Introduction

Supramolecular assembly of organic conjugated small and polymers has become one of the most active research topics during the past two decades, because the performance of organic soft devices mainly depends on the molecular design, intermolecular interaction, ordered packing and optoelectronic properties to a great extent.1, 2, 3, 4, 5, 6, 7, 8, 9 In general, these molecules can self-assemble into different microstructures/nanostructures such as spheres, bars, wires, tubes and vesicles, and these ordered aggregations were used as key active layers in material science. More recently, they also presented high selectivity and sensitivity used as bioprobes because the dimensionally controllable skeletons showed extraordinary physical properties over the corresponding single molecule.10, 11, 12, 13, 14, 15 As known, in order to intensively realize the nucleus modification and diagnostic, prognostic information, fluorescence cell imaging has matured into a novel platform in recent years.16, 17, 18, 19, 20, 21, 22 For example, Misra and co-workers prepared some highly photoluminescent semiconductor quantum dots based on ZnO that could be immobilized of HeLa cells, which was beneficial for detecting the cancer cells in cell therapy.23, 24, 25 However, the appropriate fluorescent biomaterials that can enter and deposit into the special cellular position are still limited, considering the photostability, cytotoxicity and permeability, which encourages us to prepare microstructures/nanostructures with tuning size and shape based on organic conjugated molecules.

Acenes, usually consisting of a series of fused linear benzologs, are the subjects of considerable investigations because of their extraordinary electronic structure, fascinating physical property and the potential application in organic soft electronics.26, 27, 28, 29, 30, 31 When one phenanthrene or pyrene units are grafted into the acenes in the terminal, the formed molecules can twist owing to the steric hindrance, which might be beneficial for application as active layers in organic light emitting diodes because this conformation can decrease the stacking interaction to a certain extent.32, 33, 34 Wudl, Zhang and Xiao synthesized a series of symmetric and asymmetric twistacenes containing primary color luminescence from blue to green and red through retro-Dies-Alder reaction.35, 36, 37, 38, 39, 40 It is well known that well-defined conjugated dendrimers having branched and regularly repeating molecular units possess many advantages over small molecules and the hyperbranched polymers. The optoelectronic properties of these dendrimers can be finely tuned by changing the core, decorating with various surface groups and grafting with different functional generations. For example, Müllen and co-workers synthesized some polyphenylene dendrimers with different central cores and tested their physical property.41, 42, 43 The group of Pei developed a new class star-shaped dendrimers based on truxene that could be used as promising candidates for light-harvesting materials.44, 45, 46 When twistacenes are implanted into the three-dimensional architectures, the as-formed novel dendrimers can effectively suppress intramolecular and intermolecular π–π stacking in high concentration solution and solid state without sacrificing other physical property. Nevertheless, current researches mainly focus on the synthesis and application of these derivatives in organic devices. The approach to fabricate the microstructures/nanostructures based on these π-conjugated dendrimers remains challenging and scarce until now, which strongly stimulates us to induce their self-assembly, tune the optoelectronic properties and investigate the effect of molecular topology on the cell imaging.

In this work, we describe the design, synthesis and self-assembly of the biology of several π-conjugated dendrimers (Scheme 1). TPPh, TTPy, OTPy and TOPhP can form uniform nanoparticles by using a re-precipitation method with blue, cyan, green and red luminescence under single excitation wavelength, respectively. These nanoparticles exhibited good photostability and low biological cytotoxicity. More interesting, such dendrimer-fabricated nanoparticles can selectively implant the membrane and cytoplasm of HeLa cells depending on their structural properties. Clearly, this systematic study provides a new platform for the construction of nanostructures by controlling optoelectronic property and biological affinity.

