Macrocyclic polyamine [12]aneN3modified triphenylamine-pyrazine derivatives as efficient non-viral gene vectors with AIE and two-photon imaging properties

Article abstract of DOI:10.1039/d0tb00321b

With the aim to develop a novel multifunctional gene delivery system that may overcome the common barriers of gene transfection, near-infrared fluorescent triphenylamine-pyrazine was modified with a DNA condensing triazole-[12]aneN₃moiety through different length alkyl ester linkages to afford three new non-viral gene vectors,TDM-A/B/C. All compounds showed prominent solvatochromic fluorescence (Stokes shift of up to 383 nm) and two-photon absorption properties (σ2P to 101 GM), and exhibited strong aggregation-induced emission (AIE). Gel electrophoresis demonstrated that plasmid DNA was completely condensed at a concentration of 10 μM (TDM-A), 14 μM (TDM-B) and 16 μM (TDM-C), and released in esterase and acidic environment. SEM demonstrated that the three compounds were able to self-assemble and co-aggregate with DNA to form regular nanoparticles. Experiments demonstrated thatTDM-A/B/Cwas able to integrate with DNA through electrostatic interactions and supramolecular stacking, and the short alkyl linkage favored the strong interaction with DNA. Among the three compounds,TDM-Bshowed the best luciferase and GFP transfection activities in the presence of DOPE, which were 156% and 310% higher than those of Lipo2000, respectively. The transfection process of DNA was clearly traced through one- and two-photon fluorescence microscopy imaging. Cellular uptake inhibition assay indicated that the DNA complex entered the cell mainlyviaclathrin-independent endocytosis. Furthermore, thein vivotransfection experiments ofTDM-B/DOPE were successfully implemented in zebra fish embryos, and the GFP gene expression level was superior to that of Lipo2000 (200%). Finally, this study clearly unraveled that the length of the alkyl linkage affected the DNA condensation and transfection activity, which can serve as a base for the future rational design of non-viral gene vectors.

Full text of DOI:10.1039/d0tb00321b

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ARTICLE
Macrocyclic Polyamine [12]aneN3 Modified Triphenylamine-
Pyrazine Derivatives as Efficient Non-viral Gene Vectors with AIE
and Two-photon Imaging Properties
Received 00th January 20xx,
Le-Le Ma,a Ming-Xuan Liu,a Xu-Ying Liu,a Wan Sun,a Zhong-Lin Lu,*,a Yong-Guang Gao,*,b and Lan
He *,c
With the aim to develop a novel multifunctional gene delivery system that may overcome the common barriers of gene
transfection, the near-infrared fluorescent triphenylamine-pyrazine was modified with DNA condensing moieties triazole-
[12]aneN3 through the different length alkyl ester linkage to afford three new non-viral gene vectors, TDM-A/B/C. All
compounds showed prominent solvatochromic fluorescent (stocks shift up to 383 nm), two-photon absorption properties
(σ2P to 101 GM), and exhibited strong aggregation-induced emission (AIE) property. Gel electrophoresis demonstrated that
plasmid DNA were completely condensed at a concentration of 10 μM (TDM-A), 14 μM (TDM-B) and 16 μM (TDM-C), and
released under esterase and acidic environment. SEM demonstrated the three compounds were able to be self-assembled
and co-aggregated with DNA to form regular nanoparticles. Experiments demonstrated that TDM-A/B/C were able to
integrate with DNA through electrostatic interactions and supramolecular stacking, and short alkyl linkage favored to the
strong interaction with DNA. Among the three compounds, TDM-B showed the best luciferase and GFP transfection activities
in the presence of DOPE, which were 156% and 310% higher than those of Lipo2000. The transfection process of DNA was
clearly traced through one- and two-photon fluorescence microscopic imaging. Cellular uptake inhibition assay indicated
that the DNA complex entered the cell mainly via clathrin-independent endocytosis. Furthermore, the in vivo transfection
experiments of TDM-B/DOPE were successfully implemented in zebrafish embryos, and the GFP gene expression level was
superior to Lipo2000 (200%). Finally, this study clearly unraveled that the length of the alkyl linkage affected the DNA
condensation and transfection activity which can be served as a base for future rational design of non-viral gene vectors.
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
synthesized to satisfy different needs of gene delivery.12 During
the past decades, various types of non-viral vectors have been
extensively investigated such as lipids, polymers, dendrimers
and inorganic nanoparticles, and greatly progress has also been
achieved.13.14 Nevertheless, the low transfection efficiency of
non-viral vectors remains a critical issue, the design and
synthesis of non-viral vectors are still highly needed for the
efficient transfection.15-17
1. Introduction
Gene therapy is full of promise in treating human diseases, like
cancers and gene defects ， which is strongly determined by
gene delivery systems.1-3 A desirable vector can promote the
gene cargo delivering into target cells as well as keeping the
packaged therapeutic genes from the biodegradation, and also
the efficient release of the carried genes in cytoplasm or
nucleus.4,5 Engineered viruses were first used in gene delivery
systems for the satisfactory results revealed in several initial
clinical trials.6,7 However, their intrinsic immunogenicity,
notable toxicity, and mutagenic effects hindered their wide
applications.8-11 For this reason, non-viral vectors have received
Moreover, it is also very important for vectors to effectively
elucidate DNA cellular uptake mechanisms during the delivery
process, including internalization pathway, and endosomal
release. To track gene delivery processes, coumarin,
naphthamide, tetraphenylethene and other fluorophores have
been incorporated in various non-viral gene vectors.18-21
However, those commonly used fluorophores still encounter
lots of obstacles for their poor sensitivity and high background
interference. At present, far-red and near-infrared (NIR)
fluorescent materials not only possess the characteristics of
specific targeting capability but also have aggregation-induced
emission (AIE) and strong two-photon absorption are much-
sought-after for therapeutic and bioimaging applications,
because of their high resolution and deep tissue penetration.22-
a. a Key Laboratory of Radiopharmaceutics, Ministry of Education; College of
Chemistry, Beijing Normal University, Beijing 100875, P. R. China.
b. b Lab for Bone Metabolism, Key Lab for Space Biosciences and Biotechnology,
School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi,
710072, China.
c. c China National Institute for Food and Drug Control, Institute of Chemical Drug
Control, TianTanXiLi 2, Beijing, 100050, China.
