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[12]aneN3 Modified Tetraphenylethene Molecules as High-performance Sensing, Condensing, and Delivering Agents towards DNAs Aixiang Ding, Quan Tang, Yong-Guang Gao, You-Di Shi, Alam Uzair, and Zhong-Lin Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01949 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016

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ACS Applied Materials & Interfaces

[12]aneN3 Modified Tetraphenylethene Molecules as High-performance Sensing, Condensing, and Delivering Agents towards DNAs Ai-Xiang Ding, Quan Tang, Yong-Guang Gao, You-Di Shi, Alam Uzair, and Zhong-Lin Lu* Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education; College of Chemistry, Beijing Normal University, Beijing 100875, China KEYWORDS Aggregation-induced emission, nucleic acid sensing, DNA condensation, cytotoxicity, [12]aneN3, non-viral gene vector.

ABSTRACT:

Four [12]aneN3 modified tetraphenylethene (TPE) compounds with different

numbers of polyamine units and structure configurations, namely 1, 2, 3 and 4, were designed and synthesized. All compounds showed strong aggregation-induced emission (AIE) features. Compounds 2 and 4 showed significant emission enhancement after the addition of ssDNAs and dsDNAs of different lengths as well as calf thymus DNA (ctDNA). Compounds 1 and 3 showed very poor fluorescent responses towards DNAs. Gel electrophoresis demonstrated the abilities of 1-4 to condense DNA effectively. Complete retardation of plasmid DNA can be achieved at a concentration of 25 µM (1), 8 µM (for 2 and 3) and 4 µM (4). Experiments including fluorescent contrastive titrations, scanning electron microscopy, dynamic laser scattering, EB displacement,

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and gel electrophoresis demonstrated that the four compounds were able to integrate with DNA through electrostatic interactions and supramolecular stacking. A vicinal configuration around TPE (2) and more triazole-[12]aneN3 recognition sites (4) evidently enhanced the sensing capability towards oligonucleotides, and the TPE unit played an important role in the plasmid DNA condensation process due to its strong binding. With the advantages of low cytotoxicity, effective DNA sensing, and DNA condensing properties, compound 4 was successfully applied as a nonviral DNA vector and fluorescent tracer for label-free gene delivery, which is the first example of a non-viral gene vector with AIE activity.

Introduction DNA sensing is of significant importance in fundamental biological/biomedical research including clinical disease diagnostics, drug discovery, and gene delivery monitoring.1-3 Over the past two decades, many compounds including polymers, small organic molecules, metal complexes as well as nano-scale materials were explored to meet the requirements for the efficient sensing of nucleic acid.4-10 Among the different methods of sensing DNA, fluorescence appeared highly prominent due to its outstanding selectivity, ultra-sensitivity, and real-time feasibility. Thus, fluorescent dye molecules have received much attention in sensing DNA in recent years. However, many reported nucleic acid dyes exhibited very low sensitivity and activity in water, and some of them are biohazards. Furthermore, traditional nucleic dyes such as ethidium bromide (EB), TOTO, DAPI and Hoechst dyes only functioned with double strand DNA (dsDNA) through intercalation or groove binding,11-14 and they failed to bind with single strand DNA (ssDNA) due to the absence of the secondary structure in ssDNA. Recently, Tang et al. developed several tetraphenylethene (TPE) derivatives as “switch on” DNA probes.15-16 TPE and its derivatives showed excellent aggregation-induced emission (AIE) in concentrated


2

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solution or aggregation states.17-21 Based on the AIE principle, several fluorescence “light up” DNA chemo-sensors were developed by Yang and other groups.2, 22-29 On the other hand, DNA condensation is a prerequisite for building gene delivery vehicles. In comparison with viral gene vehicles, non-viral ones have numerous advantages, such as adjustable structure, non-immunogenicity and potential for large-scale production.30-31 Different chemicals including lipids, polymers, metal complexes, and nanoparticles as non-viral gene vectors have been reported. However, mechanistic studies of the gene transferring process have not been greatly explored due to the scarcity of effective tools, although such studies will play important roles in the design of effective and applicable non-viral gene vectors. A series of [12]aneN3 based compounds as non-viral small organic molecular gene vectors were recently developed by us,32-36 which showed excellent DNA condensation and potential gene delivery ability. By incorporating the fluorescent naphthamide moiety in the [12]aneN3 compounds, we have demonstrated their tracing ability in DNA transferring processes. Considering the application of TPE compounds in DNA sensing and our work on the development of [12]aneN3 based non-viral gene vectors, it was expected that the combination of the AIE active TPE unit and the cationic [12]aneN3 moiety should result in effective DNA sensing, DNA condensation, and DNA delivering agents. In the present work, we report the synthesis of four [12]aneN3 modified TPE molecules and their application as efficient nucleic acid “light up” and condensing agents (Scheme 1). Through fluorescence spectroscopy, agarose gel electrophoresis (AGE), dynamic laser scattering (DLS), and scanning electronic microscopy (SEM) techniques, the relationships between the structures of the four derivatives and their ability towards DNA sensing and condensation were studied in detail. The excellent sensing and condensing properties of 2 and 4 revealed that the combination


3


of [12]aneN3 and TPE units is beneficial to the development of label-free DNA luminescence tracking agents for mechanistic studies of the gene transfection process and new effective nonviral gene vectors. The preliminary gene transfection and cellular uptake results of compound 4 proved its potential performance. N H NH N N N

O

N

N

O N

O

N N

4HCl

2 Boc

1 NH N H N

N

N

O

N3

9

N H NH N

i-ii)

N

8 i-ii)

