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Conjugated Polyelectrolyte/Single Strand DNA Hybrid Polyplexes

for Efficient Nucleic Acid Delivery and Targeted Protein Degradation
Yuanjie Sun, Li Jiang, Zhilin Zhang, Naihan Xu,* Yuyang Jiang, and Chunyan Tan*
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ABSTRACT: Nucleic acid-based therapeutics have gained increas-

ing attention due to their ability to regulate various genetic disorders.
Downloaded via UNIV TOWN OF SHENZHEN on March 18, 2024 at 15:41:22 (UTC).

However, the safe and effective delivery of nucleic acids to their

intended cellular sites remains a challenge, primarily due to poor cell
membrane permeation and low in vivo stability. Limitations
associated with the commonly used nucleic acid delivering agent
viral vectors such as carcinogenesis and immunogenicity have driven
scientists to develop various nonviral vectors. In this study, we
present a highly efficient nucleic acid delivery system based on
cationic conjugated polyelectrolytes and single-strand DNA poly-
plexes with further application in efficient ubiquitin-regulated
targeting protein degradation. These polyplexes, formed by 9TC,
an aptamer sequence for estrogen receptor (ERα), and cationic PPET3N2 through electrostatic and hydrophobic interactions,
demonstrate improved cellular uptake efficiency as well as enhanced stability against nuclease degradation. Furthermore, by
incorporation of 9TC into a proteolysis targeting chimera (PROTAC) molecule (P9TC), PPET3N2/P9TC polyplexes significantly
enhance the target protein ERα degradation efficiency. Collectively, our findings suggest that PPET3N2 provides a versatile, low
cytotoxicity platform for safe, efficient, and simplified delivery of nucleic acids.
KEYWORDS: conjugated polyelectrolytes, nucleic acid delivery, polyplexes, aptamers, PROTACs, targeted protein degradation

■ INTRODUCTION
The use of nucleic acid-based therapeutics has gained
increasing attention due to their ability to alter the target
genes’ expression and thus modulate a wide range of diseases at
the genetic level, including cystic fibrosis, heart disease, type 2
diabetes, Alzheimer’s, hemophilia, many types of cancers,
etc.1−3 Several strategies targeting nucleic acid delivery
challenges have evolved in order to enhance transfer efficiency,
minimize immune response, and improve the in vivo and ex
vivo stability of nucleic acid-based therapeutics.4,5 These
strategies include viral vectors, inorganic nanovectors, lipid-
based nanocarriers, polymeric nanocarriers, and peptide-based
formulations.5 Due to their biocompatibility, versatility, and
multifunctionality, polymetric materials are becoming increas-
ingly attractive as delivery platforms for nucleic acids.6 It is
common for polymetric carriers of nucleic acids to contain
positively charged groups in order to interact with negatively
charged nucleic acids and to facilitate membrane penetra-
tion,6,7 for example, polylysine (PLL),8 polyethyleneimine
(PEI),9 poly(amidoamine) dendrimer (PAMAM),10 and
poly(β-aminoesters).11 Polymer-based delivery systems exhibit
a variety of formulations,6 among which the construction of
polyplexes is a common method formed by polymetric carriers
and nucleic acids. Specifically, polyplexes refer to nano-
assemblies composed of cationic polyelectrolyte complexed
with anionic nucleic acid cargo through coulomb forces and
hydrophobic interactions.5
Conjugated polyelectrolytes (CPEs) contain delocalized π
electrons on their polymer backbones and ionic side chains
and, thus, exhibit special optical and electronical character-
istics.12 CPEs have demonstrated wide applications during the
past 30 decades in sensing,13 fluorescence imaging in vivo and/
or ex vivo,14,15 photodynamic therapy,16 photothermal
therapy,17 drug delivery,18,19 etc. The positive charges on
cationic CPEs are beneficial for transferring negatively charged
genetic molecules through the membrane barrier. Never-
theless, due to CPEs’ hydrophobic backbones and versatile side
chains, it is possible to confer amphiphilicity by designing
hydrophilic side chains, allowing them to form micelles or
vesicles by self-assembly. As compared to viral vectors, CPEs
