of APTES to the microemulsion, and the
mixture was stirred vigorously for another 4 h at RT. To purify un-
modified SNP or SNP-NH2, acetone (1.5 mL) was added in the micro-
emulsion in order to precipitate the SNPs, and the precipitate was
recovered by centrifugation and was subsequently washed twice with
ethanol and three times with water. The purified SNP or SNP-NH2 were
finally collected by centrifugation.
2.8. Cell culture for in vitro studies
Human embryonic kidney cells (i.e., HEK 293 cells) were used for in
vitro studies. HEK 293 cells were purchased from ATCC. Green fluo-
rescence protein (GFP)-expressing HEK 293 cells were bought from
GenTarget Inc. Blue fluorescence protein (BFP)-expressing HEK 293
cells generated through lentiviral transduction of a BFP dest clone was
obtained from Addgene [46]. All HEK 293 cells were cultured with
DMEM medium (Gibco, USA) added with 10% (v/v) fetal bovine serum
(FBS, Gibco, USA) and 1% (v/v) penicillin–streptomycin (Gibco, USA).
Cells were cultured in an incubator (Thermo Fisher, USA) at 37 ◦C with
5% carbon dioxide at 100% humidity.
2.5. Preparation of PEGylated SNP (SNP-PEG) and GalNAc-conjugated
SNP (SNP-PEG-GalNAc)
2.9. DNA and mRNA transfection efficiency study
The as-prepared, unmodified SNP (2 mg) was re-dispersed in 2 mL
water. An aliquot of mPEG-silane (for neutral surface charge, 200 μg), or
A red fluorescence protein (RFP)-expressing plasmid DNA (i.e., RFP-
DNA, Addgene #40260, USA) and an RFP-mRNA (Trilink Bio-
technologies #L-7203, USA) were used for DNA and mRNA transfection
studies, respectively. HEK 293 cells were placed into 96-well plates 24 h
prior to treatment, at a density of 15,000 cells/well. Cells were incu-
bated with either RFP-DNA-loaded SNPs, or RFP-mRNA-loaded SNPs. A
commercially available transfection agent, Lipofectamine 2000 (Lipo
2000), was used as the positive control. The dosage of DNA or mRNA
was 200 ng/well. The Lipo 2000-DNA (or Lipo 2000-mRNA) complex
was prepared following the protocols provided by the manufacturer,
NH2-PEG-silane (40
μ
g) + mPEG-silane (160
μ
g) (for subsequent ATRA
conjugation), or GalNAc-PEG-silane (80
μ
g) + mPEG-silane (120
μ
g) (for
SNP-PEG-GalNAc) was added to the above mixture. The pH of the so-
lution was adjusted to 8 using 0.1 M NaOH solution. The solution was
stirred at RT for 4 h. The resulting SNP-PEG, SNP-PEG-NH2, or SNP-PEG-
GalNAc was purified by washing with water for three times and
collected by centrifugation. To study the serum stability of SNP-PEG,
freshly prepared SNP-PEG was dispersed in fetal bovine serum (FBS)
at a concentration of 0.5 mg/mL. The nanoparticle dispersion was
incubated at 37 ◦C. At each time point, a portion of the SNP-PEG
dispersion (0.2 mL) was collected, the SNP-PEG was harvested by
centrifugation and re-dispersed in DI water for nanoparticle size analysis
by DLS. For stability test, mRNA-encapsulated SNP-PEG were redis-
persed in DI water with SNP concentration of 1 mg/mL and stored at
different temperatures (i.e., 4 ◦C, ꢀ 20 ◦C and ꢀ 80 ◦C); RNP encapsu-
lated SNP-PEG were redispersed in RNP storage buffer (20 mM HEPES-
NaOH pH 7.5,150 mM NaCl,10% glycerol), flash frozen in liquid ni-
trogen, and stored at ꢀ 80 ◦C.
with a final dosage of Lipo 2000 at 0.5
μ
L per well. An untreated group
was used as the negative control. After 48 h, cells were harvested with
0.25% trypsin-EDTA, spun down and resuspended in 500
μL PBS. RFP
expression efficiencies were obtained with a flow cytometer and
analyzed with FlowJo 7.6.
To study the stability of DNA encapsulated SNPs in the presence of
GSH, transfection experiments were carried out under similar conditions
as described above except GSH was intentionally added to the cell cul-
ture media with a GSH concentration ranging from 0.001 to 10 mM.
2.6. Preparation of ATRA-conjugated SNP (SNP-PEG-ATRA)
2.10. RNP genome-editing efficiency study
SNP-PEG-ATRA was synthesized via EDC/NHS catalyzed amidation.