N N N

N

N H HN

N O

N

O

N

N N

3

N N

NH N H N

N

O

HN H N

N N

NH N H N

N

O

O

O

O

O 8

7

6

HN H N

O

O

O

N

O 16HCl 4

N

O

5

HN H N

N

O 8HCl

N

N N Boc

7 i-ii)

N

N N

8HCl

5 i-ii)

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propargyl bromide DMF, K2CO3, 70°C HO

OH

O

O 7

Scheme 1. Syntheses of 1-4 and 7: i) THF/H2O, CuSO4·5H2O, sodium ascorbate, overnight; ii) CH3COCl, CH3OH. Experimental Details 1. Materials and instrumentations


4

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Agarose,

6×

and

10×

loading

buffer,

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazoliumbromide (MTT), ctDNA (calf thymus DNA), Goldview II, pUC18 and ethidium bromide (EB) were purchased from Solarbio Company (Beijing, China). Oligonucleotides (X10, Y10, X20, Y20, X30 and Y30) and pGL-3 DNA were purchased from Ruibiotech Co., Ltd. (Beijing, China). pEGFP DNA was purchased from Clontech (Palo Alto, CA, USA). Lipofectamine 2000™ was purchased from Invitrogen (Life technologies, Mauricio Minotta, USA). DOPE (Dioleoylphosphatidyl ethanolamine) was from Santa Cruz Biotechnology (USA). Analysis-level solvents and reactants were commercially available and used without further purification. Anhydrous THF was prepared by distillation from sodium/benzophenone. 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). Ultrapure milli-Q water (18.25 MΩ) was used in aggregation-induced emission (AIE) and dynamic light scattering tests. Tris-HCl buffer (pH = 7.4) was used to conduct fluorescent titrations, gel electrophoresis at room temperature. NMR spectra (1H and 13C) were recorded on a Bruker Avance III spectrometer (400 MHz) at 25 ºC using CDCl3 and D2O as solvents and calibrated using tetramethylsilane (TMS) as an internal reference. Data were reported as follows: chemical shifts (δ) in ppm, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constants J (Hz), integration, and assignment. FT-IR spectra were taken on an Affinity-1 Fourier Transform Infrared Spectrometer in the range of 4000-400 cm-1. HRMS (High resolution mass spectra) were obtained on a Waters LCT Premier XE spectrometer (USA). Agarose electrophoresis was performed using a BGsubMIDI 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. Determination of hydrodynamic diameters was performed on a


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Brookhaven Zeta Plus Partical Size and Zeta Potential Analyzer (USA). Scanning electron microscopy (SEM) images were obtained with a Hitachi S-4800 (Japan) instrument. Fluorescence measurements were carried out on a Varian Cary Eclipse spectrometer (USA) using 3.5 mL (10 × 10 mm), 100 µL and 50 µL quartz cuvettes. Varian Cary 300 UV-Vis spectrophotometer (USA) was used to record UV-Vis spectra of solutions in 1.0 cm quartz cuvettes. Caution! Due to the toxic and mutagenic natures, EB and other nucleic dyes should be used carefully under a safety precaution. 2. Fluorescence measurements Stock solutions (1 mM) of TPE dyes were prepared in deionised Milli-Q water (18.25 MΩ). Solutions of different concentrations were prepared by transferring appropriate aliquots of stock solutions to volumetric flasks and then diluting them to certain volumes using deionised Milli-Q water, anhydrous THF and Tris-HCl buffer. Trace-volume (100 or 50 µL) quartz cuvettes were used to perform DNA fluorescence titrations. All of the samples were vortex mixed and allowed to rest for 6-10 min before testing. Every fluorescent titration experiment was performed at least three times for calculating error bars. 3. Strand hybridization Equimolar amounts of X10 and Y10, X20 and Y20, X30 and Y30 were mixed in sterilized water. After annealing at a temperature 2 ºC below Tm for 15 min, the solutions were slowly cooled to 25 ºC to form the double-strand DNA. 4. Cellular uptake studies The cellular uptakes of label-free ctDNA (10 µg/mL) condensates were investigated by fluorescence microscopy in A549 cell lines. The CLSM images of the compounds or


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compounds/DOPE (molar ratio 1/1) condensed ctDNA and the controls (500 µL) at 0.5 h, 2 h and 4 h were obtained after incubation of the cells with condensates for different durations. After incubation for 4 h, the cells were washed 10 times with PBS (0.5 mL), then 10% FBS in DMEM (1 mL) was added and incubated for an additional 2 h and 20 h to capture the CLSM images of 6 h and 24 h. 5. Gel electrophoresis and other cellular experiments Other experiments including agarose gel electrophoresis, ionic strength effect, releasing the compact DNA, EB displacement assay, cell toxicity and cell transfection were performed according to the procedures reported in references.32, 36 6. Synthesis of the compounds Synthesis of 7: 4,4'-(2,2-Diphenylethene-1,1-diyl)diphenol (550 mg, 1.5 mmol) and K2CO3 (1.67 g, 12.07 mmol, 8.0 equiv.) were added to 15 mL DMF. After stirring for 1 h at 90 ºC, propargyl bromide (718 mg, 6.04 mmol, 4.0 equiv.) was added, and the mixture was kept stirring at 90 ºC overnight. After cooling, filtering and evaporating, the mixture was purified through silica gel chromatography using PE/EtOAc (V/V 30/1) as the eluent to afford 7 as a white solid with a yield of 95%. 1H NMR (CDCl3, 400 MHz): δ (ppm) 7.17-7.00 (m, 10H), 6.97 (d, J = 8.7 Hz, 4H), 6.72 (d, J = 8.7 Hz, 4H), 4.63 (d, J = 2.3 Hz, 4H), 2.51 (s, 2H). 13C NMR (CDCl3, 400 MHz): δ = 156.15, 144.07, 137.12, 132.57, 131.34, 127.70, 126.18, 113.94, 78.58, 75.42, 55.75. General procedure for the syntheses of compounds 1-4. The propargyl-modified TPE compounds 5-8 and the 1.0 ~ 4.0 equiv. of the azide compound 9 were added to the THF/H2O mixtures (V/V = 5/1). After that, CuSO4·5H2O (0.2 equiv.) and sodium ascorbate (0.4 equiv.) were added, and the mixtures were stirred at room temperature overnight under an argon atmosphere. After removing the solvent, the residues were dissolved in 30 mL EtOAc, and then