have much lower cytotoxicity and minimized immune
response.7 It has been reported that certain cationic CPEs
can serve as a stable, self-tracking, and effective means of
Special Issue: 25 Years of Conjugated Polyelectrolytes
with a Focus on Applications
Received: September 30, 2023
Revised: November 28, 2023
Accepted: November 28, 2023

© XXXX American Chemical Society https://doi.org/10.1021/acsami.3c14640

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ACS Applied Materials & Interfaces
delivering nucleic acids.20−24 Wang’s group synthesized a lipid-
modified cationic poly(fluorenylene phenylene) (PFPL) for
plasmid delivery (pCX-EGFP), and a cationic polyfluorene
(CCP) for transfection of pDNA encoding GFP with 92%
efficiency.21 Using adenine-modified conjugated oligomer 4,7-
(9,9′-bis(6-adenine hexyl)fluorenyl)-2,1,3-benzothiadiazole
(OFBT-A), Li and his colleagues created high cytocompati-
bility nanoparticles for ssDNA delivery to A549 cells.25 Feng’s
group applied cationic polythiophenes (CTs) to the surface of
polyaspartate/pGFP polyplexes and discovered an enhanced
gene delivery through light-induced ROS generation.26 In
2018, they used cationic poly(phenylene ethynylenes) (cPPEs)
as the photosensitizer in polyethyleneimine (BPEI)/DNA
complexes to increase the transfection of green fluorescent
protein gene into tumor cells.27
Herein, we investigated a CPE-based nucleic acid delivery
system utilizing a cationic CPE: PPET3N2.28,29 In traditional
gene therapy, nucleic acids are often used to influence the
expression or function of the target genes. In contrast, this
study uses an ssDNA sequence tailored specifically for
targeting protein degradation through the ubiquitin−protea-
some system, one of the most important protein degradation
pathways within cells. The transported nucleic acid P9TC
consists of an aptamer fragment (9TC) that binds specifically
to the target protein estrogen receptor ERα, and another
aptamer fragment (VH032) that binds to the ubiquitination
ligase VHL. Our method has demonstrated a successful
transportation of nucleic acids into MCF7 cells for
ubiquitination and targeted degradation of ERα.
■ EXPERIMENT SECTION
Materials and Reagents. PPET3N2 was synthesized as
reported previously and stored at 4 °C protected from light
(100 μM).29 The characterization of PPET3N2 was shown in
Supporting Information Table S2. The sequence of 9TC
(aptamer for ERα) was reported in the literature previously.30
9TC (5′-ttt ttt ttt gtc agg tca cag tga cct gat caa agt taa tg-3′, 5′
modified with Azide (N3)) and 9TC-A (5′-ttt ttt ttt gtc agg tca
cag tga cct gat caa agt taa tg-3′, 5′ modified with Alexa Fluor
568) were purchased from Sangon Biotech (Shanghai, China).
P9TC was synthesized following the procedure in the reported
literature.31 Briefly, alkyne-modified VH032 (a ligand of von
Hippel−Lindau protein) reacted with azide-modified 9TC
through CuI-catalyzed azide−alkyne cycloaddition (CuAAC)32
P9TC was further purified by ethanol precipitate, washed with
cold 75% ethanol for three times, and characterized by urea-
PAGE analysis (Figure S1). A stock solution (200 μM) of each
ssDNA was prepared in water and stocked at −20 °C. VH032-
propargyl and BTTAA were purchased from MedChemEx-
press (Shanghai, China). Dulbecco’s modified Eagle’s medium
(DMEM) and fetal bovine serum (FBS) were purchased from
Corning (New York, U.S.A.). Penicillin−streptomycin, tryp-
sin−EDTA solution (0.02% EDTA and 0.05% trypsin),
phosphine-buffered saline (PBS, pH 7.3), methyl thiazolyl
diphenyl−tetrazolium bromide (MTT), deoxyribonuclease I
(DNase I), RIPA Lysis Buffer, bicinchoninic acid (BCA)
protein assay kit, TBE Precast PAGE Gel (15%), 4′,6-
diamidino-2-phenylindole (DAPI), and DMSO were pur-
chased from Beyotime (Shanghai, China). PAGE Gel Fast
Preparation Kit (10%) was purchased from Epizyme Biotech
(Shanghai, China). Polyvinylidene Fluoride (PVDF) Transfer
Membrane and 0.22 μm sterile filter were purchased from
Millipore (Massachusetts, U.S.A.). The water was purified
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using a Milli-Q water purification system with a resistivity of
≥18.2 MΩ cm−1.