*Corresponding authors: luzl@bnu.edu.cn (Zhong-Lin Lu), helan1961@aliyun.com
(Lan He) gaoyongguang@nwpu.edu.cn (Yong-Guang Gao)
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
24
Accordingly, it is of the greatest interest to design novel
vectors with the property of far-red and NIR emission, strong
two-photon absorption and therapeutic effect. DCDPP-2TPA
increased attention because they can be tailored and
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(Scheme 2) is
a
type of aggregation-induced emission scattering (DLS), and scanning electronic microscopy (SEM)
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DOI: 10.1039/D0TB00321B
luminogen (AIEgen) with deep-red emissive, which were utilized techniques, the relationships between the linkage length of the
as contrast agents for intravital imaging of mouse cerebral three derivatives and their ability toward DNA sensing,
vasculature without craniotomy and skull-thinning.25 condensation, delivery, and transfection were studied in detail.
Structurally, the rational design of gene vectors base on the The three vectors could efficiently deliver plasmid DNAs (pGL-3
understanding of their structure
−property relationship. To and pEGFP-N1 reporter genes) into three type cells for genetic
promote the transfection efficiency, one of the most common expression, and EGFP gene was even successfully transfected in
methods is to modify different domains of the vectors, like vivo by TDM-C. The DNA delivery process were traced clearly
hydrophilic domains, linker domains and hydrophobic through one- and two-photon fluorescence microscopic
domains.26-29 Among the modifications of these domains, the imaging.
hydrophobic modification as an effective and straightforward
way has been established to promote endosomal escape and
improve gene transfection.30,31 However, the current
hydrophobic modification studies were aimed at changing the
length and number of aliphatic chains, and the effect of linker
domains on the transfection is still rarely been reported.
2. Materials and methods
2.1. Materials
Unless otherwise specified, all the chemicals and analytical
pure solvents were purchased from Energy Chemica (Shanghai,
China), and used without further purification. Anhydrous
ethanol, methanol, tetrahydrofuran (THF), dichloromethane
(DCM) and N,N-dimethyl formamide (DMF) were dried and
purified under nitrogen by using standard methods and were
distilled immediately before use. Goldview II, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT),
electrophoresis grade agarose, 6× loading buffer (30 mM EDTA,
40% glycerol, 0.03% xylene cyanol FF, and 0.05% bromophenol
blue), ethidium bromide (EtBr), ctDNA (calfthymus DNA),
pUC18 (plasmid DNA), Hochest 33342, micro BCA protein assay
Taking into account of the above considerations, we recently
designed three novel non-viral gene vectors base on DCDPP-
2TPA with different molecular structures via the esterification
procedure and subsequent linker-chain functionalization using
click chemistry, and their structure-property relationships
toward gene delivery were systematic explored. Particularly,
the strategy for exploiting ester bond break by hydrolysis to
release DNA provides a promising approach for efficient
transfection efficiency (Scheme 1). Through fluorescence
spectroscopy, agarose gel electrophoresis (AGE), dynamic laser
Scheme 1 Schematic illustration of the preparation and Characterization of liposome (TDM-B/DOPE) for Gene Delivery and transfection. (1) Self-assembly with DOPE.
(2) DNA condensation (pH 7.4). (3) Cellular uptake via Endocytosis. (4) Endosomal escape in cytoplasm. (5) Control of ester bond cleavage and DNA release by lipase
and pH 5.0. (6) Nucleus importation of DNA and DNA complex. (7) DNA release in nucleus. (8) Gene expression.
kit and luciferase assay kit were purchased from Solarbio Mauricio Minotta, USA). Ultrapure milli-Q water (18.25 MΩ)
Company (Beijing, China). DOPE (Dioleoylphosphatidyl was used in critical micelle concentration (CMC) and dynamic
ethanolamine) was from Santa Cruz Biotechnology (USA). light scattering (DLS) tests. Tris-HCl buffer (pH 7.4) was used to
Lipofectamine 2000™ was from Invitrogen (Life technologies, conduct fluorescent titrations, gel electrophoresis at room
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temperature. 6-carboxy-fluorescein FAM-labeled double- 33342 (blue), FITC filter for FAM (green), and TRITC filter for
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DOI: 10.1039/D0TB00321B
stranded oligomer 5’-GGTCGGAGTCAACGGATTTGGTCG-3’, compounds (red), respectively. The CLSM images of 0.5 h, 2 h
(FAM-DNA) and pGL-3 DNA were purchased from Ruibiotech and 4 h were obtained after incubation of the cells with
Co., Ltd. (Beijing, China). Plasmid pEGFP-N1 was purchased condensates for different durations.
from Clontech (Palo Alto, CA, USA).
2.4. Endocytosis inhibition studies
The concentrations of ctDNA were determined by recording
absorptions at 260 nm and applying Lambert−Beer’s Law (molar
absorption coefficient ε = 6600 M-1 cm-1). 1H and 13C NMR
spectra were obtained on a BrukerAvance III 400 MHz
spectrometer at 25 ºC using CDCl3 and DMSO-d6 as solvents and
calibrated using tetramethylsilane (TMS) as an internal
reference. HRMS (High resolution mass spectra) were obtained
on a Waters LCT Premier XE spectrometer (USA). Agarose
electrophoresis was performed using a BG-subMIDI sub marine
system (BayGene Biotech Company Limited, Beijing, China) and
the electrophoresis images were obtained on a UVP EC3 visible
imaging system (USA) using 254 nm UV light for visualization.