Payload-encapsulated SNP-PEG-NH2 (1 mg) was re-dispersed in 0.5 mL
For gene editing studies, GFP-expressing HEK 293 cells were used as
an RNP delivery cell model. RNP was prepared by mixing sNLS-SpCas9-
sNLS and sgRNA (GFP protospacer: 5′-GCACGGGCAGCTTGCCGG-3′) at
1:1 in molar ratio. Cells were seeded at a density of 5,000 cells per well
onto a 96-well plate 24 h before treatment. Cells were treated with RNP-
DI water. EDC (15
in 10
μg), NHS (9 μg) and a DMSO solution of ATRA (12 μg
μ
L DMSO) were added to the above solution. The solution was
stirred at RT for 6 h, and then the resulting SNP-PEG-ATRA was washed
by water three times and collected by centrifugation. The size and zeta-
potential change before and after ATRA conjugation onto the surface of
SNP is summarized in Table S1.
loaded SNP, RNP-complexed Lipo 2000 (0.5 μL/well) or RNP-complexed
Lipofectamine CRISPRMAX (CRISPRMAX). Untransfected cells, as well
as cells treated with negative control SNP (i.e., SNPs encapsulating an
RNP with a negative control sgRNA (Alt-R CRISPR-Cas9 Negative Con-
trol crRNA #1, Integrated DNA Technologies, Inc., USA)) were used as
controls. For each treatment, the RNP dosage was kept at 150 ng/well,
with an equivalent Cas9 protein dosage at 125 ng/well.
2.7. Characterization
The chemical structure of TESPIC was confirmed by nuclear mag-
netic resonance (NMR) spectroscopy (Avance 400, Bruker Corporation,
USA). The Fourier transform infrared (FTIR) spectra were recorded on a
Bruker Tensor 27 FTIR spectrometer. The hydrodynamic diameters and
zeta potentials of the SNPs were characterized by a dynamic light
scattering (DLS) spectrometer (Malvern Zetasizer Nano ZS) at a 90◦
The T7 Endonuclease I (T7E1) indel detection assay (#E3321, New
England Biolabs, USA) was performed following the protocol from
manufacturer. Briefly, genomic DNA of GFP-HEK cells was extracted
using Monarch Genomic DNA Purification Kit (#T3010, New England
Biolabs, USA) according to the manufacturer’s instructions. The sgRNA
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Y. Wang et al.
Journal of Controlled Release 336 (2021) 296–309
targeted genomic locus was amplified with Phusion Hot Start II High
Fidelity DNA Polymerase (Thermo Fisher, USA) using the designed
primers (forward primer: 5′-CGCCCCATTGACGCAAATGGGCGGTAG-3′;
Reverse primer: 5′-GCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAG-
GATG-3′). The amplicons were then treated with T7E1 and incubated at
37 ◦C for 30 min. The digested DNA was analyzed using 2% agarose gel
electrophoresis. Indel formation efficiencies were calculated using
Image J.
volume was 2
μ
L, containing 4
μ
g RNP. SNP-PEG-ATRA was injected into
the subretinal space using a UMP3 ultramicro pump fitted with a
NanoFil syringe, and the RPE-KIT (all from World Precision Instruments,
Sarasota, FL) equipped with a 34-gauge beveled needle. The tip of the
needle remained in the bleb for 10 s after bleb formation, then it was
gently withdrawn.
Four days (for mRNA delivery studies) or fourteen days (for RNP
delivery studies) post subretinal injection, mice were sacrificed, and
eyes were collected for tdTomato expression evaluation. Collected eyes
were rinsed twice with PBS and puncture was made at ora serrata with
an 18-gauge needle. The eye was opened along the corneal incisions and
the eyecup was incised radially to the center and flattened to give a final
floret shape. The RPE layer was then separated and flat-mounted on a
cover-glass slide (i.e., RPE floret). RPE florets were imaged with NIS-
Elements using a Nikon C2 confocal laser scanning microscope (CLSM).
For gene correction studies, BFP-expressing HEK 293 cells were
employed as a model cell line [46]. The RNP + ssODN mixture was
prepared by simply mixing the as-prepared BFP gene-targeting RNP
(BFP protospacer: 5′-GCTGAAGCACTGCACGCCAT-3′) and single-
stranded oligonucleotide DNA (ssODN) (BFP to GFP ssODN sequence:
5′- CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC-
CACCCTCGTGACCACCCTGACGTACGGCGTGCAGTGCTTCAGCCGC-
TACCCCGACCACATGA-3′, changing BFP to GFP via alternation of
histidine to tyrosine) donor template at 4 ◦C for 5 min at a 1:1 molar
ratio [46,47]. Gene correction will lead to the replacement of three
nucleotides in the genome converting BFP to GFP, and thus the per-
centage of GFP positive cells can be used to evaluate the genome editing
efficiency [47,48]. BFP-expressing HEK 293 cells were seeded at a
density of 5,000 cells per well onto a 96-well plate 24 h before treat-
ment. Cells were treated with RNP + ssODN-loaded SNP or Lipo 2000
2.13. Intravenous injection
Ai14 mice (6–8 weeks; three mice in each group) were injected with
Cre-mRNA (20 μg per mouse) or RNP (100 μg per mouse)-encapsulated
SNP-PEG or SNP-PEG-GalNAc through retro-orbital injections; PBS
injected Ai14 mice were used as controls. The SNP-injected and control
mice were sacrificed 3 days (Cre mRNA) or 7 days (RNP) post-injection.
Organs and tissues (liver, heart, lung, spleen, kidney and muscle) were
then collected and analyzed.
(0.5 μL/well) carrying RNP + ssODN as the positive control. For each
treatment, the RNP dosage was kept at 150 ng/well (i.e., an equivalent
Cas9 protein dosage of 125 ng/well), and the ssODN dosage was 25 ng/
well. The precise genome editing efficiencies were quantified six days
after treatment using flow cytometry by counting the percentage of
green fluorescence positive cells. Data were analyzed with FlowJo 7.6.