Instrumentation. UV−visible absorption spectra were
collected on an Agilent Cary 100 UV−visible spectrometer.
Fluorescence spectra were obtained on a TECAN Infinite
M1000 Pro multiwell plate reader with black flat-bottomed
polystyrene 96-well microplates (Corning 3916). Circular
dichroism (CD) was measured using an Applied Photophysics
qCD. Polyplex sizes and ζ potentials were measured by
dynamic light scattering (DLS) using a Malvern Zetasizer
Nano ZS (MDTC-EQ-MO2-01). Transmission electron
microscopy (TEM) images were obtained by using an FEI
Tecnai T12 transmission electron microscope. Fluorescence
images of cells were recorded using an Olympus A1R HD25
confocal laser scanning biological microscope with 405, 488,
and 561 nm excitation according to different fluorescent
probes.
Cell Culture. MCF7 cells were grown in DMEM and added
with 10% FBS and 1% penicillin−streptomycin. Cells were
grown in 10 cm dishes and then cultured in a 37 °C incubator
with 5% humidified CO2. A subculture was performed every
2−3 days using trypsin−EDTA solution in PBS.
Preparation of CPE/ssDNA Polyplexes. Polyplexes were
prepared at different CPE/ssDNA molar ratios (μM/μM). (1)
For the nuclease resistance assay and circular dichroism assay,
the molar ratio of PPET3N2/9TC is 1:1. PPET3N2 (10 μL, 100
μM) was added to 9TC (5 μL, 200 μM) and mixed by up−
down pipetting. (2) For DLS, ζ potential, and TEM
measurements, the molar ratio of PPET3N2/9TC is 5:3.
PPET3N2 (50 μL, 100 μM) was added to 9TC (15 μL, 200
μM) and mixed by up−down pipetting. (3) For cell imaging,
the molar ratio of PPET3N2/9TC-A is 5:3. PPET3N2 (50 μL,
100 μM) was added to 9TC (15 μL, 200 μM) and mixed by
up−down pipetting. (4) For the ERα degradation assay, the
molar ratio of PPET3N2/P9TC is 5:3. PPET3N2 (50 μL, 100
μM) was added to P9TC (15 μL, 200 μM) and mixed by up−
down pipetting. After 3 s vortex mixing, all polyplexes were
incubated for 20 min at 4 °C. Then, the polyplexes were
diluted to different concentrations by H2O, PBS, or DMEM,
depending on the experiment.
Fluorescence Emission Measurement. The fluorescence
emission measurement was conducted at 25 °C. In black 96-
well microplates (Costar), various concentrations of 9TC-A
were incubated with PPET3N2 in the dark for 30 min followed
by recording the fluorescence spectra with an excitation
wavelength of 470 nm. All solutions were added in the
following order: H2O (solvent), cationic PPET3N2, and 9TC-
A. The final concentration of PPET3N2 was 5 μM, while the
final concentration of 9TC-A was between 0 and 5 μM. Each
sample was replicated three times.
Circular Dichroism (CD). Samples of 9TC (1 μM in 2
mL), PPET3N2 (1 μM in 2 mL), and PPET3N2/9TC
polyplexes (molar ratio = 1:1; [9TC] = 1 μM in 2 mL)
were placed in quartz cuvettes of 0.1 cm path length,
respectively. Spectra were recorded in a continuous scan
mode between 180 and 300 nm. In all cases, spectra were
measured in three scans, subtracted from the base and
averaged using the Chirascan program.
Determination of Hydrodynamic Diameters and ζ
Potentials of Polyplexes. The DLS measurement was
carried out at a detection angle of 173° and temperature of
25 °C. The hydrodynamic diameters of polyplexes were
calculated by the cumulants method and applying the Stokes−
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Scheme 1. Schematic Representation of CPE/ssDNA Polyplexes for Efficient Delivery of Nucleic Acids and Targeted Protein
Degradation
Einstein equation. Each report value was the average of five
measurements (4 × 30 times). The ζ potentials of the
polyplexes were measured in a folded capillary cell DTS1070.
The number of measurements was set to five for a sample with
10 runs.