The average particle sizes and zeta potentials of lipoplexes/DNA
complex were detected by dynamic light scattering (DLS) on a
Brookhaven Zeta Plus Partical Size and Zeta Potential Analyzer
(USA). Fluorescence quantum yield was measured with an
Absolute PL Quantum Yield Spectrometer QY C11347-11. Flow
cytometry assays were performed on a CytoFLEX (Beckman-
coulter, USA). Scanning electron microscopy (SEM) images were
Hela cells were first preincubated with 50 µM CPZ, 2.5 mM
MβCD or 75 µM AM for 1h, then 1 mL of complexes in DMEM
o
was added to each well. After 2 h incubation at 37 C, FAM
positive cells were compared, determining which inhibitor
played a critical role in decreasing the cellular uptake.
Additionally, to study whether the cellular uptake of TDM-
B/DOPE is energy-dependent, the polyplex was added to the
cells, and cultured at 4 oC for 2h.
2.5. GFP transfections in vivo.
Zebrafish embryos were provided by the China Zebrafish
Resource Center. The TDM-B/DOPE-DNA complexes formed by
10 μg/mL eGFP DNA and 20 μM of TDM-B/DOPE in water for 30
min. Zebrafish embryos were incubated with 1 mL solutions
containing the complexes for 5 h, then 1 mL fresh water was
added and further incubated for 24 h. The fluorescence
intensity of GFP was measured by confocal imaging, and
Lipo2000 was used as a control. The relevant ethical protocols
used for the in vivo study for zebrafish embryos were followed
by the relevant laws. All experimental protocols were approved
by the animal care committee of Beijing Normal University.
obtained with
a
Hitachi S-4800 (Japan) instrument.
Fluorescence spectra were determined on a Hitachi F-4600
spectrophotometer. Absorption spectra were determined on a
Shimadzu UV-1601PC UV-Visible spectrophotometer. Confocal
2.6. Gel Electrophoresis and Other Cellular Experiments.
fluorescence imaging was performed with Nikon A1R MP Other experiments including agarose gel electrophoresis, ionic
multiphoton microscopy with a 60×oil-immersion objective strength effect, releasing the compact DNA, EB displacement
lens. Two-photon excitation fluorescence cross section was assay, cell toxicity, and cell transfection were conducted
measured by two-photon excitation fluorescence method using according to the procedures in reference. 14,19
rhodamine B as a reference. All the solutions were measured in
a 3.5 mL (10 × 10 mm), 100 and 50 μL quartz cuvettes.
2.2. Fluorescence Measurements
3. Results and discussion
3.1. Molecular design, synthesis and characterization
Stock solutions (1 mM) of TDM-A/B/C were prepared in
deionized Milli-Q water (18.25 MΩ). Stock solutions were
diluted to certain volumes in volumetric flasks with appropriate
aliquots and deionized Milli-Q water, and Tris-HCl buffer, and
solutions of different concentrations were obtained. The DNA
fluorescence titrations were performed using race-volume (100
or 50 μL) quartz cuvettes. After vortex mixed, all the samples
allowed to rest for 6 - 10 min before testing.
As depicted in Scheme 2, three 5,6-bis(4′-(diphenylamino)-[1,1′-
biphenyl]-4-yl)pyrazine-2,3-diester
modified
[12]aneN3
derivatives were originally designed and synthesized. In the
molecules, the fluorophore structure were connected with
triazole-[12]aneN3 units through various length of alkyl chain.
The AIE fluorophore DCDPP-2TPA (compound 1) were prepared
according to literature methods.25,32 The conversion of cyano
groups to carboxylic acids was achieved by hydrolysis in a strong
base (6M KOH aq.) and further acidification to afford
compounds 2. Compound 2 were esterified with alcohols
containing different length alkyl chain and an azido ending to
give compounds 3a-3c. The Propargyl [12]aneN3 was
2.3. Cell-uptake of liposome
The cellular uptakes of FAM-DNA (9 μg/mL) condensates were
observed by fluorescence microscopy. Hela cells were cultured
in DMEM medium supplemented with 10% FBS in a humid
atmosphere containing 5% CO2 at 37 °C. The cells were seeded
in Glass Bottom Cell Culture Dishes at 1000 cells per dish and
cultured for 24 h. After washing three times with DMEM, the
cells were treated with freshly prepared compounds condensed
FAM-DNA and the controls (500 μL) of DMEM. The Hochest
33342 (5 μg/mL) was also added to the cells for nuclear staining
o
at 37 C for 15 min. Finally, the cells were washed for 5 times
with PBS buffer, observed using a confocal laser scanning
microscope with a 40× objective and DAPI filter for Hoechst
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attributed to the charge transfer (CT) transition absorption of
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the large conjugate system (Fig. S1). ThDeOwI: a10v.e10le3n9/gDt0hTsBo00f3t2h1Be
absorption maximum of TDM-A/B/C changed
a
few
nanometers along the increase of the solvent polarity. On the
contrary, the fuorescence spectra of the three compounds
exhibited a very pronounced bathochromic-shift up to 383nm
(from 547 nm to 748 nm). Simultaneously, the emission color
changed from green to orange under 365 nm UV irradiation.