Fresh organs/tissues were imaged using the in vivo imaging system
(IVIS Lumina system, Perkin Elmer) for tdTomato expression. A portion
of the liver samples were weighed and homogenized with cell lysis
buffer as reported previously [50]. Briefly, the liver tissues were washed
with chilled 1× PBS, cut into small pieces and accurately weighed. RIPA
buffer with protease inhibitor (cOmplete™ Protease Inhibitor Cocktail,
Sigmaaldrich, USA) was added to the liver tissue with 1 mL RIPA buffer
for every 50 mg tissue. The liver tissue samples were homogenized on ice
by a sonicate homogenizer for 5 min at a power of 150 watts with 10 s
sonication/10 s rest cycles, and kept on ice for 1 h with occasional
vortex. The tissue lysate was then centrifuged at 15,000 xg for 30 min at
4 ◦C to yield pellet of cell debris. The supernatant was transferred to a
fresh microfuge tube without disturbing the pellet. The protein con-
centration of the lysate was determined by the BCA protein assay and
kept the same prior to IVIS analysis. The homogenized liver samples
were then added to a 96-well black/clear flat bottom Imaging Micro-
plate (Corning Life Science, USA). Thereafter, the tdTomato fluores-
cence was measured and analyzed by the IVIS system.
2.11. Cell viability assay
The cytotoxicity of SNPs was studied by an MTT assay. Cells were
treated with complete medium, DNA-complexed Lipo 2000 (0.5 μL/
well), and DNA-loaded SNP-PEG with concentrations ranging from 10 to
1000 g/mL. Cell viability was measured using a standard MTT assay 48
h after treatment (Thermo Fisher, USA). Briefly, cells were treated with
media containing 500 g/mL MTT and incubated for 4 h. Then, the MTT-
containing media was aspirated, and the purple precipitate was dis-
μ
μ
solved in 150 μL of DMSO. The absorbance at 560 nm was obtained with
a microplate reader (GloMax® Multi Detection System, Promega, USA).
2.12. Subretinal injection
2.14. Immunofluorescence staining
All animal research was approved by UW-Madison animal care and
use committee. Ai14 reporter mice (obtained from The Jackson Labo-
ratory) were used to assess the mRNA delivery/genome editing effi-
ciency induced by mRNA- or RNP-encapsulated SNP-PEG-ATRA,
respectively. Cre-mRNA was purchased from Trilink Biotechnologies,
USA (#L-7211). RNPs were prepared using either a sgRNA targeting the
stop cassette composed of 3× SV40 polyA blocks (protospacer: 5′-
AAGTAAAACCTCTACAAATG-3′) in Ai14 mice, or a mouse negative
control sgRNA (Alt-R CRISPR-Cas9 Negative Control crRNA #1, Inte-
grated DNA Technologies, Inc., USA). Subretinal injection and subse-
quent RPE tissue collection were performed as reported previously [49].
Mice were maintained under tightly controlled temperature (23 ± 5 ◦C),
humidity (40–50%), and light/dark (12/12 h) cycle conditions in a 200-
lx light environment. The mice were anesthetized by intraperitoneal
injection of ketamine (80 mg/kg), xylazine (16 mg/kg) and aceproma-
zine (5 mg/kg) cocktail. For mRNA delivery studies, right eyes of mice
Tissues were fixed in 4% paraformaldehyde (PFA) at RT for 24 h,
then switched to PBS solution containing 30% sucrose and stored at 4 ◦C
for 72 h. Thereafter, the tissues were embedded in Tissue-Tek® Optimal
Cutting Temperature Compound (Sakura Finetek, USA), and frozen in
dry ice. The blocks were sectioned using a cryostat machine (CM1900,
Leica Biosystems, USA) at 8 μm thickness and mounted on microscope
slides. The sections were incubated in 10% goat serum and 0.3% Trixon
X-100 in PBS at RT for 1 h. For immunofluorescence staining, the sec-
tions were first incubated with a rabbit anti-tdTomato primary antibody
(ab152123, 1:5000, Abcam, USA) for 1 h at RT. The primary antibody
was then detected by a fluorescence-conjugated secondary antibody
(goat anti-rabbit IgG H&L (Alexa Fluor® 594), ab150080, 1:1000,
Abcam, USA). Finally, the slides were mounted with DAPI and covered
with microscope cover glasses. All of the images were acquired using
CLSM.
were injected with mRNA-encapsulated SNP-PEG-ATRA (2 μL with 4 μg
mRNA), and left eyes were injected with PBS. For RNP delivery studies,
right eyes of mice were injected with SNP-PEG-ATRA encapsulating RNP
with a sgRNA targeting the Ai14 stop cassette (i.e., Ai14 SNP), left eyes
of mice were injected with SNP-PEG-ATRA encapsulating RNP with a
negative control sgRNA (i.e., negative control SNP). The injection
2.15. Blood biochemical profile
Blood samples were immediately collected from the orbital sinus of
each mouse from the SNP-treated groups or PBS control groups and
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Journal of Controlled Release 336 (2021) 296–309
centrifuged at 1500 xg and 4 ◦C for 10 min for serum preparation.