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washed with water (3 × 20 mL) three times. The organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to afford the crude products, which were purified by silica gel chromatography using DCM/MeOH (V/V from 5/1 to 3/1) as the eluent to afford the Boc moieties as white solids. The obtained Boc moieties were dissolved in 10 mL methanol, then 5 mL acetyl chloride was added slowly at 0 ºC, and the mixtures were kept stirring for 30 min to afford the white solids 1-4 with yields of 60-70%. 1: FT-IR (KBr, cm-1): 3423 (N-H stretching), 2953 (=C-H stretching), 2750 (CH2 asymmetrical stretching), 2638 (CH2 symmetrical stretching), 1597 (C=C and N=N stretching), 1502, 1462 (Ar stretching). 1H NMR (D2O, 400 MHz): δ (ppm) 7.46 (s, 1H, C=CH-N), 7.237.25 (m, 19H, ArH), 4.51 (s, 2H, OCH2), 4.18 (brs, 2H, N-CH2-CH2), 3.44-2.70 (m, 14H, CH2), 2.41-1.50 (m, 8H, CH2). 13C NMR (D2O, 101 MHz): δ 155.99, 143.13, 142.66, 139.73, 136.06, 132.02, 130.56, 127.25, 125.88, 124.21, 113.53, 60.46, 52.39, 47.50, 46.91, 46.60, 42.04, 41.03, 23.72, 19.96, 17.90. HR-MS: calcd. for [C41H52Cl4N6O+H–4HCl]+ 641.3968, found 641.3971. 2: FT-IR (KBr, cm-1): 3423 (N-H stretching), 2953 (=C-H stretching), 2744 (CH2 asymmetrical stretching), 2638 (CH2 symmetrical stretching), 1597 (C=C and N=N stretching), 1502, 1450 (Ar stretching). 1H NMR (D2O, 400 MHz): δ 7.58-7.37 (m, 2H, C=CH-N), 7.23-6.17 (m, 18H, ArH), 4.65-4.41 (m, 4H, OCH2), 4.30-4.10 (m, 4H, CH2), 3.35-2.99 (m, 28H, CH2), 2.28-1.86 (m, 16H, CH2). 13C NMR (D2O, 101 MHz): δ 156.21, 143.86, 143.70, 143.15, 139.76, 136.94, 132.37, 131.05, 127.94, 126.52, 124.95, 114.12, 60.93, 51.65, 48.89, 47.58, 47.22, 42.15, 41.13, 24.34, 20.42, 17.84. HR-MS: calcd. for [C56H84Cl8N12O2+H–8HCl]+ 949.6292, found 949.6284. 3: FT-IR (KBr, cm-1): 3425 (N-H stretching), 2956 (=C-H stretching), 2748 (CH2 asymmetrical stretching), 2634 (CH2 symmetrical stretching), 1597 (C=C and N=N stretching),


8

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1502, 1462 (Ar stretching). 1H NMR(D2O, 400 MHz): δ 7.46 (s, 1H, C=CH-N), 7.09-5.82 (m, 9H, ArH), 4.44 (s, 2H,OCH2), 4.23 (s, 2H, CH2), 3.40-3.11 (m, 14H, CH2), 2.25-2.03 (m, 8H, CH2).

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C NMR (D2O, 101 MHz): δ 156.12, 143.89, 143.81, 143.11, 139.60, 136.84, 132.35,

130.92, 127.85, 124.94, 114.08, 60.90, 51.23, 48.81, 47.55, 42.11, 41.07, 24.24, 20.35, 17.74. HR-MS: calcd. for [C56H84Cl8N12O2+H–8HCl]+ 949.6292, found 949.6284. 4: TF-IR (KBr, cm-1): 3421 (N-H stretching), 2954 (=C-H stretching), 2738 (CH2 asymmetrical stretching), 2628 (CH2 symmetrical stretching), 1593 (C=C and N=N stretching), 1504, 1458 (Ar stretching). 1H NMR (D2O, 400 MHz): δ 7.79 (s, 1H, C=CH-N), 6.99-6.18 (m, 4H,ArH), 4.88 (s, 2H, OCH2), 4.37 (s, CH2), 3.38-3.06 (m, 14H, CH2), 2.36-1.98 (m, 8H,CH2). 13

C NMR (D2O, 400 MHz): δ 156.24, 143.81, 143.10, 139.74, 139.60, 136.78, 132.39, 131.03,

127.82, 126.28, 124.90, 114.01, 60.93, 51.93, 47.46, 47.21, 42.05, 41.02, 24.54, 20.44, 17.69. HR-MS: calcd. for [C86H148Cl16N24O4+H–16HCl]+ 1566.0928, found 1566.0897.