The change of the fluorescent maximum with the solvent
polarity parameter (ET(30)) were studied (Fig. 2S),22,33 the linear
relationship was founded with the slopes to be 16.970, 17.660,
and 17.445 for TDM-A/B/C, respectively. The above results
indicate that the emission properties of TDM derivatives were
strongly dependent on the solvent polarity, which could be
likely ascribed to the TICT processes in these D-π-A structure
molecules.22 The absorption and fluorescent spectra of the
three compounds are similar to those of DCDPP-2TPA in
Scheme 2 Schematic route for TDM-A, TDM-B and TDM-C: (i) NaN3, DMF, 65 ºC, 8 h; (ii) literatures,33 but both the spectra showed a blue-shift relative
NaOH (6 M), Reflux, 24 h; (iii) HCl (2 M); (iv) NMM, EDCI, HOBT, TEA, r.t., 8 h; (v) Propargyl
[12]aneN3, CuBr, CHCl3, r.t., 30 min; (vi) HCl-EtOAc, DCM, r.t., 2h.
to that of DCDPP-2TPA, which can be attributed to the weaker
electro-withdrawing property of the ester groups in the three
synthesized compounds when comparing with cyano groups in
DCDPP-2TPA.
prepared according to literature method we reported.14
Through the highly efficient alkyne
−azide click reactions
TDM-A/B/C with strong push −pull dipolar structures are
characterized by interesting nonlinear optical properties, in
particular, their two-photon absorption properties can be
expected.35,36 Thus, the two-photon excitation spectra of three
compounds were measured in THF, and rhodamine B was used
as the standards (Fig. S3 and Fig. S4). The emission signals were
collected upon excitation from 720 to 800 (at 20 nm intervals).
Impressively, the slopes of the linear relationships between
laser power and the logarithm of fluorescence intensity were
calculated to be 1.87, 1.88, and 1.82 (Fig. 2). In addition, they
exhibit a maximum two-photon absorption cross-section (σ2P)
of 109, 101, and 94 GM at 800 nm for TDM-A/B/C, respectively,
which are beneficial to their biological application.
catalyzed by Cu(I) and subsequent deprotections, the target
compounds, TDM-A/B/C, were successfully synthesized as their
hydrochloride salts. All the synthesized compounds were
purified and further confirmed by 1H NMR, 13C NMR, FT-IR, and
HR-MS (shown in the Supporting Information).
3.2. Solvatochromism and two-photon properties
The two triphenylamine groups (D, donor) were introduced into
pyrazine-2,3-diester skeleton (A, acceptor) with phenyl unit to
constitute the D-π-A conjugation. Thus, the photo-physics
property of these compound is strongly dependent on the
solvent
polarity.
Therefore,
the
absorption
and
photoluminescence (PL) spectra of TDM-A/B/C in solvents with
different polarities were investigated, the results were
summarized in Fig. 1 and Table S1. The maximum absorption
wavelength of the three compounds were found to locate at ca.
400 nm in the various frequently used solvents, it can be
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Fig. 1 (A−C) Fluorescence spectra of TDM-A (A), TDM-B (B) and TDM-C (C) in different solvents. (D−F) Images of TDM-A (D), TDM-B (E) and TDM-C (F) in different solvents
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DOI: 10.1039/D0TB00321B
taken under a UV 365 nm lamp. Concentrations: 10 µM, λex = 410 nm
.
108-fold higher than those in the pure water solution,
respectively (Fig.
Fig. 2 Relationship between logarithm integrated intensity and logarithm laser power of
TDM-A (A), TDM-B (B), and TDM-C (C) in THF. (D) Image of two-photon absorption (2PA)
of the three TDM derivatives in THF solution. Concentration: 10 μM. Rhodamine B used
as the standards.
Fig. 3 Fluorescence spectra of TDM-A (A), TDM-B (B), and TDM-C (C) in water/THF
mixtures with different THF fractions (fo). Inset: Photographs of TDM-A/B/C in
THF/water mixtures under the UV 365 nm lamp. (D) Plots of fluorescent intensities
versus THF fractions for TDM-A/B/C in water/THF mixtures. Concentration: 10 μM; λex
410 nm.
=
TDM-A/B/C with strong push −pull dipolar structures are
characterized by interesting nonlinear optical properties, in
particular, their two-photon absorption properties can be
expected.35,36 Thus, the two-photon excitation spectra of three
compounds were measured in THF, and rhodamine B was used
as the standards (Fig. S3 and Fig. S4). The emission signals were
collected upon excitation from 720 to 800 (at 20 nm intervals).
Impressively, the slopes of the linear relationships between
laser power and the logarithm of fluorescence intensity were
calculated to be 1.87, 1.88, and 1.82 (Fig. 2). In addition, they
exhibit a maximum two-photon absorption cross-section (σ2P)
of 109, 101, and 94 GM at 800 nm for TDM-A/B/C, respectively,
which are beneficial to their biological application.
S6). These are very typical phenomena for compounds bearing
AIE characteristic.37,39 Clearly, the aggregation-caused
restriction rotational motions of molecular rotors (such as the
phenyl rings of the triphenylamine moiety and pyrazine moiety)
was responsible for the observed emission enhancement. These
results demonstrated that TDM-A/B/C are typical AIE-active
molecules. Fluorescent emission images displayed the
immediate visualization fluorescent changes under 365 nm UV
light.
Scanning electron microscopy (SEM) and dynamic light
scattering (DLS) were further investigated to confirm the
formation of aggregated nanoparticles with high THF fraction.
The samples for the SEM measurement were prepared by
adding THF to aqueous solution of the three compounds, and
the well-assembled particles were formed after the THF/water
mixtures stand for over 12 h, the regular nanoparticles were
obtained with sizes of about 70−150 nm (Fig. S7). The results
indicated the molecules undergo aggregation into a kind of
particular arrangement in the THF/water mixtures.40 The
average diameters of the nanoparticles measured in solution by
using DLS were 99, 141, and 194 nm for TDM-A, TDM-B, and
TDM-C, respectively (Fig. S8), which were conformity with the
SEM measurements.