Clinical biochemical assessment of levels of blood urea nitrogen (BUN),
creatinine (CRE), alanine aminotransferase (ALT), aspartate amino-
transferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL),
glucose (GLU), Calcium (CA), total protein (TP), albumin (ALB), glob-
ulin (GLOB), Na+, K+, Clꢀ and total carbon dioxide (tCO2) was per-
formed using VetScan Preventative Care Profile Plus rotors (Abaxis,
USA) in a VetScan VS2 chemistry analyzer (Abaxis, USA).
bis[3-(triethoxysilyl)propyl]-disulfide (BTPD), was incorporated into
the silica network to yield the GSH-responsive SNP. To enhance endo-
somal escape, a imidazole-containing silica reagent, N-(3-(triethox-
ysilyl)propyl)-1H-imidazole-4-carboxamide (TESPIC), was added to the
microemulsion. The Fourier transform infrared (FTIR) absorption
spectra confirmed the successful incorporation of imidazole groups and
disulfide crosslinkers (Fig. S2) [51,52]. The SNP surface can be modified
with amine groups by addition of (3-aminopropyl)triethoxysilane
(APTES) to yield positively-charged SNP (i.e., SNP-NH2). The as-
prepared, unmodified SNP or SNP-NH2 were purified by precipitation
in acetone, and washed by ethanol and deionized (DI) water. Unmodi-
fied SNP can be PEGylated to obtained neutral surface charge. The liver-
targeting SNP (i.e., SNP-PEG-GalNAc) was prepared by simultaneous
addition of silane-PEG-GalNAc and silane-mPEG. The RPE-targeting
ligand, ATRA, was conjugated to the distal ends of the surface PEG
through amidation.
2.16. Statistical analysis
Results are presented as mean ± standard deviation (SD). One-way
analysis of variance (ANOVA) with Tukey’s multiple comparisons was
used to determine the difference between independent groups. Statisti-
cal analyses were conducted using GraphPad Prism software version 6.
The morphology of the DNA-loaded SNP-PEG was characterized by
transmission electron microscopy (TEM). Fig. 3A shows a TEM image of
the PEGylated SNP with a spherical structure and an average size of 40
nm. The GSH-responsiveness of SNP-PEG was further studied by TEM,
SNP-PEG was dispersed in 2 mM GSH solution and incubated at 37 ◦C.
The morphology of the SNP changed significantly after incubation in 2
mM GSH for 1 h, indicating the GSH-responsive cargo release capability
of the SNPs. The hydrodynamic diameter of DNA-loaded SNP-PEG was
45 nm, as measured by dynamic light scattering (DLS) (Fig. 3B). The
3. Results
3.1. Design and characterization of GSH-responsive SNPs
Silica nanoparticles (SNPs) were synthesized via a water-in-oil
microemulsion method (Fig. 1A and B). A mixture of payload aqueous
solution and silica reagents was emulsified in a continuous oil phase.
Tetraethyl orthosilicate (TEOS) was used as a basic building block that
constructs the silica network. A disulfide bond-containing crosslinker,
Fig. 1. Design and synthesis of SNPs conju-
gated with either ATRA or GalNAc. (A)
Illustration of the multifunctional SNP for
the delivery of nucleic acids (e.g., DNA and
mRNA) and CRISPR genome editor (e.g.,
RNP, RNP + ssODN). (B) Synthesis scheme
for the ATRA- or GalNAc-conjugated SNPs.
SNP: silica nanoparticle; TEOS: tetraethyl
orthosilicate; TESPIC: N-(3-(triethoxysilyl)
propyl)-1H-imidazole-2-carboxamide; BTPD:
bis3-[(triethoxysilyl) propyl]-disulfide; PEG:
polyethylene glycol; GalNAc: N-acetylga-
lactosamine; ATRA: all-trans-retinoic acid.
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Journal of Controlled Release 336 (2021) 296–309
zeta-potential of DNA-loaded SNP-PEG was 6.4 mV, indicating a nearly
neutral surface charge after PEGylation.
shown in Fig. 3C, SNP-NH2 exhibited a 1.6-fold higher DNA transfection
efficiency and a 1.8-fold higher mRNA transfection efficiency than
negatively charged SNP. This finding is consistent with previous reports
[59–61]. In comparison with negatively charged NPs, positively charged
NPs can induce higher cargo-delivery efficacy by promoting the rate of
nanoparticle internalization, increasing the total number of internalized
nanoparticles, and enhancing the endo/lysosomal escape capability.
SNP-PEG with a neutral surface charge exhibited similar DNA and
mRNA transfection efficiencies as SNP-NH2, indicating that moderate
surface PEGylation did not significantly affect the SNP uptake by HEK
293 cells.
The SNP formulation was then optimized in HEK 293 cells to achieve
high transfection efficiencies, using DNA and mRNA as payloads, sepa-
rately. Imidazole groups can be quickly protonated in acidic endocytic
compartments, and thus enhance the endo/lysosomal escape capability
of the SNP-PEG due to the proton sponge effect (Fig. 2) [53,54].
Therefore, the ratio of imidazole in the SNP is an essential factor for
efficient nucleic acid delivery. The optimal ratio of imidazole-containing
reactant TESPIC in the SNP was investigated by fixing the feed molar
ratio of TEOS and BTPD. As shown in Fig. 3C, SNP-PEG with 10 mol%
imidazole-containing TESPIC exhibited higher DNA transfection effi-
ciency (1.3-fold) than the one without TESPIC, but further increase in
the TESPIC molar ratio did not lead to higher DNA transfection effi-
ciency. Therefore, the TESPIC ratio was set at 10 mol% for further
studies. The TESPIC ratio in mRNA-encapsulated SNP-PEG was also
optimized, as shown in Fig. S4. Different from DNA delivery, the mRNA
delivery efficiency was independent of the TESPIC ratio. This discrep-
ancy may be attributed to the difference in cytoplasm stability of DNA
and mRNA [55,56]. One major limiting step for mRNA delivery is its
poor cytosolic stability [55,57,58]. In other words, although imidazole-
containing SNP can induce rapid endo/lysosomal escape and efficiently
transport mRNA payload into the cytosol, due to mRNA’s poor cytosolic
stability, it could result in less durable transfection. As a result, incor-
poration of imidazole did not significantly enhance mRNA delivery
efficiency.