Materials and methods

Materials and characterization

The chemicals were utilized as received from commercial companies. 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were recorded on Bruker AVANCE 600 MHz digital NMR spectrometer (Fällanden, Switzerland) with CDCl3 as a solvent without any internal standard. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric measurements were measured on Bruker Biflex III MALDI-TOF (Billerica, MA, USA). UV-Vis absorption and fluorescence spectra were carried out on Shimadzu UV-2550 and RF5300PC spectrometers (Kyoto, Japan). Scanning electron microscopy (SEM) characterization was achieved by a field-emission SEM (FESEM) (JSM-7500F, JEOL, Tokyo, Japan) at an accelerating voltage of 10 kV. The size distributions and zeta potential of the nanoparticles were also characterized by dynamic light scattering method with a Nano ZS90 system (Malvern Instruments Ltd, Malvern, UK). Thermalgravimetric analysis was recorded on a Perkin-Elmer thermal analyzer (Pyris 6, Boston, MA, USA). X-ray crystallographic data were obtained by using a Bruker smart APEX ІІ CCD diffractometer (Karlsruhe, Germany) with a graphite-monochromatic Mo Kα radiation (λ=0.71073 Å) at 296 K. Ultrapure water was prepared by Millipore S.A. 67120 appratus with a resistivity of 18.2 MΩ cm−1.

Compound 3: 2,7-Di-tert-butyl-pyrene-4,5-dione (1, 700 mg, 2.03 mmol) and 1,3-diphenylacetone (2, 475 mg, 2.26 mmol) were dispersed into ethanol (30 ml). A mixture solution of KOH (105 mg) in methanol (2.0 ml) was added dropwise slowly. The solution was stirred and heated in refluxing temperature for 15 min. The reaction mixture was cooled to room temperature, successively washed with brine solution and extracted with methylene chloride (30 ml × 3). The organic phase was collected and concentrated under reduced pressure. The obtained residue was further purified by column chromatography with methylene chloride/petroleum ether (v/v, 1:7) to afford compound 3 (black solid, 449 mg, 43%). 1H NMR (600 MHz, CDCl3, 298 k): δ=7.92 (d, 2 H), 7.66 (d, 2 H), 7.60 (s, 2 H), 7.50–7.46 (m, 8 H), 7.42–7.39 (m, 2 H), 1.13 (s, 18 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=200.59, 149.60, 148.57, 133.11, 131.98, 130.03, 128.79, 128.00, 127.12, 127.09, 126.71, 126.52, 124.84, 123.50, 34.61, 30.84. MS (MALDI-TOF): Calc. for C39H34O: [M] 518.26, found: [M+1] 519.2, [M+Na+] 541.2.

Compound 6 (DBPy): 2-Amino-5-methylbenzoic acid (4, 320 mg, 2.1 mmol) dispersed in 1,2-dichloroethane (DCE) solution and isoamyl nitrile (2.5 ml) were added alternatively into a mixture solution of 2,7-di-tert-butyl-9,10-diphenyl-10H-cyclopenta[e]pyren-10-one (3, 1.0 g, 1.9 mmol), which was preheated at 90 °C in DCE (40 ml). The reaction mixture was stirred and kept at 90 °C for 12 h. After cooling to room temperature, brine and methylene chloride was added. The organic phase was collected and concentrated under reduced pressure. The crude product was further purified through column chromatography on silica gel using petroleum ether to give compound 6 (light yellow solid, 120 mg, 11%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.13 (s, 2 H), 7.84 (s, 2 H) 7.83 (s, 2 H), 7.76 (d, J=12.0 Hz, 1 H), 7.61–7.55 (m, 9 H), 7.47–7.45 (m, 2 H), 7.29 (d, J=12.0 Hz, 2 H), 2.46 (s, 3 H), 1.12 (s, 18 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=147.21, 147.17, 142.88, 142.86, 136.05, 135.51, 135.27, 132.52, 132.43, 132.28, 130.54, 130.32, 130.30, 129.82, 129.78, 129.26, 128.85, 127.99, 127.67, 127.47, 127.40, 127.39, 126.92, 126.91, 126.73, 125.47, 123.92, 123.82, 122.12, 121.99, 34.78, 31.44, 31.43, 32.05. MS (MALDI-TOF): Calc. for C45H40: [M] 580.31, found: [M] 580.3.