3.3. Aggregation Induced Emission
To confirm whether the three compounds are AIE-active, their
emission properties was studied in THF/water mixtures with
different THF fractions (fo) in volume (vol %) under an excitation
of 410 nm. Unlike most reported typical AIEgens which are poor
soluble in water, TDM-A/B/C are soluble in water, but they are
virtually nonluminescent in aqueous solution. Their emission
intensity is subjected to organic solvent content (fo, THF
fraction). As shown in Fig. 3 and Fig. S5, with increasing fo in
THF/water mixtures from 0 to 20%, the weak fluorescence of
TDM--A/B/C (around 590 nm) enhanced gradually. When the
THF fraction was increased to 30%, the maximal fluorescence
intensities of TDM--A/B/C were decreased accompanied by a
red-shift from 590 to 660 nm, which can be attributed to the
change of the aggregation morphology.18,37,38 The emission
intensity of compounds further enhanced by the addition of THF
fractions from 30% to 99% with a blue-shift from 650 to 610 nm.
When the proportion of THF reached 99%, the maximum
emission intensity of compounds TDM-A/B/C were 72, 81 and
3.4. Gel Electrophoresis Assays
In the course of gene transfection, it is very important to
transform the negatively charged DNA into nanosized particles,
which can reduce the electrostatic repulsion between DNA and
cell surface. Furthermore, the formation of particles can also
protect DNA to resist the degradation by nucleases in cytoplasm
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or serum. Efficient condensation ability is the prerequisite for TDM-A/B/C. As shown in Fig. 4, the concentrationVfioewr AcroticmlepOlnelitnee
non-viral gene vectors. Thus, gel retardation assays were retardation with plasmid DNA (pDNA) byDTODIM: 10-.1039/D0TB00321B
performed to test the DNA condensation ability of compound
Fig. 4 Agarose gel electrophoresis assay of pUC18 DNA retardation induced by different concentrations (labeled on the top, μM) of TDM-A/B/C in the absence (A,C, E) and presence
of DOPE (B, D, F) in 50 mM Trips-HCl (pH 7.4). The molar ratio of TDM derivatives/DOPE was 1:2, [DNA] = 9 μg/mL, 37 °C; SEM images of condensed pUC18 DNA (9 μg/mL) by TDM-
A/B/C in Tris-HCl buffer (5 mM, pH 7.4): (A) TDM-A, 10 μM; (B) TDM-B, 14 μM; (C) TDM-C, 16 μM. Scale bars: 1 μm.
A/B/C were 10, 14, and 16 μM, respectively. The effective potentials of the nanoparticles were lower than most report
condensation ability can be ascribed to the electrostatic gene carriers, which can enhance the DNA stability in serum and
neutralization between the two triazole-[12]aneN3 units and decrease the cytotoxicity.42
the negatively charged DNA phosphate groups, as well as the
At present, numerous tactics for triggering the dissociation
hydrophobic interactions of the two triphenylamine moiety and of DNA from the condensing state have been developed.
the aliphatic chain linkage. It was observed that TDM-A Herein, we investigated the pH effect or the presence of
containing shorter alkyl chain linkage showed lower esterase in the release of the compacted DNA. As shown in Fig.
concentrations for the complete condensation. Furthermore, it S11, the condensed DNAs were mostly released after treated at
was found that the complexes formed by mixing the three pH5 for 1h, suggesting that the condensation process by the
compounds with DOPE in a molar ratio of 1:2 further improved three compounds was reversible. Similarly, the addition of the
the condensation ability (Fig. 4D-F).41 To be noticed, choosing lipase also resulted in the release of the condensed DNAs. The
the ratio of 1:2 of the three compounds with DOPE was derived hydrolysis of ester bonds in the vectors should be responsible
from their good transfection performance (see 3.8).
for the release of DNA and subsequent transfection studies.
The condensing behaviors were further characterized by 3.5. Fluorescence response to ctDNA
SEM and DLS measurements. As shown in Fig. 4, all of TDM-
The calf thymus DNA (ctDNA) is a natural dsDNA which has been
A/B/C effectively condensed DNA into the regular square
nanoparticles with high dispersity and narrow size distributions,
which were suitable for cell endocytosis and subsequent gene
transfection.18,41 The average sizes of these condensates
obtained by DLS were 112, 103 and 111 nm (Fig. S9),
respectively, which were similar to the SEM results. Moreover,
the zeta potentials of the nanoparticles formed at the
concentration for the complete DNA condensation were found
to be 4.81, 8.92 and 6.18 mV, respectively (Fig. S10). The zeta
widely used in the research of biochemical, chemical and
medical science.43-46 As shown in Fig. S12, the emission
intensities of the three compounds gradually increased upon
the addition of ctDNAs, their fluorescent intensities were
almost linear-dependent to the ctDNA concentration with the
correlation coefficients of 0.999, 0.972 and 0.988, respectively
(Fig. S13). At the ctDNA concentration of 8 μg/mL, the
fluorescence enhancements were 6.5-, 4- and 4-fold of the
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original values for the three compounds (Table S2), indicating recognition of ctDNA by TDM-A/B/C showed a higher value of
View Article Online
that the shorter linker of hydrophobic chain is beneficial to the (I/I0 − 1) at the same concentration, whDicOhI:m10a.1y03b9e/Dc0aTuBs0e0d32b1By
interactions with DNA. Compared to the reported literature, the the multiple interactions between triazole[12]aneN3
Fig. 5 Cytotoxicity of HeLa (A), HepG2 (B), and HEK293T (C) cells cultured with different concentrations of TDM-A, TDM-B and TDM-C.
moieties and ctDNAs.19,21 The morphologies and sizes of the indicated that the three compounds are suitable as safe carriers
condensed nanoparticles were further characterized by SEM for delivering DNAs.
and DLS measurements. As shown in Fig. S14, SEM images 3.8. Cell Transfection
clearly demonstrated that ctDNA was effectively condensed
At first, quantitative evaluation of the transfection of the three
into regular nanoparticle by the three compounds. The mean
compounds were determined by luciferase assay in the absence
diameters of the nanoparticles measured by DLS measurements
or presence of DOPE, which is a naturally occurring lipid
frequently used as auxiliary lipid to elevate the endosomal
escape, and effectively enhance the transfection efficiencies. It
can be seen that the three compounds were able to transport
(Fig. S6) are 79 nm (TDM-A), 135 nm (TDM-B), and 175 nm
(TDM-C) (Fig. S15), which are consistent with those obtained by
SEM.