Disulfide bonds were integrated into the SNP to facilitate payload
release in the cytosol with a high GSH concentration (2–10 mM). The
extracellular GSH (0.001–0.02 mM), although much lower, can still
raise stability concerns or induce premature cargo release. Hence, to
study the GSH-responsive behavior of SNP, DNA encapsulated SNP was
incubated with HEK 293 cells in culture media containing intentionally
added GSH with a GSH concentration ranging from 0 to 10 mM. As
shown in Fig. 3D, the DNA transfection efficiency was not affected at
GSH concentrations equal to or lower than 0.1 mM, suggesting that the
SNP is stable in the extracellular space. However, a significant decrease
in the DNA transfection efficiency was observed at a GSH concentration
of 1 mM or higher, suggesting that the SNP are not stable at high GSH
concentrations, therefore, they can effectively break down in the cytosol
to release the payload. The serum stability of SNP-PEG was investigated.
SNP-PEG was dispersed in fetal serum albumin (FBS) and incubated at
37 ◦C, the hydrodynamic diameter was monitored by DLS at each time
point. As shown in Fig. S5, the size of SNP-PEG remained unchanged
after 48 h and only slight size change was observed after 72 h, indicating
excellent serum-stability of SNP-PEG for in vivo applications. The sta-
bility of mRNA-loaded SNP-PEG after long-term storage was studied.
The mRNA transfection efficiency of SNP-PEG was intact after 60-day
To investigate the influence of SNP surface charges on nucleic acid
delivery efficiencies, we prepared DNA- and mRNA-encapsulated SNPs
with different surface charges (Fig. 3C and Fig. S4). The as-prepared,
unmodified SNP had
a strong negative zeta-potential; positively
charged SNP (i.e., SNP-NH2) and neutral PEGylated SNP (SNP-PEG)
were prepared by APTES and mPEG-silane conjugation, respectively. As
Fig. 2. Schematic illustration of the intracellular trafficking pathways of SNP.
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Journal of Controlled Release 336 (2021) 296–309
Fig. 3. Characterization of SNP and formulation optimization. (A) Size distribution of SNPs measured by DLS with an average hydrodynamic diameter of 46 nm. (B)
TEM image of SNP. (C) The effect of (1) molar ratio of TESPIC, and (2) surface charge in DNA-delivery by SNP. The transfection efficiency was quantified by the
percent of RFP-positive HEK 293 cells 48 h post treatment. (D) Effects of GSH concentration in cell culture medium on the DNA transfection efficiency of SNP-PEG.
(E) mRNA delivery efficiency of SNP after storage at different conditions. NS: not significant; *: p < 0.05; **: p < 0.01; ****: p < 0.0001; n = 3.
storage at ꢀ 80 ◦C, or 25 days at 4 ◦C or ꢀ 20 ◦C (Fig. 3E), indicating SNP-
Table 1
PEG is desirable for future biomedical applications.
Summary of the SNP-PEG size, zeta-potential, loading content and loading ef-
ficiency of different payloads. Hydrodynamic diameter and zeta-potential data
are presented as mean ± SD.
3.2. In vitro nucleic acid and genome editor delivery by SNPs
Payload
Hydrodynamic
Zeta-
Loading
content (wt
%)
Loading
diameter (nm)
potential
(mV)
efficiency (%)
A variety of biomacromolecules were encapsulated into SNP,
including plasmid DNA, mRNA, RNP and the mixture of RNP and donor
oligonucleotide for gene correction (i.e., RNP + ssODN). The hydrody-
namic diameter, zeta-potential, loading content and loading efficiency
of PEGylated SNP with different payloads were summarized in Table 1.
The size and zeta-potential of SNP-PEG remained relatively constant for
the various payloads investigated. As shown in Table 1, the loading
contents varied between 9.0 and 9.4 wt%, with an overall high loading
efficiency of >90%. In particular, there was no significant difference in
DNA
45 ± 2
46 ± 4
52 ± 4
49 ± 3
6.4 ± 0.6
3.0 ± 0.3
6.5 ± 0.4
5.9 ± 0.7
9.0
9.2
9.1
9.4
90
91
90
93
mRNA
RNP
RNP +
ssODN
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Y. Wang et al.
Journal of Controlled Release 336 (2021) 296–309
loading content and loading efficiency between payloads, indicating
that the SNP is a versatile nanoplatform for encapsulation of nucleic
acids, proteins, and gene editors.
encapsulated SNP-PEG, which is higher than the mutation frequency
of cells treated with Lipo 2000 (49%) and CRISPRMAX (56%). To
investigate gene correction capability of SNP, a BFP-expressing HEK 293
cell line was used. Precise gene editing by HDR will lead to the
replacement of three nucleotides in the genome, thereby altering one
histidine to tyrosine (Fig. 5D), which leads to the BFP to GFP conversion
[47,48]. RNP targeting the BFP gene and a donor ssODN were co-
encapsulated into SNP-PEG. The genome-editing efficiency was evalu-
ated by the percentage of GFP-positive cells. As shown in Fig. 5D, SNP
exhibited a statistically higher (1.1-fold) gene-correction efficiency than
Lipo 2000. These results demonstrate the capability of SNP as an effi-
cient nanoplatform for genome editor delivery.