Compound 7: 2-Amino-5-bromobenzoic acid (5, 250.0 mg, 1.16 mmol) dispersed in DCE solution and isoamyl nitrile (1.2 ml) in DCE (25 ml) were added alternatively into a mixture solution of compound 3 (580.0 mg, 1.12 mmol), which was preheated at 90 °C in DCE. After 12 h, the reaction mixture was cooled to room temperature, washed with brine and extracted with methylene chloride (30 ml × 3). The organic phase was separated and concentrated under reduced pressure. The crude product was further purified through column chromatography using petroleum ether as eluent to give compound 7 (light yellow solid, 203 mg, 28%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.12 (t, 2 H), 7.98, (S, 1 H), 7.84 (S, 4 H), 7.71 (d, J=12.0 Hz, 1 H), 7.57–7.55 (m, 8 H), 7.49–7.47 (m, 3 H), 1.11 (s, 18 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=147.36, 147.35, 142.05, 136.21, 135.25, 133.23, 132.33, 130.86, 130.57, 130.37, 130.34, 130.07, 129.55, 129.44, 129.34, 129.28, 128.84, 128.78, 128.65, 127.84, 127.76, 127.73, 127.61, 126.98, 123.89, 122.62, 122.51, 120.22, 34.80, 31.42. MS (MALDI-TOF): Calc. for C44H37Br: [M] 645.67, found: [M+1] 646.5.

Compound 9: Pd(dppf)2Cl2 (25 mg, 0.031 mmol) was added to a mixture of compound 7 (400 mg, 0.62 mmol), bis(pinacolato)diboron (8, 253 mg, 1 mmol), potassium acetate (365 mg, 3.72 mmol) and dry DMF (20 ml) in nitrogen atmosphere. The reaction solution was stirred and kept at 90 °C for 24 h. After cooling to room temperature, brine and methylene chloride were added. The organic phase was collected and dried with anhydrous sodium sulfate. After removal the solvent, the residue was further purified with on silica gel with ethyl acetate/petroleum ether (v/v, 1:50) to afford compound 9 (yellow solid, 321 mg, 75%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.41 (s, 1 H), 8.13 (s, 1 H), 8.08 (s, 1 H), 7.84 (d, J=6.0 Hz, 4 H), 7.79 (s, 2 H), 7.61–7.54 (m, 8 H), 7.49–7.45 (m, 2 H), 1.35 (s, 12 H), 1.12 (s, 18 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=147.23, 147.20, 142.65, 142.24, 136.95, 135.85, 134.96, 132.71, 132.50, 130.30, 130.27, 130.06, 129.57, 129.25, 129.16, 127.66, 127.57, 127.44, 126.91, 125.68, 123.98, 123.82, 122.34, 122.12, 83.75, 34.75, 31.39, 24.88. MS (MALDI-TOF): Calc. for C50H49BO2: [M] 692.38, found: [M] 692.4.

Compound 11: A mixture of compound 7 (92 mg, 0.15 mmol), 9 (105 mg, 0.15 mmol), Pd(PPh3)4 (10 mg, 0.008 mmol) and Na2CO3 (300 mg) was stirred in tetrahydrofuran (THF)/H2O (25 ml per 4 ml) at 70 °C under nitrogen atmosphere. After 2 d, the solution was cooled to room temperature. The solution was diluted with dichloromethane (80 ml), washed with brine (50 ml × 3) and dried over sodium sulfate. The organic extracts were removed under reduced pressure. The formed residue was further purified through column chromatography on silica gel by using petroleum/dichloromethane (v/v, 25:1) to afford 11 (green solid, 68%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.17 (t, J=1.2 Hz, 4 H), 8.15 (d, J=1.8 Hz, 2 H), 7.92 (s, 1 H) 7.90 (s, 1 H), 7.85–7.84 (m, 8 H), 7.73 (d, J=1.8 Hz, 1 H), 7.72 (d, J=1.8 Hz, 1 H), 7.63–7.57 (m, 12 H), 7.55 (t, J=7.8 Hz, 4 H), 7.50–7.46 (m, 4 H), 1.13 (s, 18 H), 1.12 (s, 18 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=147.30, 147.27, 142.71, 142.54, 137.77, 136.48, 135.98, 132.46, 132.41, 130.33, 130.25, 129.87, 129.64, 129.35, 129.33, 127.71, 127.61, 127.57, 127.52, 127.49, 126.93, 125.10, 124.91, 123.94, 123.90, 122.27, 122.22, 34.78, 31.41. MS (MALDI-TOF): Calc. for C88H74: [M] 1130.58, found: [M+1] 1130.7.