Results and Discussion Synthesis and Characterization: The synthetic procedures for compounds 1-4 are shown in Scheme 1. The mono-(O-propargyl)-modified, vicinal di-(O-propargyl)-modified and tetra-(Opropargyl)-modified TPE compounds 5, 6 and 8 were prepared according to literature methods.37-38 The geminal di-(O-propargyl)-modified TPE 7 was prepared through a multi-step synthesis

involving

titanium-catalyzed

McMurry

coupling,

demethylation,

and

final

propargation. The azide compound, 9, an important building block for the preparation of multi[12]aneN3 compounds, was obtained following the literature procedure.39 In the synthesis of the target molecules, highly efficient copper-mediated alkyne-azide click reactions were carried out between the O-propargy-modified TPE compounds 5-8 and azide 9.


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After de-protection of the Boc moieties with acetyl chloride in methanol, compounds 1-4 were obtained as their hydrochloride salts. All the newly synthesized compounds were characterized by 1H NMR,

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C NMR, FT-IR, and high-resolution mass spectrometry (see supporting

information). To be noticed, both 2 and 3 contain two triazole-[12]aneN3 units on the double bond of TPE, but they are arranged in the different configurations. 2 is a vicinal isomer composed of a mixture of 1:1 ratio of cis- and trans-configuration isomers, and 3 is a geminal isomer. Aggregation Induced Emission: TPE and its derivatives are famous for their AIE-active fluorescent nature17-21. Most reported TPE derivatives are soluble in organic solvents such as acetonitrile and methanol, they showed AIE effects upon the addition of water as a poor solvent. However, four [12]aneN3 modified TPE derivatives in this work, 1-4, are soluble but virtually non-luminescent in dilute water solution. It can be seen that the addition of THF to the water solutions of 1-4 effectively enhanced their fluorescent emissions under an excitation of 320 nm (Figure 1A and S1). The wavelengths of the emissions were accompanied by a blue-shift from 419 nm to 366 nm when the THF fraction in the water-THF mixtures was increased to 30%, which can be attributed to a change of the aggregation morphology.37,

40-41

The maximal

fluorescence emission (FL) intensities of compounds 1-4 were reached when the THF fraction was increased to about 60-70%, which were 110, 108, 107 and 109-fold higher, respectively, than those in the pure water solutions. Nevertheless, after addition of excess THF (more than 70%), the FL intensity did not increase but started to decrease because of the formation of amorphous aggregates.42 It is noteworthy, however, the FL intensities of the TPE derivatives were still enhanced by about 67, 83, 73, and 88-fold in a water-THF mixture at the ratio of 1:99 by volume, respectively. Apparently, the fluorescent emissions of compounds 1-4 were induced


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by their aggregation due to the addition of the poor solvent THF to their water solutions. The aggregation resulted in the restriction of intramolecular rotation (RIR) of the TPE units, which prohibited energy dissipation via nonradiative channels and enhanced the fluorescent emissions.37 Fluorescent emission images of the four derivatives with THF fractions from 0% to 99% volume in water-THF mixtures provided an immediate visualization of the fluorescent changes under 365 nm UV light (Figure S2). To be noticed, the AIE performance of these TPE derivatives are quite different from the tetraalkylammonium-modified TPE compounds, which are soluble in water but did not show clear AIE effects due to their amphiphilic nature.15 To prove the formation of the fluorescent particles in the THF/water mixtures with high THF fraction, scanning electron microscopy (SEM) and dynamic light scattering (DLS) measurements were conducted. In the SEM measurements, all the samples were prepared by adding THF to water solutions of the four derivatives, whereby well-assembled particles formed by allowing the THF/water mixtures to stand for over 12 h (Figure 1B). Ordered and regular particles with sizes of about 70-150 nm were found, indicating a kind of particular arrangement of the molecules.43 The average diameters (d) of the nanoparticles confirmed by DLS were 105, 112, 105, and 101 nm for 1, 2, 3 and 4, respectively (Figure S3), which were consistent with the SEM measurements. Based on the above phenomena, it can be definitely confirmed that these TPE derivatives are AIE-active.


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Figure 1. (A) The changes of FL peak maxima with different THF fractions, [Dyes] = 10 µM, λex = 320 nm. (B) SEM images of 1 (a), 2 (b), 3 (c), and 4 (d) in THF/water mixtures with 70% THF fraction. Scale bars: 1 µm. Error bars are ± SD. Fluorescence Response to DNA in aqueous solution: To explore their DNA sensing ability, the FL measurements of 1-4 with the addition of ssDNA and dsDNA were carried out in Tris-HCl (pH = 7.4) media. The sequences of the synthetic oligonucleotides (X10, X20, X30 and Y10) are shown in Table S1. The measurements demonstrated that both 2 and 4 showed significant FL enhancement with increasing amounts of all the DNAs (Figure S4 and S5). At 0.3 equiv. of X10, Y10, X10&Y10 (hydridized dsDNA), X20 and X30, the FL magnifications were 23.0, 25.0, 27.6, 20.7 and 19.7-fold enhancements for 2, and 28.8, 30, 32.6, 39.1 and 29-fold enhancements for 4, respectively. More manifest data (I/I0-1) were summarized in Table S2. Compounds 1 and 3 showed no fluorescent signals even when the equivalents of these DNAs were up to 0.5 (Figure S6). The poor sensing ability of 1 and 3 can be attributed to the weak interactions between shorter ssDNAs and one triazole[12]aneN3 recognition unit in 1 or two triazole-[12]aneN3 units in a geminal geometry in 3, which resulted in a less effective rotation restriction of the hydrophobic TPE units. Although no obvious fluorescent enhancement was observed, 1 and 3 were able to combine with theses DNAs through electrostatic interactions, generating an evident red-shift of the emission peak from 419 nm to 479 nm. And further evidences could be obtained by the small bathochromic shifts in the UV-Vis absorption spectra of 1 and 3 upon the addition of 0.1 equivalent DNAs (Figure S7). The sensing properties of 2 and 4 are thus discussed in more detail (Figure 2). Firstly, the DNA concentration greatly affected the emission enhancement. At a lower DNA concentration range (< 0.1 equiv.), 2 displayed a superior performance than 4. The reason might be attributed to the