The intracellular trafficking of RNP-encapsulated SNP-PEG was
studied by confocal laser scanning microscopy (CLSM) in HEK 293 cells
(Fig. 4). RNP was prepared by mixing the NLS-tagged Cas9 and ATTO-
550-tagged guide RNA. After the cells were incubated with the RNP-
loaded SNP-PEG for 0.5 h, RNP was mainly co-localized with endo/ly-
sosomes, indicating the internalization of SNP-PEG via endocytosis.
Endo/lysosomal escape of the SNP-PEG assisted by imidazole was
observed 2 h post-treatment, indicated by a decrease in the co-localized
RNP and endo/lysosome signals. The RNP signal showed considerable
overlap with the nucleus and further decreased co-localization with
endo/lysosomes 6 h post-treatment, indicating the successful nuclear
transportation of RNP induced by the NLS tags on the RNP.
We next evaluated the biocompatibility of SNP. HEK 293 cells were
treated with DNA-encapsulated SNP-PEG at different SNP-PEG concen-
trations, and the cell viability was studied by an MTT assay. As shown in
Fig. 5E, SNP-PEG did not induce significant cytotoxicity in HEK 293 cells
To investigate the versatility of the SNP for biomacromolecule de-
livery, HEK 293 cells were used for nucleic acid delivery/genome edit-
ing efficiency studies, and flow cytometry was used to quantify the
delivery efficiency. The DNA and mRNA transfection efficiency by SNP-
PEG were tested in HEK 293 cells (Fig. 5A and B). SNP-PEG exhibited
statistically higher DNA and mRNA transfection efficiency (1.3-fold and
1.1-fold, respectively) than the commercially available transfection re-
agent, Lipofectamine 2000 (Lipo 2000), indicating the superior nucleic
acid delivery capability of SNP.
with concentrations up to 1000
μg/mL, 45-times higher than the
working concentration used for our delivery efficiency studies. How-
ever, at the working DNA concentration, DNA-complexed Lipo 2000
showed only 77% cell viability, indicating a significantly higher cyto-
toxicity than SNP-PEG. Together with the delivery efficiency results, we
show that the SNP is a desirable nanoplatform for efficient delivery of
various biomacromolecules.
The CRISPR-Cas9 RNP is a fast, efficient and accurate genome editor.
Cas9 as a nuclease can cause double-stranded DNA break in a specific
genomic locus under the guidance of gRNA, achieving gene editing by
the non-homologous end-joining (NHEJ) DNA repair pathway [6,62].
Moreover, with a donor DNA template (e.g., single-stranded oligonu-
cleotide DNA (ssODN)) delivered together with RNP, gene correction or
insertion can be achieved through the homology-directed repair (HDR)
pathway [46]. We first investigated the genome-editing efficiency of
SNP-PEG by delivering the RNP targeting the GFP gene in a transgenic
GFP-expressing HEK 293 cell line. As shown in Fig. 5C, RNP-
encapsulated SNP-PEG exhibited a significantly higher gene-knockout
efficiency (1.3-fold) than Lipo 2000 and CRISPRMAX. T7E1 assay was
performed to further investigate the indel formation (Fig. S6). This assay
revealed a mutation frequency of 72% for cells treated with RNP-
3.3. Genome editing in RPE via subretinal injection
Nucleic acid delivery/genome editing efficiency of SNP were further
investigated in transgenic Ai14 mice (Fig. 6). Ai14 mouse genome
contains a CAGGS promoter and a LoxP-flanked stop cassette with three
repeats of the SV40 polyA sequence that prevents the expression of the
downstream tdTomato fluorescent protein gene. The gain-of-function
fluorescence can be achieved by: 1) Cre-Lox combination via the de-
livery of Cre recombinase or Cre-encoding DNA/mRNA (Fig. 5A), or 2)
excision of 2 of the SV40 polyA blocks by Cas9 RNP (Fig. 6C). The
tdTomato fluorescence signal in edited cells provides a robust and
quantitative readout of nucleic acid delivery/genome editing in Ai14
mice [49,63,64].
To study the mRNA delivery efficiency by SNP, eyes of Ai14 mice
Fig. 4. Intracellular trafficking of SNP. Colocalization of ATTO-550-tagged RNP and endo/lysosomes was studied at 0.5 h, 2 h, and 6 h post-treatment.