Compound 12: A mixture of compound 9 (321 mg, 0.468 mmol), 1,3,6,8-tetrabromopyrene (10, 42 mg, 0.081 mmol), Pd(PPh3)4 (50 mg, 0.043 mmol) and K2CO3 (110 mg, 0.79 mmol) in toluene/ethanol/H2O (15 ml, v/v/v, 8:1:1) was stirred in refluxing temperature for 16 h in nitrogen atmosphere. After cooling to room temperature, brine was added to the reaction solution, which was further extracted with methylene chloride (50 ml × 3). The organic phase was collected, dried with anhydrous sodium sulfate and then concentrated under reduced pressure. The obtained product was further purified through silica gel column chromatography using methylene chloride/petroleum ether (v/v, 1:100) as the eluent to afford 12 (TTPy, yellow solid, 86 mg, 45%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.34 (s, 4 H), 8.23 (d, 4 H), 8.20 (s, 8 H), 8.14 (s, 2 H), 8.12 (d, J=12.0 Hz, 4 H), 7.87–7.85 (t, 16 H), 7.81 (d, J=12.0 Hz, 4 H), 7.75 (d, J=6.0 Hz, 8 H), 7.69 (d, J=6.0 Hz, 8 H), 7.62 (t, 8 H), 7.52 (t, 4 H), 7.46, (t, 8 H), 7.33 (t, 4 H), 1.15 (s, 36 H), 1.12 (s, 36 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=147.32, 147.30, 142.71, 142.43, 138.21, 137.42, 136.48, 136.08, 132.54, 132.45, 132.06, 131.89, 131.28, 131.26, 130.36, 130.34, 130.04, 129.91, 129.64, 129.40, 128.81, 128.45, 128.43, 128.41, 127.73, 127.62, 127.58, 127.01, 126.96, 126.00, 125.60, 125.59, 123.96, 123.95, 122.32, 122.30, 34.81, 34.79, 31.43. MS (MALDI-TOF): Calc. for C192H154: [M] 2459.21, found: [M+1] 2460.7.

Compound 15: 2-Amino-5-iodobenzoic acid (14, 162 mg, 0.6 mmol) dispersed in DCE (10 ml) solution and isoamyl nitrile (1 ml) were added alternatively into a mixture solution of 13 (333 mg, 0.6 mmol), which was preheated at 90 °C in DCE (10 ml). The reaction mixture was stirred and kept at 90 °C overnight. After cooling to room temperature, brine (60 ml) was added and the mixture was extracted with methylene chloride (30 ml × 3). The organic phase was collected and concentrated. The obtained crude product was purified through column chromatography on silica gel using petroleum ether/CH2Cl2 (v/v: 8:1) to give 15 (light yellow solid, 348 mg, 81%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.30 (d, J=2.4 Hz, 2 H), 8.27 (d, J=1.8 Hz, 1 H), 7.70 (dd, J1=9.0 Hz, J2=1.8 Hz, 1 H), 7.62 (d, J=9.0 Hz, 1 H), 7.57–7.52 (m, 6 H), 7.49–7.47 (m, 4 H), 7.33 (t, J=9.0 Hz, 2 H), 7.10 (dd, J1=9.0 Hz, J2=1.2 Hz, 2 H). 13C NMR (150 MHz, CDCl3, 298 K): δ=140.56, 140.41, 135.88, 135.39, 134.64, 134.61, 133.42, 132.66, 132.59, 132.33, 132.29, 132.01, 131.91, 130.71, 130.20, 130.18, 129.47, 129.46, 129.36, 128.18, 128.47, 128.29, 128.19, 126.21, 126.20, 121.66, 121.59, 92.75. MS (MALDI-TOF): Calc. for C34H19Br2I: [M] 714.2, found: [M] 714.0.

Compound 17: A mixture of compound 15 (619 mg, 0.87 mmol), 16 (100 mg, 0.14 mmol), Pd(PPh3)4 (190 mg, 0.164 mmol) and NaHCO3 (300 mg) in THF/H2O (20 ml per 4 ml) was stirred at 70 °C for 8 days under nitrogen atmosphere. After cooling to room temperature, brine was added to the reaction solution, which was further extracted with methylene chloride (60 ml × 3). The organic phase was collected and then removed under reduced pressure. The resulting residue was further purified through silica gel column chromatography using methylene chloride/petroleum ether (v/v, 1:8) as the eluent to give 17 (light yellow solid, 135 mg, 37%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.32 (s, 8 H), 8.21 (s, 4 H), 8.19 (s, 3 H), 8.12 (d, J=9.0 Hz, 3 H), 8.05 (s, 2 H), 7.79 (d, J=7.8 Hz, 4 H), 7.62–7.55 (m, 29 H), 7.42–7.35 (m, 21 H), 7.14–7.08 (m, 8 H). MS (MALDI-TOF): Calc. for C152H82Br8: [M] 2547.51, found: [M+2] 2549.0.