3.6. EB Assay Experiments
pGL-3 plasmid successfully into HeLa, HpeG2 and HEK293T for
The binding capabilities of TDM-A/B/C with DNAs were further genetic expression in the absence of DOPE. Clearly, the
evaluated by ethidium bromide (EB) exclusion assays.47-49 As concentrations largely influenced the transfection efficiency,
shown in Fig. S16, upon addition of TDM-A/B/C to the ctDNA- and the optimal concentrations were all located at 20-30 μM
bounded EB solution, the fluorescent intensities of the solution (Fig. S18). However, the overall transfection efficiencies were
were gradually diminished. The apparent binding constants poor, the best transfection efficiencies of TDM-A/B/C in Hela
(Kapp) of TDM-A/B/C were deduced from the quenching plots cell lines were only 1.1, 12 and 0.3% compared with those of the
(Fig. S17) to be 1.10 × 107, 0.99 × 107 and 0.86 × 107 M − 1
respectively. The result clearly proved the strong binding
,
commercial transfection reagent Lipo2000.
Then, the transfections of the liposome formed by TDM-
capabilities of these molecules interacted with ctDNA, the A/B/C and DOPE were explored. The cell transfections in Hela
shorter alkyl linkage is slightly beneficial to the binding affinity. cell lines with the liposomes of the three compounds and DOPE
Compared to reported literatures, the high Kapp value of the were investigated in detail (at different concentrations and
three compounds should be attributed to the synergetic different molar ratios). It was found that the liposome formed
contributions of the electrostatic interaction between the by mixing the three compounds with DOPE in a molar ratio of
positively charged compounds and the negatively charged DNA 1:2 afforded the best transfection efficiency, and the
phosphate backbones as well as the supramolecular stacking of transfection efficiencies of TDM-B/DOPE was 156% of that
TDM units with DNA through hydrophilic interactions, π−π achieved by Lipo2000 (Fig. 6). Results from the transfection
stacking, and hydrogen-bond interactions.18,50
3.7. Cytotoxicity
assays in Hela cells clearly demonstrated that the three
compounds hold great promise as non-viral gene carriers, and
TDM-B showed the best transfection efficiencies among the
three compounds.
To assess the biocompatibility of the three compounds, we
measured the cytotoxicity by 3-(4,5-dimethythiazol 2yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. As shown in Fig. 5,
the cellular viabilities of HeLa, HpeG2 and HEK293T cells were
investigated with increasing concentrations of the three
compounds over 4 h, respectively. It can be seen that the
increased cytotoxicity was observed with increasing feeding
dosages, but all of the cells still maintained above 70%
metabolic viabilities even at the concentrations up to 30 μM,
which could be attributed to presence of the biocompatible
triazole groups and the bio-reducible ester linkage in the
molecules. In addition, the cytotoxicity of the three compounds
were somewhat lower in Hela than in HpeG2 and HEK293T, and
TDM-B showed lower toxicity in all cell lines. The above results
Under optimal conditions, transfection assays mediated
were also carried out in HepG2 and HEK293T cells. As shown in
Fig. 7, among the three types of cancer cells tested, the best
transfection efficiencies are observed with the HeLa cell lines,
then the HEK293T cell lines, followed by the HepG2 cell lines.
The optimal transfection efficiencies compared with Lipo2000
were summarized in Table S3. The transfection efficiencies of
TDM-B/DOPE in HepG2 and HEK293T cells were 87, and 130%
that of Lipo2000, respectively. The above results indicated that
the three compounds with rigid TDM frameworks can act as
generally applicable non-viral gene vectors.
The visual transfections of the compounds were obtained
through the expression of pEGFP-N1 reporter gene in the Hela
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cell lines. The results indicated that the GFP genes were showed higher TEs than TDM-A and TDM-C. NotVeiewwoArrttichley,Ontlhinee
DOI: 10.1039/D0TB00321B
successfully delivered to the expression of GFP by the three transfection efficiency of TDM-B /DOPE was much better than
compounds (Fig. 8 and Fig. S19). It can be seen that TDM-B still
Fig. 6 Luciferase expressions transfected by liposomes TDM-A/DOPE(A), TDM-B/DOPE(B), TDM-A/DOPE(C), with molar ratios of 1:1-1:3 at varying concentrations in Hela cell lines.
Luciferase expressions transfected by liposomes TDM-A/DOPE, TDM-B/DOPE, TDM-A/DOPE, with molar ratios of 1:2 in HepG2 cell lines (D) and HEK293T cell lines(E). [pGL-3] = 10
μg/mL.
Based on the transfection results, several points can be
concluded. First, the liposomes of the three compounds with
DOPE efficiently delivered plasmid DNAs (pGL-3 and pEGFP-N1
reporter genes) into HepG2, HEK293T and Hela cell lines, the
Fig. 7 Result histogram of luciferase expressions mediated by TDM-A/B/C in the absence
and presence of DOPE under respective optimal conditions with Lipo2000 as control.
[pGL-3] = 10 μg/mL (A). GFP expression for TDM-A/B/C in the presence of DOPE in Hela
cells under respective optimal conditions with Lipo2000 as control (B). Concentrations
for the three compounds were 20 μM (TDM-A and TDM-B) and 30 μM (TDM-C); [pEGFP-
N1] = 10 μg/mL.