Examination of Polyplexes Morphologies by TEM. A
10 μL drop of the polyplexes (molar ratio = 5:3; [9TC] = 30
μM) was placed on a 300 mesh copper grid covered with a
perforated carbon film. The polyplexes were allowed to absorb
for 5 min. Excess liquid was removed with filter paper, and the
samples were negatively stained by phosphotungstic acid (1%,
pH 6.7) for 4 min. After the removal of the excess dye, the
sample was dried in 50 °C for 20 min before being subjected to
TEM.
Evaluation of Cytotoxicity. MCF7 cells were preseeded
in 96-well plates at 8000 cells/well density and then cultured in
complete DMEM medium at 37 °C for 12 h to form cell
adhesion. Then, PPET3N2 or PPET3N2/9TC-A polyplexes
were diluted using a complete DMEM medium to a series of
specific concentrations to form drug suspension. Then, the
medium in each well was replaced by a 200 μL/well drug
suspension. After an additional 24 h of incubation, the solution
in each well was discarded and the cells were carefully washed
with 1 × PBS for 3 times. Each well was added with 100 μL of
0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) solution diluted by complete DMEM
medium, followed by an additional 4 h of incubation at 37 °C
in a CO2 incubator. Further, the MTT solution was carefully
discarded and 150 μL of DMSO was added to each well. The
plate was shaken on an orbital shaker for 15 min to ensure the
solubilization of MTT formazan. Then, the absorbances were
immediately read at 562 nm in a microplate reader, and
cytotoxicity was expressed as a percentage relative to control
cells (cells without any treatments).
Assessment of Stability against Nuclease Degrada-
tion. Naked 9TC (1 μM) and PPET3N2/9TC polyplexes
(molar ratio = 1:1; [9TC] = 1 μM) were treated with 0.01 U/
μL DNase I at 37 °C for various time periods (0, 1, 5, 10, 30,
60, or 120 min). After being denatured by heating the samples
at 95 °C for 1 min, samples were quickly put on ice to stop
DNase I digestion and then analyzed through urea
polyacrylamide gel electrophoresis (Urea PAGE).
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Endocytosis of CPE/ssDNA Polyplexes. MCF7 cells
were seeded in glass bottom cell culture dishes at a density of 1
× 105 cells/well and then cultured in a complete DMEM
medium at 37 °C for 12 h to form cell adhesion. Cells were
treated with complete DMEM medium containing PPET3N2
(5 μM), 9TC-A (3 μM) or PPET3N2/9TC-A polyplexes
(molar ratio = 5:3; [9TC-A] = 3 μM), respectively. After the
wells were incubated for 2, 6, 12, or 24 h, the medium of each
well was removed. Next, the cells were washed twice with 1 ×
PBS and then fixed for 15 min in 4% paraformaldehyde. After
another two PBS washing steps, cells were treated with PBS-
diluted DAPI solution (0.5 μg/mL) for 15 min in the dark to
stain the nucleus. The solution was carefully aspirated, and
cells were washed three times. Fluorescence images were
recorded by a confocal laser scanning microscope (CLSM).
Protein Degradation and Western Blot Analysis.
MCF7 cells in 24-well plates were treated with naked P9TC
(3 μM) or PPET3N2/P9TC polyplexes (molar ratio = 5:3;
[P9TC] = 3 μM) for 3, 6, 12, or 24 h. For Western blot
analysis, cells were lysed with RIPA buffer supplemented with
protease and phosphatase inhibitors on ice for 30 min. Samples
were measured for total protein quantification by the BCA
assay. The cell lysates were separated through 10% sodium
dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-
PAGE) and blotted onto PVDF membranes. The images
were visualized using Clarity Western ECL Substrate from Bio-
Rad and recorded through Bio-Rad imager.