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four triazole-[12]aneN3 recognition sites in 4, which resulted in the poor hydrophobic aggregation of the TPE units due to the electrostatic interference among the unbound protonated triazol-[12]aneN3 units.27 When the concentration of DNA exceeded more than 0.2 equiv., a fluorescent enhancement was exhibited by 4, which can be explained by its larger number of recognition sites and superior intrinsic binding ability with DNA. However, the emission enhancements started to be reduced at the higher concentrations of X20 and X30 (> 0.15 equiv. for 2 and 0.2 equiv. for 4, respectively), which may be caused by a different aggregation behavior (see later results in SEM, Figure 4). Secondly, the structure of DNA also affected the fluorescence enhancements, longer ssDNA generally favoring a stronger fluorescence emission. When being exposed to the same amount of nucleotides in X10&Y10 and X20, both 2 and 4 showed a slight higher sensitivity towards X20, indicating that the relatively higher steric hindrance of the hybridized dsDNA (X10&Y10) reduced the binding interaction with 2 and 4 and lowered their fluorescent response. These results are consistent with those in the literatures.27-29 To be noticed, compound 2 is a mixture of cis- and trans-configuration isomers, which we found very difficult to separate. According to literatures,27,28 the cis-isomer showed a higher sensitivity to stain ssDNAs.


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Figure 2. Plot of I/I0 − 1 of (A) 2 and (B) 4 at 479 nm versus DNA equivalents. I0 = FL intensity in the absence of DNA. [Dyes] = 10 µM, λex = 320 nm, λem = 479 nm. Insets: Plot of I/I0 – 1 of addition of DNA less than 0.15 equiv. for 2 (A) and 0.2 equiv. for 4 (B). Error bars are ± SD.

Figure 3. (a) 2 and (b) 4: fluorescence titrations of X30 in Tris-HCl buffer (pH = 7.4). (c) Plot of I/I0 − 1 at 479 nm versus the DNA equivalents of 2 and 4. I0 = FL intensity in the absence of DNA. (d) 2 and (e) 4 photographs recorded under a UV 365 nm lamp with addition of gradually increasing equivalents of X30 (0 ~ 0.05 equiv.) from left to right. [Dyes] = 10 µM, λex = 320 nm, λem = 479 nm. Error bars are ± SD. The fluorescence titrations of DNAs under 0.05 equiv. to 2 and 4 gave us a more unambiguous comparison between the two probes (Figure 3). Meanwhile, the varying fluorescence intensities can be easily recorded by the visualized variation of the fluorescence images under 365 nm UV light. The quenching effects of the excess DNAs to the emission of 2 and 4 were also observed through the titration of X30 in the range of about 0.5-1.0 equiv. (Figure S8). It can be seen that the emission intensity of 2 gradually decreased along with the increased concentration of X30. Compound 4 caused a rapid emission decline until the addition of 0.8 equiv. X30 and then plateaued. The results suggested that 4 had a stronger binding ability towards DNA than 2.


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To gain insight into the fluorescent response behaviors of 2 and 4 with X30, SEM measurements were carried out. The samples were prepared by adding corresponding equivalents of X30 to the solutions of the two probes. Then the mixtures were kept for over 12 h to allow for a sufficient interaction and effective assembling between the probes and X30. As seen in Figure 4, only a small portion of 2 co-aggregated with X30 into square nanoparticles when 0.01 equiv. X30 were added. Then the nanoparticles grew regularly and intensively after introducing 0.15 equiv. X30. Therefore, the emission intensity was enhanced dramatically. However, upon addition of 1.0 equiv. X30, the excessive DNA molecules deposited onto the initially formed aggregates, the nano-particles grew bigger, with a notable change of shape, from square to spherical globular. Because of the formation of the bulkily packed particles, the emission intensity was lowered. Concerning the behavior of 4, the growing tendency of the co-aggregates was similar to that of 2. The formed particles were initially small and separate, then they continued to grow into larger and interconnected star-like assemblies, and finally condensed into compact sheet-like structures. Thanks to the co-assembled aggregates of the probes with DNA, which caused a restriction of the TPE molecules, the fluorescence was “lighted up”.


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Figure 4. SEM images of 2 (a) and 4 (b) (10 µM) after addition of different equivalents of X30: (a1) 0.01 equiv., (b1) 0.05 equiv. (a2) 0.15 equiv., (b2) 0.2equiv., (a3, b3) 1.0 equiv., respectively, scale bars: 5 µm. Insets are the magnifications of a selected region for clarity, scale bars: 1 µm. Sensing of double stranded DNA Furthermore, the sensing ability of double stranded DNA by compound 1-4 was investigated by using ctDNA (calf thymus DNA) as a model. The ctDNA is a natural dsDNA which has been widely used in chemical, biochemical and medical science research.44-50 Consequently, a fluorescent study of the four [12]aneN3-modified TPE derivatives with ctDNA would be meaningful and instructive. As shown in Figures 5 and S9, the four compounds showed various fluorescence enhancements upon the addition of ctDNA. It can be seen that both 2 and 4 showed a remarkable emission enhancement upon addition of ctDNA. At a ctDNA concentration of 12 µg/mL, the fluorescence enhancements were 58 and 41-fold of the original values for 2 and 4, respectively. For compounds 1 and 3, only weak emission enhancements were observed, which implied that partial rotations of the hydrophobic TPE benzene rings were still permitted.15 As the concentration of ctDNA increased from 0 to 12 µg/mL, the FL intensities at 479 nm of 3 and 4 were almost linear-dependent with correlation coefficients of 0.971 and 0.998, respectively, while those of 1 and 2 showed saturation curves. These results further confirmed that the number and spatial location of the recognition sites are the critical factors for restricting the TPE units. Compared to the recognition of ctDNA by amino-modified and tetraalkylammonium-modified TPE derivatives,27 compounds 2 and 4 showed a higher value of (I/I0-1) at the same concentration, which may be attributed to the multiple interactions between triazole-[12]aneN3 moieties and DNAs.