Compound 18: A mixture of dendron 17 (100 mg, 0.04 mmol), 9 (444 mg, 0.64 mmol), Pd(PPh3)4 (289 mg, 0.25 mmol) and NaHCO3 (300 mg) in THF/H2O (20 ml per 4 ml) was stirred at 70 °C for 15 days under nitrogen atmosphere. After cooling to room temperature, brine (100 ml) was added, and the reaction solution was extracted with dichloromethane (40 ml × 3). The organic solvent was collected and dried with sodium sulfate. After the solvent was removed in vacuum, the obtained residue was further purified through silica gel column chromatography using methylene chloride/petroleum ether (v/v, 1:8) as the eluent to give 18 (light yellow solid, 55 mg, 21%). 1H NMR (600 MHz, CDCl3, 298 k): δ=8.57 (s, 7 H), 8.34 (s, 4 H), 8.30 (s, 4 H), 8.24–8.15 (m, 29 H), 8.02 (t, J=9.0 Hz, 8 H), 7.86-7.81 (m, 43 H), 7.75 (d, J=6.0 Hz, 9 H), 7.70–7.55 (m, 75 H), 7.51-7.41 (m, 36 H), 7.37–7.29 (m, 19 H), 1.14 (s, 36 H), 1.12 (s, 36 H), 1.07 (s, 36 H), 1.05 (s, 36 H). MS (MALDI-TOF): Calc. for C504H378: [M] 6428.96, found: [M+2] 6431.

Preparation of TPPh, TTPy, OTPy and TOPhP nanoparticles

TPPh in a stock THF (5.125 ml, 0.024 mM) solution was rapidly injected into ultrapure water (12 ml) under ultrasonic. After 5 min, the formed solution was bubbled with nitrogen for 1 h, and then the resulting solution was heated. After about 3 h, only 5 ml of water was observed. The nanoparticles were formed and characterized. Accordingly, the other nanoparticles of TTPy, OTPy and TOPhP were prepared at the same conditions.

Cytotoxicity assays

HeLa cells were cultured and seeded at a density of 5 × 103 cells per well in 96-well plates in Dulbecco’s modified Eagle’s medium incorporated 10% fetal bovine serum. After incubated overnight, the culture medium was replaced with 100 μl fresh cell culture medium containing various concentrations of nanoparticles. After 24-h incubation, the medium was replaced with 100 μl 0.5 mg ml−1 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide (MTT) and incubated for 3 h at 37 °C. The MTT solution was discarded and then dissolved with 100 μl dimethyl sulfoxide. The absorbance at 570 nm was measured using Synergy HT microplate reader (BioTek, Winooski, VT, USA). All experiments were conducted in triplicate and presented as mean±s.d.

Photostability

To confirm the photostability, 25 μM nanoparticles solution were added dropwise on slide and then covered by coverslip. The samples were continuously irradiated by a mercury lamp (100 W) with 380/30 nm excitation filter (for TPPh, TTPy and OTPy nanoparticles) and 540/40 nm excitation filter (for TOPhP nanoparticles). Then fluorescence intensities were measured from Olympus IX 71, Tokyo, Japan, fluorescence microscopy with the software.

Cell imaging

HeLa cells were seeded at a density of 1 × 104 cells per well in 35-mm glass dishes. After incubation overnight, the cells were incubated with fresh culture medium containing various nanoparticles at a concentration of 1 μM for 18 h at 37 °C. Then the cells were washed with phosphate-buffered saline (pH=7.4) and stained by nuclear localization dye (4′,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI)) for intracellular localization analysis. The cells were washed three times with phosphate-buffered saline again before recording by confocal laser scanning microscopy (FV1000-IX 81, Olympus, Tokyo, Japan).