■ RESULTS AND DISCUSSION
Design and Characterization of the CPE/ssDNA
Polyplexes. The previously designed and synthesized
PPET3N2 has a poly(p-phenylene ethynylene terthiophene)
backbone and incorporates quaternary ammonium salt groups
on its side chains, which provide two positive charges per
repeating unit.28 These positive charges enable effective
complexation with negatively charged targets. The inclusion
of a terthiophene unit in a polymer backbone has been
reported to provide exceptional optical properties, which is
attributed to the charge delocalization along the polymer
backbone through the overlapping π-orbitals of the thiophene
units. Additionally, the presence of terthiophene imparts
greater flexibility to the polymer’s backbone. This flexibility
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Figure 1. Characterization of CPE/ssDNA polyplexes. (a) UV−visible absorption spectrum (red) and fluorescence emission spectrum (gray) of
9TC-A in the aqueous solution. (b) UV−visible absorption spectrum (red) and fluorescence emission spectrum (gray) of PPET3N2 in the aqueous
solution. All spectra were normalized. (c) Fluorescence emission spectra and (d) fluorescence photographs of PPET3N2/9TC-A polyplexes at
different concentrations of 9TC-A. [9TC-A] = 0−5 μM; [PPET3N2] = 5 μM. The excitation wavelength was 470 nm. (e) FRET efficiency of the
PPET3N2/9TC-A polyplexes as a function of the 9TC-A concentration. The FRET efficiency was determined by quantifying the fluorescence
quenching of PPET3N2 at 556 nm. Each value is the average of three independent measurements, and error bars represent the standard deviation of
the mean. (f) CD spectra of 1 μM PPET3N2 (gray), 1 μM 9TC (red), and PPET3N2/9TC polyplexes (molar ratio = 1:1; [9TC] = 1 μM; blue).

allows for conformational changes to occur more readily upon
binding with nucleic acids.33,34 PPET3N2 has also demon-
strated successful application to intracellular ATP detection.29
In addition, we found that PPET3N2 is capable of penetrating
the cell membrane and cytolyzing to lysosomes. Scheme 1
provides an illustration of the proposed principle behind the
CPE-based nucleic acid delivery system. In this method, CPE
and single-stranded DNA (ssDNA) are directly mixed to form
CPE/ssDNA polyplexes. As depicted in Scheme 1, PPET3N2
contains a flexible side chain with a high positive charge, which
contributes to its strong affinity for binding to nucleic acids
while remaining soluble in water. Furthermore, there is a
hydrophobic interaction between the aromatic polymer
backbone of CPE and the nucleic acid bases.
To investigate the capability of CPE to enhance ssDNA
transfection efficiency, we selected a specific oligonucleotide
sequence called 9TC as a model ssDNA to form the CPE/
ssDNA polyplexes. This choice was based on the fact that 9TC
serves as an aptamer for ERα, a protein closely associated with
breast cancer carcinogenesis. By utilizing the CPE/ssDNA
polyplexes with 9TC, we aimed to validate the effectiveness of
CPE in enhancing the transfection efficiency of ssDNA.
Förster resonance energy transfer (FRET) is a highly
distance dependent phenomenon in which an excited state
donor transfers energy to a ground state acceptor through a
nonradiative process.35 This technique is an essential tool for
analyzing the dynamics of biological molecules on the
nanoscale. In order to facilitate the study of the interaction
between PPET3N2 and ssDNA, we modified the 5′ side of 9TC
with Alexa Fluor 568 to form 9TC-A. The photophysical
D https://doi.org/10.1021/acsami.3c14640
characterization of PPET3N2 and 9TC-A was demonstrated.
Panels a and b of Figure 1 show the absorption spectrum and
fluorescence emission spectrum of 9TC-A and PPET3N2,
respectively. PPET3N2 shows a broad fluorescence emission
peak, while 9TC-A exhibits a narrow and robust absorption
signal. We measured the overlap integral between the donor
(PPET3N2) emission and acceptor (9TC-A) absorption that
satisfied the requirement for FRET. As a result, upon excitation
at the PPET3N2 absorbance band of 470 nm, we conducted the
fluorescence titration experiment (Figure 1c,d), utilizing the
highly efficient FRET pair of PPET3N2/9TC-A polyplexes.
The fluorescence intensity of PPET3N2 quickly drops while
sensitized emission at 610 nm from 9TC-A increases; the
corresponding FRET efficiency data are shown in Figure 1e.
Using the fluorescence titration data, we can calculate the
distance information between PPET3N2 and 9TC-A; the
detailed calculation process is shown in the Supporting
Information (SI). Table S1 shows the calculated results of
the spectral overlap (J), Förster distance (R0), and donor−
acceptor distance (R). When the FRET efficiency is at its
maximum (92%), the distance between the donor and acceptor
is 24.52 Å, indicating a very close proximity between PPET3N2
and 9TC-A.