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Figure 5. (A) Plot of emission intensity at 479 nm vs ctDNA concentration. [Dyes] = 10 µM; λex = 320 nm, error bars are ± SD. (B) Fluorescent pictures of 10 µM 1 (b1), 2 (b2), 3 (b3), and 4 (b4) with increased concentrations of ctDNA, 0 ~ 12 µg/mL. Error bars are ± SD. Given the different emission enhancement with dsDNA, the fluorescent titrations of 2 and 4 with varied length of dsDNA (X10&Y10, X20&Y20 and X30&Y30) were further performed (Figure 6 and Figure S10). As shown in the experiments of sensing ssDNA, both compounds showed greatly enhanced fluorescence after the addition of these dsDNA, and they exhibited larger fluorescent amplifications towards longer dsDNA. Moreover, the FL intensity decreased when the equivalents of dsDNA (X20&Y20 and X30&Y30) exceeded 0.2 (for 2) and 0.1 (for 4).

Figure 6. Plot of I/I0 − 1 of (A) 2 and (B) 4 at 479 nm versus different dsDNA equivalents. I0 = FL intensity in the absence of DNA. [Dyes] = 10 µM, λex = 320 nm, λem = 479 nm. Error bars are ± SD.


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DNA condensation: Gel Electrophoresis Gel retardation assays were carried out to appraise the condensation ability of the four compounds toward pUC18 DNA (Figure 7). With increasing concentrations of the four derivatives, a gradually diminishing amount of the supercoiled DNA was observed. Even though 1 contained only one [12]aneN3 unit, it also expressed excellent condensation ability, and the concentration for complete retardation of DNA in electrophoresis was 25.0 µM. 2 and 3 containing two [12]aneN3 units showed similar, but enhanced condensing abilities. In these cases 8.0 and 10.0 µM of dyes were enough for complete retardation, respectively. The four[12]aneN3-modified TPE 4 displayed the best DNA agglomerated behavior. In this case the concentration at which complete condensation was reached was 4.0 µM. The neutralization of the negatively charged DNA phosphate groups by the positively charged triazole-[12]aneN3 units is the primary driving force to induce the plasmid DNA condensation. Thus, the condensing activities were greatly enhanced by increasing the number of cationic moieties in the molecules, which were in line with those reported results in the literatures.32, 35-36 On the other hand, the TPE unit in the above molecules also made a great contribution in the condensation process, as their completely retardant dosages were obviously less than those of the previously reported compounds with the same numbers of triazole-[12]aneN3 units (Table S3), especially for compounds 1-3.32-36 Ionic Effects on DNA Condensation The role of the TPE units was explored by studying ionic strength effects. As seen in Figure S11, the four compounds expressed efficient agglomeration towards pUC18 even in the presence of high concentrations of sodium chloride. For our previously reported [12]aneN3 derivatives,32-


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a slightly subdued agglomeration was observed when the concentration of NaCl was more than

300 mM. Generally, the presence of high concentrations of mineral salt could distinctly weaken the electrostatic interactions between the positively charged species and negative DNA chains, resulting in an evidently diminished condensation. The results here clearly implied a significant contribution of the TPE units in driving the effective condensation of plasmid DNA. SEM Study of DNA Condensation To obtain furtehr insight into the condensing behaviors of the four compounds toward DNA, SEM measurements were carried out. The samples were prepared by adding these compounds into aqueous solutions of DNA, and allowing them to stand for 12 h. After that, 10 µL samples were dropped onto the surface of silicon, and allowed to dry fully for 12 h. In Figure 8, SEM images clearly showed that pUC18 was effectively condensed by the four TPE derivatives. Due to the co-assembling effect between the TPE unit and the DNA molecules, pUC18 was condensed into neat and long nanobelts instead of traditional spherical nanoparticles.


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Figure 7. Condensation of pUC18 DNA with different concentrations (lane number labeled in yellow font, µM) of 1 (a), 2 (b),3 (c) and 4 (d) in 50 mM Tris-HCl buffer (pH = 7.4). [DNA] = 9 µg/mL, 37 ºC. Lane 1 is the DNA control.

Figure 8. SEM images of condensed pUC18 DNA (9 µg/mL) by 1-4 in Tris-HCl buffer (5 mM, pH 7.4): (a) 1, 20 µM; (b) 2, 12 µM; (c)3, 12 µM; (d) 4, 8 µM. Scale bars: 1 µm.