The visual transfections of the compounds were obtained
through the expression of pEGFP-N1 reporter gene in the Hela
cell lines. The results indicated that the GFP genes were
successfully delivered to the expression of GFP by the three
compounds (Fig. 8 and Fig. S19). It can be seen that TDM-B still
showed higher TEs than TDM-A and TDM-C. Noteworthy, the
transfection efficiency of TDM-B /DOPE was much better than
that acquired by Lipo2000. We also quantitatively evaluate the
GFP genes transfection efficiency of these compounds and their
liposomes by flow cytometry. Under optimal conditions, the
transfection efficiencies of TDM-A/DOPE, TDM-B/DOPE and
Fig. 8 Fluorescence microscope images (10×) of pEGFP-transfected in Hela cells. GFP was
observed with FITC filter (A); The inherent red fluorescence of TDM-A/B/C were
observed with the TRITC filter (B); Bright field (C); Merge of A, B, C. GFP (D). Lipo2000
TDM-C/DOPE were 1.8, 3.1 and 2.3 times of that by Lipo2000,
respectively. Simultaneously, the weak red fluorescence in the
red channel were produced by the AIE phenomenon after the
release of DNA from the liposomes.
and naked pEGFP-N1 were used as control. Concentration = 20 μM; [pEGFP-N1] = 10
μg/mL, scale bar: 100 μm.
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transfection efficiencies in HeLa cells were much higher than visualized through the red channel and green channel,
View Article Online
those in A549 and HepG2 cells. Considering that different cell respectively. In order to locate the DNADcOoIm: 1p0l.1e0x3,9t/hDe0TnBu00c3le2u1Bs
types have distinct pathways, the different transfection was stained by Hoechst 33342, a nucleus-specific blue
performance of the three compounds among various cell lines fluorescent dye. As shown in Fig. 10, only a small fraction of
is reasonable. Secondly, the best transfection efficiencies blue-emission condensates entered into the cells at 0.5 h, and
obtained for TDM-B/DOPE in a molar ratio of 1:2 were the condensates mainly accumulated near the cell membrane.
significantly higher than that of Lipo2000, which came up to
After 1 h of incubation, more condensates transferred into the
Fig. 9 Cellular uptake of TDM-B/DOPE-DNA complexes incubated at 37 and 4 °C (A). The
internalization of TDM-B/DOPE-DNA complexes in the presence of different inhibitors
(B). [TDM-B] = 20 μM, [pEGFP-N1] = 10 μg/mL.
1.54 times for luciferase expressions and 3.1 times GFP
expressions, respectively. Thirdly, it clearly demonstrated that
the transfection efficiencies of TDM-A/B/C are closely related
to the alkyl length of the linkage between the TDM unit and the
triazole-[12]aneN3, the eight carbon alkyl linkage exhibited
good transfection efficiencies.
3.9. Cellular uptake mechanism study
The vectors’ performance is closely related to the intracellular
kinetics, the detailed polyplex cellular uptake level,
internalization pathway, and DNA release need further
definition. As a gene delivery system, the internalization
pathway of TDM-B/DOPE with DNA is a basic parameter to be
analyzed. To confirm which endocytosis is decisive in the
cellular uptake, the active transport process was investigated
with a temperature change. In consequence, the cellular uptake
efficiency decreased to 46% at 4 °C compared to that of 85% at
37 °C (Fig. 9 and Fig. S20), which clearly indicated that the
cellular uptake of TDM-B/DOPE was greatly through the energy-
dependent endocytosis. Chemical inhibitors such as
chlorpromazine (CPZ), amiloride hydrochloride (AM), and
methyl-β-cyclodextrin (MβCD) may inhibit the cellular uptake
Fig. 10 CLSM images (60×) of TDM-B/DOPE (1:2) at 0.5, 1, 1.5, 2, 3, and 4 h after delivery
into Hela cells. The nucleus (blue channel) (A). The FAM-labeled DNA (green channel) (B).
TDM-B/DOPE (red channel) (C). Bright field (D) and Merge of A, B, C (E). [TDM-B] = 20
μM, [FAM-DNA] = 10 μg/mL, scale bar: 10 μm.
cells, and the majority of them were found to locate in
cytoplasm. It is noteworthy that weak fluorescence of the
nucleus revealed that some condensates had already
translocated into the nucleus. Upon further incubation for 2 and
4 h, the green fluorescence intensity of the nucleus became
stronger, proving more condensates entered into the nucleus.
These results demonstrate that this unique gene vector can
indeed enter into the nucleus together with DNAs rapidly.
Furthermore, this delivery behavior could effectively prevent
degrading the released DNA in cytoplasm from the DNase,
which might be responsible for the decent transfections shown
by the TDM-B/DOPE liposome. To be noticed, some spots can
be seen in cells under Hoechst staining, which can be attributed
to the DNA complex condensates remained.
pathways
of
clathrin-mediated
endocytosis
(CME),
micropinocytosis, and clathrin-independent endocytosis (CIE),
respectively.42,51,52 It was found that the cellular uptake efficacy
decreased slightly to 84% and 81% in the presence of CPZ and
AM, respectively (Fig. 9 and Fig. S21). Sharply differently, the
cellular uptake efficacy was reduced to 5% with MβCD
preincubation. Accordingly, we can firmly believe that the
ultrahigh cellular uptake efficacy of TDM-B/DOPE formulated
with DNA mainly proceed via a CIE pathway.