CD spectroscopy was used to observe the formation of
polyplexes. Figure 1f illustrates the CD spectra of the 9TC,
PPET3N2, and PPET3N2/9TC polyplexes. PPET3N2 does not
possess chirality and thus no signal in its CD spectrum. A CD
spectrum of 9TC exhibits characteristic elliptic peaks at 245
nm (negative) and 278 nm (positive) caused by the helicity
and base stacking of ssDNA. The positive peak of 9TC was
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slightly decreasing and red-shifted the band from 278 to 282

nm in the mixture of PPET3N2/9TC, suggesting polyplexes
formation between the polymer and the ssDNA due electro-
static attraction and hydrophobic interaction. There was also
an increase in the magnitude of the band near 220 nm, which
might be due to the disruption of the aggregation of ssDNA.
The size and morphology of PPET3N2/9TC polyplexes were
characterized by using DLS and TEM (Figure S2). DLS
indicates the presence of polyplexes with a Z-averaged
hydrodynamic diameter of 135.3 ± 11.6 nm. TEM revealed
that the PPET3N2/9TC polyplexes were spherical nano-
particles with diameters ranging from 40 to 120 nm. Notably,
this size was significantly smaller than the hydrodynamic size
measured in DLS, due to the different nature of the
measurements, including the contributions from the solvation
layer in DLS and the direct imaging of the nanoparticle core in
TEM. Additionally, aggregation of polyplexes may occur in
aqueous solutions. The ζ potential of the PPET3N2/9TC
polyplexes was increased from −30.23 mV (9TC) to 8.86 mV
after being complexed with PPET3N2 (21.67 mV), indicating a
successful formation of polyplexes (Figure S2).
Biocompatibility of CPE/ssDNA Polyplexes. We first Figure 2. Stability of 9TC against nuclease degradation. (a) Urea-
evaluated the in vitro biocompatibility of the PPET3N2/9TC PAGE results for PPET3N2/9TC polyplexes (molar ratio = 1:1,
[9TC] = 1 μM) treated with DNase I for various times at 37 °C. (b)
polyplexes using the MTT assay to determine the CPE/ssDNA
Urea-PAGE results for naked 9TC treated with DNase I for various
delivery and transfection capability. After adding PPET3N2 to times at 37 °C. [9TC] = 1 μM. (c) Relative degradation ratios of
the incubation medium for 24 h, the viability remains greater ssDNA by normalization to the untreated 9TC control in (a) squares
than 70% (Figure S3). This indicates that PPET3N2 exhibits and (b) dots, respectively. [Dnase I] = 0.01 U/μL.
low cytotoxicity, rendering it suitable for various biomedical
applications. Considering the necessity of performing follow-
up experiments to evaluate the viability of MCF7 cells
incubated with different ratios of PPET3N2/9TC, we opted Intracellular Distribution of the CPE/ssDNA Poly-
for a PPET3N2 concentration of 5 μM. By maintaining low plexes. To assess the cellular uptake of the CPE/ssDNA
toxicity on MCF7 cells, the PPET3N2/9TC polyplexes polyplexes, we conducted CLSM imaging on MCF7 cells
showcase their compatibility and safety for subsequent cell transfected with PPET3N2/9TC-A polyplexes (molar ratio =
culture experiments. 5:3; [9TC-A] = 3 μM) and 3 μM 9TC-A. A control group
It is challenging to develop nucleic acids as therapeutics using 3 μM 9TC-A and 5 μM PPET3N2 for transfection was
also included. The PPET 3 N 2 /9TC-A polyplexes were
owing to the susceptibility of natural nucleic acids to
incubated with MCF7 cells in the medium for 2, 6, 12, and
degradation by nucleases. Several approaches have been
24 h before CLSM imaging. The CLSM images demonstrate
proposed to overcome this obstacle, such as chemical
the entry of both the PPET3N2/9TC-A polyplexes and 9TC-A
modifications on natural nucleic acids, construction of self-
into MCF7 cells (Figure 3). With increasing incubation time,
assembled DNA nanostructures, and integration with various the red puncta signal from both groups intensified, indicating
types of nanomaterials (e.g., lipid-based vectors, inorganic ongoing cell uptake. Notably, observations from the red
nanoparticles, or polymetric materials).5 To assess this, we channel and merged images revealed a stronger fluorescence of
performed a nuclease degradation experiment using both 9TC-A in the cytoplasm of cells treated with PPET3N2/9TC-A
naked 9TC and PPET3N2/9TC polyplexes (Figure S4, Figure polyplexes compared to those treated with 9TC-A alone. This
2). Both samples were exposed to 0.01 U/μL DNase I at 37 °C highlights the superior efficiency of the CPE/ssDNA trans-
for various time intervals. Subsequently, aliquots were collected fection system. By comparing the results from the two groups
and analyzed using a denaturing urea-PAGE assay, which at the same time intervals, it is evident that the PPET3N2/
separates unstructured ssDNA molecules based on their 9TC-A group consistently exhibits a slightly stronger red
molecular weight. The results showed that naked 9TC was fluorescence signal than the 9TC-A group, indicative of a
completely degraded after 5 min of incubation. However, the higher cell entry efficiency for the PPET 3 N 2 /9TC-A
PPET3N2/9TC polyplexes demonstrated significant stabiliza- polyplexes. These findings collectively demonstrate the
tion and resistance to nuclease degradation compared to naked enhanced cell uptake efficacy of the PPET3N2/9TC-A polyplex
9TC (Figure 2). These findings indicate that PPET3N2 system compared to 9TC-A alone.