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Journal of Controlled Release 336 (2021) 296–309
Fig. 5. Delivery efficiency of nucleic acids and CRISPR-Cas9 genome editors by SNP. (A) and (B) The transfection efficiency of (A) DNA- and (B) mRNA-loaded SNP-
PEG in HEK293 cells. (C) Gene editing efficiency of RNP-loaded SNP-PEG in GFP-expressing HEK 293 cells. (D) Illustration of HDR at a BFP reporter locus induced by
the RNP + ssODN. Sequences of unedited (BFP) and edited (GFP) loci are shown. The protospacer adjacent motif sequence of RNP is underlined and the RNP cleavage
site is marked by a red arrow. (E) Gene-correction efficiency of RNP + ssODN co-encapsulated SNP-PEG in BFP-expressing HEK 293 cells. NS: not significant *: p <
0.05; **: p < 0.01; n = 3. (F) Viability of HEK 293 cells treated with DNA-loaded SNP-PEG with different concentrations and DNA-complexed Lipo 2000. NS: not
significant; ****: p < 0.0001; n = 7. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
were subretinally injected with a Cre-mRNA-encapsulated SNP-PEG-
ATRA (Fig. 6B); subretinal injection of PBS was used as a control. Four
days post injection, the mice were sacrificed and eyes were enucleated.
The eyes were then dissected to remove anterior region leaving the
posterior eye cup of an eye. Retina was later removed from the RPE-
choroid and flat-mounted, tdTomato expression in the flattened RPE
tissue (i.e., RPE floret) was studied by CLSM. As shown in Fig. 6D, strong
tdTomato fluorescence was visualized in the RPE florets with SNP-PEG-
ATRA injection, indicating efficient delivery of Cre-mRNA by SNP. The
ratio of tdTomato positive area to the total area of the RPE floret was
6.9% (Fig. S7). Moreover, the genome editing efficiency of SNP was
studied by subretinal injection of Cas9 RNP encapsulated SNP. Mice
were subretinally injected with a SNP-PEG-ATRA encapsulating the RNP
targeting the SV40 polyA block (i.e., Ai14 RNP), or a SNP-PEG-ATRA
encapsulating the RNP with the negative control sgRNA (i.e., negative
control). The tdTomato expression was evaluated 14 days post-injection.
As shown in Fig. 6E, Ai14 RNP-loaded SNP induced very strong tdTo-
mato expression in the RPE surrounding the injection site. The ratio of
tdTomato positive area to the total area of the RPE floret was calculated
as 4.5% (Fig. S8). No tdTomato signal was found in eyes injected with
negative control SNP. These results suggest that SNP is a reliable
nanoplatform for in vivo biomacromolecule delivery.
3.4. Genome editing in liver via intravenous injection
The nucleic acid and RNP delivery efficiency of intravenously
injected SNP was also evaluated in vivo using Ai14 mice. Two types of
SNPs were involved in this study: (1) SNP-PEG and (2) liver-targeting
SNP-PEG-GalNAc. We chose liver as the target organ because liver is
an important target for therapeutics development. Nanoplatforms
capable of safe and efficiency gene/gene editor delivery to liver can be
powerful tools for the treatment of liver diseases (e.g., nonalcoholic fatty
liver disease, liver cancer and hereditary tyrosinemia) [65–67].
Cre-mRNA delivery was first investigated, with a mRNA dosage of
20 μg per mouse. Major organs were collected 3 days post injection, and
the tdTomato fluorescence was analyzed by IVIS (Fig. S9A). Although
tdTomato signal was mainly detected in the liver for both non-targeted
and targeted SNPs, the SNP-PEG-GalNAc injected mice exhibited a
stronger liver tdTomato signal than SNP-PEG (Fig. 7A). In fact, ho-
mogenized liver tissue showed a 2-fold increase of tdTomato signal in
the liver of SNP-PEG-GlaNAc injected mice than the SNP-PEG group
(Fig. 7B), indicating GalNAc conjugation on the SNP surface can further
enhance liver targeting efficiency. To confirm the tdTomato expression,
liver sections were immunofluorescence stained with anti-tdTomato
antibody and then fluorescein-tagged secondary antibody. The immu-
nostained liver sections were examined using CLSM. As shown in Fig. 7C
and Fig. S9B, tdTomato-positive cells were found in liver tissue, while
tdTomato positive cells were not detected in the PBS-injected mice,
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Journal of Controlled Release 336 (2021) 296–309
Fig. 6. Nucleic acid and RNP delivery efficiency of SNP in Ai14 mice via subretinal injection. (A) The tdTomato locus in the Ai14 reporter mouse. tdTomato
expression can be achieved by Cre-Lox recombination. (B) Illustration of subretinal injection targeting the RPE tissue. (C) The stop cassette containing 3 Ai14 sgRNA
target sites prevents downstream tdTomato expression. Excision of two SV40 polyA blocks by Ai14 RNP results in tdTomato expression. (D) Efficient delivery of Cre-
mRNA by SNP-PEG-ATRA in mouse RPE. D1, RPE floret of eyes subretinally injected with Cre-mRNA-encapsulated SNP; D2, 20× magnification images of tdTomato+
RPE tissue; D3, RPE floret of PBS controls. (E) Efficient delivery of RNP by SNP-PEG-ATRA in mouse RPE. E1, RPE floret of mouse eyes subretinally injected with Ai14
RNP-encapsulated SNP; E2, 20× magnification images of tdTomato+ RPE tissue; E3, RPE floret of Ai14 mice injected with negative control SNP-PEG-ATRA (SNP-
PEG-ATRA encapsulating RNP with negative control sgRNA). The whole RPE layer was outlined with a white dotted line.
indicating that SNPs, with or without GalNac, can deliver mRNA into
liver via systemic administration.
can further enhance the biomolecule delivery efficiency in targeted
tissues/cells.