Results and Discussion

Synthesis

The synthetic approach to DBPy, DPyT, TTPy and OTPy is depicted in Figure 1. Note that compounds TPPh,47 13,48 1649 and TOPhP50 were prepared according to the reported methods. 2,7-Di-tert-butylpyrene-4,5-ketone51 (1) was first reacted with commercially available 1,3-diphenylacetone (2) in the presence of potassium hydroxide and ethanol to give black solid 3, which was further converted to 6 (DBPy) and 7 in 11% and 28% isolated yield through a Diels-Alder [4+2] cycloaddition reaction by treatment with 2-amino-5-methylbenzoic acid (4), 2-amino-5-bromobenzoic acid (5) and isoamyl nitrile in refluxing DCE. Bromo-substituted derivative 7 was converted into the corresponding single pinacol boronic ester (9) under Pd(dppf)Cl2, dry KOAc and bis(pinacolato)diboron (8). The Suzuki coupling between the intermediate 9 and 7/1,3,6,8-tetrabromopyrene (10) using Pd(PPh3)4 and K2CO3 as the catalysts in THF/water or toluene/ethanol/water solution afforded the target molecules 11 (DPyT) and 12 (TTPy) in 68% and 45% yield, respectively. Compound 15 (81%) was generated from 13 and 2-amino-5-iodobenzoic acid (14) by treatment with isoamyl nitrile in DCE at 90 °C, which was selectively reacted with 16 to afford 17 in 37% yield. Under the similar Suzuki reaction, the larger dendrimer 18 (21%) was successfully achieved through the further cross-coupling of 17 and 9. The introduction of tert-butyl groups and twisted structures in DBPy, DPyT, TPPh and OTPy made them easily dissolve in common organic solvents such as toluene, 1,2-dichlorobenzene, dichloromethane, chloroform and THF, which was in favor of full characterization through 1H NMR, 13C NMR, MALDI-TOF (or EI) mass spectroscopes (Supplementary Figures S3–S27).

Figure 1
figure 1

Synthetic route to DBPy (6), DPyT (11), TTPy (12) and OTPy (18).

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Single crystal for compound DBPy

The twisted structure was confirmed by X-ray diffraction analysis. Single crystals of DBPy, DPyT, TTPy and OTPy were grown through slow evaporation of a mixture solution of CH2Cl2/methanol, and the formed crystals of DBPy were suitable for the test. The molecular structure of DBPy and its packing model are shown in Figure 2 and the detailed crystal data are presented in Supplementary Table S1. DBPy adopts a triclinic unit cell, space group P-1 (CCDC number: 999488) and the crystal parameter data are a=15.5265 (14) Å, b=10.2258 (9) Å, c=20.7939 (18) Å, α=90°, β=98.997 (2)°, γ=90°. In the molecular structure, the terminal pyrene unit is almost coplanar, and the near naphthalene moiety is twisted out of this plane, which was similar to our reported molecules.35, 36, 37, 38, 39, 40 It should be noted that the twisted torsion angle of plane C1-C2-C24 and plane C10-C12-C14 is 27.69°. Furthermore, the intermolecular distance is more than 3.8 Å, inferring that π–π stacking interaction is absent between intermolecules based on the packing model (Figure 2b). This distortion model was in favor of decreasing the aggregation in the solid state. Taking TPPh, TTPy, OTPy and TOPhP into consideration, one do believe that the twisted structures and three-dimensional architectures could effectively suppress π–π interaction, which might be beneficial to decreasing the fluorescent quenching in the self-assembled aggregation to a great extent. Thermal gravimetric analysis was measured under nitrogen atmosphere, and the onset of 5% weight loss of DBPy, DPyT, TPPh,48 TTPy, OTPy and TOPhP are 386, 467, 232, 325, 246 and 181 °C, respectively, indicating that the obtained compounds have high thermal stability (Supplementary Figure S1).

Figure 2
figure 2

(a) Single crystal of DBPy and (b) its corresponding packing model.