provides essential protection to ssDNA against nuclease The improved nuclease resistance and membrane pene-
digestion, prolonging its lifetime for more than 60 min. This tration efficiency observed with the PPET3N2/9TC polyplexes
might attribute to the conjugated structure and positive are mainly attributed to the protective effect of PPET3N2
charges of PPET3N2, which can form a protective barrier against nuclease degradation and its ability to form stable
around the ssDNA by means of hydrophobic interaction and polyplexes with the nucleic acids. These factors enhance the
electrostatic attractions, thus to shield it from enzymatic stability and integrity of the nucleic acids, allowing them to
degradation, and prolong lifetime of ssDNA in the presence of successfully cross the cell membrane and improve the cellular
PPET3N2. uptake.
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Figure 3. CLSM images of MCF7 cells incubated with PPET3N2/9TC-A polyplexes (molar ratio = 5:3, [9TC-A] = 3 μM) and 9TC-A (3 μM) for
various times (2, 6, 12, and 24 h). Green channel (PPET3N2), λex = 488 nm and λem = 500−600 nm; blue channel (DAPI), λex = 405 nm and λem =
425−475 nm; red channel (9TC-A), λex = 559 nm and λem = 575−675 nm. Scale bars are shown in the images.

Figure 4. Degradation of ERα in the MCF7 cells. Western blot evaluation of ERα levels in response to (a) PPET3N2/P9TC polyplexes (molar ratio
= 5:3; [P9TC] = 3 μM) or (c) P9TC (3 μM) after 0, 3, 6, 9, 12, and 24 h of incubation. (b, d) Quantification of ERα levels by normalization to the
β-actin (columns), and relative ERα degradation ratio (dots) in panels a and c, respectively. Values are means ± standard error of the mean of three
independent measurements.

Validation of Protein Degradation by PPET3N2/P9TC aptamer−PROTAC molecule (P9TC) by connecting a ligand

Polyplexes. Since we have demonstrated the high transfection for ubiquitin E3 ligase (VH032) and 9TC sequence via the
efficiency of the CPE/ssDNA system, we speculated that this CuAAC reaction (see the SI). A PROTAC molecule is a
system may promote the efficiency of ssDNA biofunctionality hetero-bifunctional compound that can recognize target
realization. As shown in Scheme 1, we constructed an proteins and result in the degradation of the target by
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ACS Applied Materials & Interfaces
ubiquitination.36,37 Ubiquitination is a post-translational
modification process in which the small protein ubiquitin is
attached to other proteins. This modification plays a crucial
role in regulating various cellular processes such as protein
degradation, signal transduction, DNA repair, and the immune
response. Ubiquitination can occur in different cellular
compartments, depending on the specific targets and functions
involved. For example, the proteasome is the organelle
primarily responsible for ubiquitination in protein degradation.