3.10. Single Photon Fluorescent Tracing the Delivery of DNA
3.11. Two-Photon Fluorescent Tracing of the Delivery of DNA
Inspired by the performance of the transfection and its optical
property, TDM-B/DOPE was singled out as representative for
tracing the gene delivery process by single-photon fluorescence
microscopy. The condensates of TDM-B/DOPE with FAM-
labeled DNA were incubated in vitro with Hela cell lines for
varying times. The TDM-B/DOPE and FAM-labeled DNA were
Based on the excellent two-photon characteristics of the TDM
derivatives, the DNA delivery process with the liposome TDM-
B/DOPE were further explored under an excitation of 800 nm.
Parallel results were shown in Fig. 11, the DNA delivery process
were observed more clearly through two-photon fluorescence
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microscopy. The liposome-loaded DNA was efficiently delivered of condensates had already got into the
into the cytoplasm by endocytosis in two hours, and a portion
View Article Online
DOI: 10.1039/D0TB00321B
about 2-fold higher than that of Lipo2000 in zebrafish embryos.
The effectiveness to enhance the transfection effect of GPF in
Fig. 12 In vivo GFP transfection: confocal image (A) and fluorescence intensity (B) of GFP
expression in zebrafish embryos. [TDM-B/DOPE] = 20 μM, [EGFP-DNA] = 10 μg/mL;
Lipo2000 was used as a control.
physiological environment proved that TDM-B/DOPE was an
effective non-viral gene vector in this in vivo work.
4. Conclusions
In this study, the near-infrared fluorescent unit with the
aggregation-induced emission and two photon absorption
properties triphenylamine-pyrazine with DNA condensing
moieties triazole-[12]aneN3 through the different length alkyl
ester linkage to afford three new non-viral gene vectors, TDM-
A/B/C. The three compounds showed strong AIE features upon
the addition of THF as a poor solvent with the fluorescent
emission enhancements to be 72, 81, and 108-fold higher than
those in pure water solutions, respectively. The largest stokes
shift of the three compounds up to 383 nm and the large 2PA
cross section up to 101 GM for TDM-B were achieved. Gel
electrophoresis demonstrated that TDM-A/B/C were able to
completely retard the movement of plasmid DNA at the
concentration of 10, 14, and 16 μM, respectively.
Simultaneously, under esterase and acidic environment, the
condensed DNA were able to be reversely released due to the
hydrolysis of ester bonds in the three compounds. From SEM
study, the compounds themselves, their liposome and their
lipoplexes can be assembled into regular cubic nanoparticles,
the sizes of the lipoplexes nanoparticles were in the range of
100-200 nm, which were further confirmed by DLS
measurements, and the lipoplexes showed zeta potential to be
4-9 mV for three compounds. The cytotoxicity of the three
compounds were also good for the gene transfection. Among
the three compounds, TDM-B showed the best transfection
efficiencies, and the expression of luciferase and GFP was 146%
and 310% of those achieved by Lipo2000 in Hela cells. The
One/two-photon fluorescence microscopic image with TDM-B
clearly revealed the transfection process of DNA, the DNA were
efficiently delivered into the nucleus in 4 hours. Moreover,
TDM-B/DOPE were successfully implemented gene transfection
in zebrafish embryos, and the EGFP gene expression level was
about 2-fold higher than that of Lipo2000. The work also
demonstrated that the length of aliphatic chain linkage affected
the DNA delivery and transfection, the chain with eight carbon
Fig. 11 CLSM images (60×) of TDM-B/DOPE (1:2) at 0.5, 1, 2, 4, 6, and 12 h after delivery
into Hela cells. The nucleus (blue channel) (A). The FAM-labeled DNA (green channel) (B).
TDM-B/DOPE (red channel) (C). Merge of a and c (D). Merge of A, B, C (E). [TDM-B] = 20
μM, [FAM-DNA] = 10 μg/mL, λex = 800 nm, scale bar: 10 μm.
nucleus. As the incubation time was increased to 4 h, more and
more condensates gradually transferred into the nucleus. When
the incubation lasted to 6 hours, it was clearly observed that
most of the DNA were released from the liposomes and the
vector were discarded into cytoplasm reassembled into micelles
(white arrows) with weak red-emission. Upon further
incubation for 12 hours, the red fluorescence of the vector was
nearly completely separated from FAM-DNA, and the vector
was excreted in the metabolic process. Considering the
spatiotemporal transition of the intracellular fluorescence and
the distribution, the fate of the internalized condensates can be
easily monitored, which would contribute to the further
comprehension of the transfection mechanism.
3.12. GFP transfections in Vivo
Encouraged by the transfection results in vitro, the gene
expressions experiments of TDM-B/DOPE in vivo were further
studied by introducing the reporter gene EGFP-DNA in
developing zebrafish embryos. Zebrafish is an ideal vertebrate
model for biological in vivo imaging, owing to the exceptionally
high optical clarity in embryonic and larval stages.53 After
treatment of the TDM-B/DOPE-DNA complexes into zebrafish,
the fluorescence intensity of GFP was measured by confocal
imaging, and Lipo2000 was used as a control (Fig. 12). It was
found that the GFP gene expression level of TDM-B/DOPE was
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24 Z. Zheng, T. Zhang, H. Liu, Y. Chen, R. T. K. Kwok, C. Ma, P.
atoms gave the best transfection results. This study provides
information regarding improvements of real-time tracking
during gene delivery, transfection and the rational design of
multifunctional non-viral vectors.
View Article Online
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This study was supported by the National Natural Science
Foundation of China (21372032, and 21778012), and the Drug
Innovation Major Project (2018ZX09711001002-005). L.H. thanks the
government of the Guangxi Autonomous Region for being the fifth
batch of distinguished experts.
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J. Name., 2020, 00, 1-3 | 11
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Journal of Materials Chemistry B
Page 12 of 12
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DOI: 10.1039/D0TB00321B
Graphical Abstract
[12]aneN3 Modified triphenylamine-pyrazines as non-viral gene vectors with AIE and two-photon
imaging properties.