EB Assay Experiments An ethidium bromide (EB) displacement assay was carried out to further evaluate the binding capability of the four compounds with DNA. Upon intercalating into or groove binding to dsDNA, the emission intensity of the well-known nucleic dye, EB, could be enhanced effectively. However, the EB molecules can be displaced by other cationic molecules. Accordingly, quenching of EB emission at 608 nm was widely used to evaluate the binding ability of target molecules.51-53 As shown in Figure S10, the fluorescent emission of the EBbound ctDNA was monotonically decreased with increasing concentrations of four TPE


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derivatives. Furthermore, the peak at approximately 545 nm was heightened. Deduced from the quenching plots (Figure 9 and Table S3), the apparent binding constants (Kapp) of compounds 1-4 were calculated to be 0.61 × 107, 1.18 × 107, 0.98 × 107 and 1.85 × 107 M-1, respectively. The data clearly indicated that these molecules interacted with ctDNA through strong binding. Compared to reported compounds with similar structures (Table S2), the exceptionally high Kapp value of the four derivatives might be the result of the synergetic contributions of the electrostatic interaction of the positively charged compounds with the negatively charged DNA backbone and the superamolecular stacking of TPE units with DNA through co-assembly. Because of the superamolecular stacking, a twisted phenyl ring was forced to adopt a more coplanar conformation, leading to the characteristic red-shift in the fluorescent spectra.15 From the Kapp data, it can be seen that electrostatic interactions did not strongly contribute to the binding strength of the molecules with DNA, since Kapp increased only about 3 times when the number of the positively charged moieties (triazole-[12]aneN3, which is tetra-protonated at physilogical pH 7.4) is increased from 1 to 4. Secondly, the binding constants were not affected by the geometry of the positively charged moieties in the molecules, since the binding constants of 2 and 3 were very close.


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Figure 9. Plot of 1- I/I0 (deduced from the quenching spectra upon addition of [12]aneN3modified TPE agents to EB-bonded ctDNA solution) vs concentrations of the four [12]aneN3modified TPE derivatives, λex= 537 nm, [EB] = 20 µM, [DNA] = 100 µM, 25 °C, 5 mM TrisHCl, 50 mM NaCl, pH = 7.4. Condensation Reversibility For a non-viral gene carrier, effective release of compact DNA is of equal importance. The tactics for the release of condensed DNA included linkage breaking by means of a pH jump, addition of additives, etc.54-58 In our present work, a highly concentrated solution of sodium chloride was used to assess the reversibility of condensed DNA. As shown in Figure S13, the condensed DNA was released upon treatment with 800 mM NaCl solution, indicating the condensing process was reversible. The DNA release should be attributed to the electrostatic competition between the condensing agents and sodium ions with the negatively charged phosphate backbone of DNA. Although such highly concentrated NaCl is fatal to the living cell, the experiments indicated a possibility that the potential reversibility could occur in the complicated process of cellular metabolism. The results were also consistent with the conclusion that the effective DNA condensation of the four compounds mainly resulted from the intercalative binding and that the electrostatic interactions were relatively weak. Cytotoxicity Considering their potential applications in the development of non-virus gene vectors and tracers, the cytotoxicity of compounds 1-4 was measured by using an MTT assay. Figure 10 shows the cellular viability of HpeG2, A549 and HeLa cells treated with increasing concentrations of the four compounds over 4 h, respectively. At 5.0 µM, all of the derivatives showed a lower toxicity, the viabilities of HpeG2 were 0.92, 0.82, 0.82, 1.06, those of A549


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were 0.83, 0.83, 0.90 and 0.98, and those of HeLa were 0.88, 0.79, 0.86 and 0.99, respectively. However, with increasing dosage 1, 2, and 3 exhibited increased toxicity, especially 3. It is unexpected that 4 showed a high cell viability, with 90% of the cultured cells remaining active at the concentration of 30 µM. The performance of compounds 1-4 in the cytotoxicity assay is hard to explain according to our knowledge. However, the above results clearly demonstrated that 4 is a superior biocompatible DNA sensing and condensing agent, and has great potential to be developed as an effective gene delivery and tracking agent. Considering its excellent ability for sensing traces of DNA, 2 has also the potential for applications at lower concentration (10 µM of 2).

Figure 10. Relative viabilities of HepG2 (a), A549 (b) and HeLa (c) cells in the presence of different concentrations (µM) of compounds 1-4 after incubation at 37 ºC for 4 h. Each point represents the means of three experiments. Error bars are ± SD. Cell transfection To obtain a visual comparison of the transfection abilities of the four compounds, the in vitro transfection experiments were initially carried out in the A549 cell lines by using the GFP (green fluorescent protein) reporter gene. The transfection experiments with Lipofectamine 2000™, pEGFP and DOPE mixed pEGFP were also conducted as controls. As can be seen in Figure 11A, the density of the transfected cells by 4 was much higher than those obtained by 1 ~ 3. Despite the fact that the reason for the low transfection activities of 1 ~ 3 was complicated, the high


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cytotoxicity of them might be an important factor. Furthermore, the transfection of the liposome formed by 4 and DOPE was also taken into consideration. Excitingly, the transfected cells were even more intensive than the commercially available Lipofectamine 2000™. Based on the above results, compound 4 was singled out for the quantitative evaluation of the transfection efficiency (TE) in HepG2, A549 and Hela cells by luciferase transfection assay (Figure 11B). The results showed that compound 4 itself was able to transport plasmid DNA successfully into these cells for genetic expression, whereby the formation of liposomes greatly improved the transfection efficiency. The optimal transfection concentrations of 4 in HepG2 is 40 µM, TE was 62% of Lipofectamine 2000™, while the liposome of 4 exhibited 2.15-fold higher TE than that of Lipofectamine 2000™ at its optimal concentrations (30 µM). Among the three types of cancer cells examined, compound 4 resulted in the highest TE in A549, and the liposome of 4 brought the best TE in Hela cells. Through the transfection assays, we confirmed that 4 and its liposome can be successfully applied as non-viral gene vectors, the liposome of 4 showed high performance in gene transfection, even better than commercially available Lipo2000. This is the first example of non-viral gene vectors with AIE property according to our knowledge.