Figure S5 indicates that polyplexes were internalized into cells
through endocytosis pathway and accumulated in lysosome
within 4 h of incubation, which is similar to our previous work
on the polymer PPET3N2.28 The 9TC sequence can recognize
and specifically bind to ERα, while VH032 engages the E3
ubiquitin ligase to ERα. Recruitment of the E3 ligase to ERα
results in the ubiquitination and subsequent degradation of
ERα by the proteasome. We evaluated the rate of ERα
degradation facilitated by PPET3N2/P9TC polyplexes via a
time course experiment. We incubated MCF7 cells with the
polyplexes for various times and measured the abundance of
ERα by Western blot; cells treated with naked P9TC were
used as controls. Compared with cells treated with naked
P9TC, very potent degradation was observed in the group
treated with PPET3N2/P9TC polyplexes (Figure 4). The
group treated by PPET3N2/P9TC polyplexes gets a satisfactory
ERα degradation effect (95.3%) after only 9 h of treatment. In
comparison, naked P9TC reached only 69.4% degradation
after 24 h of treatment. The polyplexes showed significantly
enhanced ssDNA delivery capacity and high protein
degradation efficiency. All of these results were aligned with
our hypothesis, indicating the potential of PPET3N2 as a
powerful enhancer for a simple, efficient, and biocompatible
nucleic acid delivery platform.
■ CONCLUSIONS
In summary, we present an efficient nucleic acid delivery
system based on CPE/ssDNA polyplexes, further extending
the application in efficient protein degradation (utilizing the
PROTAC technique). PPET3N2 and 9TC (ssDNA) are
directly mixed to form PPET3N2/9TC polyplexes through
electrostatic and hydrophobic interaction. We investigated the
distance between PPET3N2 and 9TC-A through FRET
calculation, short separate distance indicates good affinity
between PPET3N2 and 9TC-A. Both MTT assay, and nuclease
resistance experiments indicated CPE/ssDNA polyplexes
possess good biocapacity. CLSM imaging revealed that
polyplexes exhibited enhanced cellular internalization com-
pared to naked ssDNA. Highly positive charged side chains of
PPET3N2 enable neutralization of negatively charged nucleic
acids and facilitate their permeation across the negatively
charged cell membrane. In addition, remarkable acceleration of
protein degradation was observed when we utilized PPET3N2
as P9TC-delivery enhancer; the polyplex group takes 12 h less
than the naked P9TC group to reach the same degradation
degree. This delivery platform exhibits facile construction, low
toxicity, and high efficiency and demonstrates promising
potential for application in a wide range of nucleic acid
delivery systems.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsami.3c14640.
G https://doi.org/10.1021/acsami.3c14640
www.acsami.org Forum Article
Synthesis of P9TC; FRET efficiency and separate
distance calculation; summary of FRET efficiency and
separate distance calculation; characterization of
PPET3N2; characterization of P9TC by urea-PAGE
analysis; hydrodynamic diameter, ζ potential and TEM
characterization; MTT assay results for cell viabilities;
protection of PPET3N2/9TC polyplexes against nuclease
degradation; colocalization of lysosome and PPET3N2/
9TC polyplexes (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Chunyan Tan − The State Key Laboratory of Chemical
Oncogenomics, Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, People’s Republic of
China; Open FIESTA, Shenzhen International Graduate
School, Tsinghua University, Shenzhen 518055, People’s
Republic of China; orcid.org/0000-0002-4263-2175;
Email: tancy@sz.tsinghua.edu.cn
Naihan Xu − The State Key Laboratory of Chemical
Oncogenomics, Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, People’s Republic of
China; School of Food and Drug, Shenzhen Polytechnic
University, Shenzhen 518055, People’s Republic of China;
Email: xu.naihan@sz.tsinghua.edu.cn
Authors
Yuanjie Sun − The State Key Laboratory of Chemical
Oncogenomics, Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, People’s Republic of
China
Li Jiang − State Assets Management Office, Shenzhen
Polytechnic University, Shenzhen 518055, People’s Republic
of China
Zhilin Zhang − The State Key Laboratory of Chemical
Oncogenomics, Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, People’s Republic of
China
Yuyang Jiang − The State Key Laboratory of Chemical
Oncogenomics, Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, People’s Republic of
China; orcid.org/0000-0003-3904-4855
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsami.3c14640
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work is supported by the State Key Laboratory of
Chemical Oncogenomics, and the Shenzhen Science and
Technology Innovation Commission (JCYJ
20220530142812029).
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