We further investigated the in vivo systemic RNP delivery efficiency
using SNP. Adult Ai14 mice were retro-orbitally injected with RNP
4. Discussion
encapsulated SNP or SNP-PEG-GalNAc (100 μg RNP per mouse), and
major organs were collected 7 days post-injection. Similar to Cre mRNA,
tdTomato signal were mainly found in the liver (Fig. 7D and Fig. S10A),
and SNP-PEG-GalNAc showed a 2-fold higher gene editing efficiency
than SNP-PEG, as quantified by the fluorescence intensity of homoge-
nized tissue (Fig. 6E). Immunofluorescence staining of sectioned liver
showed strong tdTomato expression induced by RNP delivery (Fig. 7F
and Fig. S10B). To evaluate the potential systemic toxicity of SNP, blood
biochemistry test was performed for all the injected mice (Fig. S11). The
key elements of the blood biochemical profile (e.g., total CO2 (tCO2),
ALT, AST, BUN, etc.) showed no significant difference between SNP-
injected groups and the PBS control group, indicating that the SNP
possessed good biocompatibility. This proof-of-principle data indicates
that intravenous administration of SNP can achieve gene delivery/gene
editing in vivo. Furthermore, SNP conjugated with targeting moieties
Intracellular delivery of nucleic acids (e.g., DNA, mRNA) and
genome editors (e.g., RNP, RNP + ssODN) plays an important role in
revolutionizing the treatment of genetic diseases. Although a number of
non-viral vectors have recently been developed, delivering these pay-
loads in their active forms to the target site in a safer and efficient
manner remains a challenge.
The key properties of non-viral vectors including in vitro and in vivo
stabilities, biocompatibility, payload loading content and encapsulation
efficiency, payload types, payload release profile, and surface charac-
teristics (e.g., zeta potential and ligand conjugation) are largely deter-
mined by the composition (formulation) and fabrication process of the
non-viral vectors. Lipid- or polymer-based self-assembled nanocarriers
with stimuli-responsive capabilities have been previously reported
[20,68,69]; however, such self-assembled nanosystems often lack of
305

–
Fig. 7. SNP enabled nucleic acid (A-C) and RNP (D F) delivery in vivo via systemic administration. Ai14 mice were injected with Cre-mRNA or RNP encapsulated SNP-PEG or SNP-PEG-GalNAc, and the tdTomato
fluorescence of the whole liver (A and D) and homogenized liver tissue (B and E) was detected and analyzed by IVIS imaging. Liver sections were studied by immunofluorescence staining and confocal laser scanning
microscopy (C and F). *: p < 0.05; ***: p < 0.001; n = 3.

Y. Wang et al.
Journal of Controlled Release 336 (2021) 296–309
sufficient in vivo and in vitro stabilities because the interactions be-
tween the carrier materials (i.e., lipids or polymers) and payloads are
merely physical interactions (e.g., electrostatic interaction) [70,71].
Moreover, biomacromolecules such as DNA, mRNA and RNP are very
different payloads in terms of structure, charge, and functionality,
therefore, it is not surprising to see some of the reports suggesting that
minor changes in the chemical structure of the lipids or polymers can
affect the delivery efficiency of different payloads in very distinctive, or
even opposite manners [56,72], creating potential obstacles for co-
delivery of multiple types of biomolecules (e.g., RNP + ssODN for
HDR) in vivo. Co-delivery of multiple types of biomolecules was re-
ported using a functionalized gold NP core for payload absorption, fol-
lowed by a cationic polymer surface coating [73]. However, this type of
non-viral vector still encounters potential instability issue, the resulting
~500-nm nanoparticle with positive surface charges is not desirable for
systemic administration. To address these challenges, we have devel-
Data availability
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
Acknowledgements
We acknowledge the financial support from the National Institute for
Health
(1-UG3-NS-111688-01,
R01HL129785,
R01HL143469,
R01EY024995, and 1R24 EY032434).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jconrel.2021.06.030.
oped
a versatile strategy to encapsulate various types of bio-
References
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a GSH-responsive silica nanoparticle (SNP) via a microemulsion process.
We demonstrated that a silica-based network is constructed around the
payload molecule in the nano-sized water droplet, forming a covalently
crosslinked, yet GSH-responsive SNP entrapping the payload inside. It is
noted that our GSH-responsive SNP is distinctively different from pre-
viously reported mesoporous silica NP (MSN) systems [74,75]. Macro-
molecular payloads were loaded onto the MSN surface via electrostatic
interaction only, leading to poor NP stability (aggregation) and low
payload loading content (<5 wt%) [76,77]. Our GSH-responsive SNP
possessed excellent stability due to its covalent nature. The SNP also
offered a high payload loading content (>9 wt%), and high encapsula-
tion efficiency (>90%), regardless of payload type. It is expected that the
SNP can also facilitate the delivery of other CRISPR variant payloads,
such as epigenomic editors, RNA editors and base editors [78].
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cells [79]. For example, A study found that 60 nm PEGylated NPs
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mucus-penetration efficiency [82,83]. The PEGylated SNP used for the
studies presented in this paper had a hydrodynamic diameter around 50
nm that allows for both local and systemic delivery. However, the
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and the microemulsion fabrication process allow us to conveniently
change the size of the SNP in the range of 40 to 200 nm by varying the
emulsification conditions (e.g., water to oil ratio and type/ratio of sur-
factant) [34,73], making it desirable for various target tissues/cells in
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