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Figure 11. (A) Fluorescence microscope images (10×) of pEGFP-transfected A549 cells. (a) 1; (b) 2; (c) 3; (d) 4; (e) 4 + DOPE; (f) Lipofectamine 2000™, (g) pEGFP control; (h) DOPE + pEGFP control. Concentrations = 20 µM, [pEGFP-N1] = 10 µg/mL. (B) Luciferase gene expressions transfected by DNA complexes of 4 and 4/DOPE (1/1) at varied concentrations in HepG2 (i), and by 4 (40 µM) and its liposome (30 µM) in three cell lines (j). [pGL-3] = 9 µg/mL. Cellular uptake studies Considering the good fluorescence responses of 2 and 4 to DNAs, the possibilities of using them as self-traceable vectors for gene delivery were examined. The condensates of 2 and 4 with ctDNA were incubated in vitro with A549 cell lines for varying times to observe their intercellular distributions. Confocal laser scanning microscopy (CLSM) images (Figure 12) showed that the fluorescence of the condensates in the cells were very weak at 0.5 h, indicating that only a very small amount of the condensates were internalized into the cells. And close survey of the results discloses that the condensates mainly accumulated near the cell membrane


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(Figure S14). Upon further incubation for 2 h and 4 h, the fluorescence of cell-internalized condensates became stronger, proving more condensates entered into the cells. The fluorescence is only visible in the cytoplasm, but not in the nucleus. This implied that the condensates did not translocate into the cell nucleus. After 4 h incubation with the condensates, the cells were washed with PBS (0.5 mL) 10 times and cultured in fresh mediums for another 2 h and 18 h without the condensates, and the subcellular fluorescence of the condensates was again investigated by CLSM. At 6 h, the intense fluorescence indicated that the vectors could effectively prevent the backflow of the condensed DNAs from the cell interiors to the extracellular medium. The further observed fluorescence at 24 h, however, was much weaker than those observed at 4 and 6 h. We assume that the lowered emission was caused by the escape of DNA from the condensates. Thus, by tracking the transition of the time-dependent fluorescence intensity, we concluded that 2 and 4 condensed DNAs could be validly taken up by cells and remain in the cytoplasm for certain period, which lighted up the cells. At last, the observed fluorescence was suppressed, which may be contributed to the detachment of DNAs from the condensates in the metabolic process and the DNA uptake by the nucleus, which reduced the AIE effect of compounds 2 and 4.


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Figure 12. Confocal laser scanning microscopy (CLSM) images (20×) of A549 cells incubated with 2 (A) and 4 (B) carrying label-free ctDNA (10 µg/mL) for 0.5 h, 2 h, 6 h and 24 h. Concentrations = 10 µM. Conclusion In this work, four triazole-[12]aneN3 modified TPE derivatives were designed and synthesized as novel DNA sensing and condensing agents. The four compounds showed strong AIE features in water solution upon the addition of THF as a poor solvent. At a THF fraction of about 60-70%, the maxima fluorescent emission intensities of 1-4 were achieved to be 110, 108, 107 and 109fold higher than those in pure water solutions, respectively. In the presence of different length ssDNA and hybridized dsDNA as well as calf thymus DNA (ctDNA), compounds 2 and 4 showed significant emission enhancement, which can be attributed to the aggregation-induced restriction of intramolecular rotation (RIR). Their emission enhancements increased along the increase of the DNA concentration at a lower concentration range and showed some deduction at


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higher DNA concentrations, and they showed better sensitivity towards longer DNA. Compounds 1 and 3 showed very low fluorescence responses towards DNAs. Gel electrophoresis demonstrated that 1-4 were able to completely retard the movement of plasmid DNA at the concentration of 25 µM (1), 8 µM (for 2 and 3) and 4 µM (4). Through fluorescent contrastive titrations, scanning electronic microscopy, dynamic laser scattering, EB displacement, and gel electrophoresis at different conditions, the following points were demonstrated: the four compounds were able to interact with DNA through the electrostatic interactions and supramolecular stacking; a vicinal configuration around TPE (2) and more triazole-[12]aneN3 recognition sites (4) caused more tight interactions with DNA and resulted in better sensing and condensation behaviors; the TPE unit played an important role in the plasmid DNA condensation process due to its promoted binding capabilities; a high concentration of DNA changed the shape and structure of nanoparticles formed and their fluorescent enhancements. Further assays including cytotoxicity, cell transfection and cellular uptake study indicated that compound 4 can be successfully applied as a DNA vector and fluorescent tracer for label-free gene delivery. This work clearly showed that the combination of macrocyclic polyamine [12]aneN3 and tetraphenylethene functional units provided novel DNA sensing, DNA condensation, and DNA delivery agents. Further work on the development of effective non-viral gene vectors with good AIE properties is undergoing in our group.

Supporting Information. Oligonucleotide sequences, additional spectra and tables for fluorescent, condensing and cellular uptake measurements, and characterizations of newly synthesized compounds. These materials are available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author * Corresponding authors, [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This article was supported by the financial assistance from the Nature Science Foundation of China (21372032 and 91227109), the Fundamental Research Funds for the Central Universities, Beijing Municipal Commission of Education, the Program for Changjiang Scholars and Innovative Research Team in University. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors also thank Prof. Andreas Mayr from the State University of New York at Stony Brook in the USA for his kind help with language and discussion. REFERENCES (1)

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[12]aneN3 Modified Tetraphenylethene Molecules ... - ACS Publications

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