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Fabrication and characterization of organic nanoparticles

The nanoparticles of TPPh, TTPy, OTPy and TOPhP were prepared by using a re-precipitation method.52, 53 Compounds TPPh–TOPhP dissolved in THF solution were quickly injected into pure water under sonication. The resulting nanoparticles were subsequently deposited on silica wafers and characterized by FESEM. It should be pointed out that the obtained nanoparticles can remain suspended in water solution. In comparison, under the similar condition, both DBPy and DPyT emitted strong blue light in water, and disordered aggregation was observed. This confirms that the twisted structure and three-dimensional architecture might be beneficial for the formation of nanoparticles. As shown in Figure 3, the formed nanoparticles are spherical shapes with the diameters of 60–200 nm for TPPh, 40–120 nm for TTPy, 150–500 nm for OTPy and 76–300 nm for TOPhP. Moreover, hydrodynamic diameters of TPPh (102 nm), TTPy (204 nm), OTPy (169 nm) and TOPhP (81 nm), determined by using dynamic light scanning, are close to those from SEM analysis (Figure 3 and Supplementary Table S2). Zeta potentials (ζ) of the nanoparticles solutions were observed to be −36.6 mV for TPPh nanoparticles, −1.7 mV for TTPy nanopartices, −29.3 mV for OTPy nanoparticles and −25.6 mV for TOPhP nanoparticles, implying that the surface of the nanoparticles were negatively charged.

Figure 3
figure 3

SEM images of TPPh (a), TTPy (b), OTPy (c) and TOPhP (d) nanoparticles. The hydrodynamic diameters of TPPh (e), TTPy (f), OTPy (g) and TOPhP (h) nanoparticles recorded by using dynamic light scattering.

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Optical property

The optical properties of the obtained molecules in THF solution and their corresponding nanoparticles in pure water were investigated, and the results are shown in Figure 4 and Supplementary Figure S2. Small dendron TPPh in THF displayed the maximum absorption band at 350 nm, with other two peaks at 336 nm and 430 nm owing to the π–π* transition. TPPh in THF showed azure fluorescence with the emission maximum at 445 and 470 nm. When the central unit benzene was substituted with pyrene unit, the formed compound TTPy presented broad absorption peak at 350 and 414 nm, which might be assigned to the increase of conjugated length. More interesting, TTPy exhibited strong emission at 484 nm, falling into the cyan-emission region. The larger dendron OTPy showed a broad structureless absorption between 320 and 480 nm, with a maximum around 380 nm and emission peak at 490 nm. Note that it also emits cyan light in THF solution. The known compound TOPhP exhibited the absorption bands at 575, 533 and 442 nm. The red emission spectra of TOPhP displayed structured features with the maximum peak at 608 nm and a shoulder peak at 655 nm. In addition, the quantum yields of TPPh, TTPy, OTPy and TOPhP were calculated to 0.35, 0.82, 0.79 and 0.80, respectively, by using 9,10-diphenylanthracene (Φf=0.95 in ethanol) or N,N′-di(2,6-diisopropylphenyl)-perylene-3,4:9:10-tetracarboxylic acid bisimide (Φf=1.00 in chloroform) as the standards.54, 55

Figure 4
figure 4

(a) UV-Vis absorption and (c) fluorescence spectra of TPPh, TTPy, OTPy and TOPhP in THF (1 × 10−5 M) solution. (e) Absorption spectra and (g) fluorescence spectra of TPPh, TTPy, OTPy and TOPhP nanoparticles in water. Panels (b) and (f) show the corresponding absorption colors in THF and water, panels (d) and (h) show the corresponding emission colors in THF and water under the UV light (λ=365 nm).

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The photophysical properties of the desired nanoparticles in pure water were further measured. TPPh, TTPy and TOPhP nanoparticles presented the similar absorption profiles compared with those in diluted THF solution. However, OTPy nanoparticles showed the same absorption maximum at 380 nm but much broader absorption contour than that in THF, which might result from the weak electronic coupling of molecule OTPy.56, 57 As shown in Figure 4g, a 27-nm bathochromic shift in fluorescence spectra was observed between TTPy (484 nm) and OTPy nanoparticles (511 nm), which was larger than that (6 nm) in THF. More importantly, one can notice that the full width at half maximum became smaller than those in THF. Apparently, TPPh, TTPy, OTPy and TOPhP nanoparticles in pure water emitted blue, cyan, green and red color with the quantum yields of 0.11, 0.10, 0.04 and 0.06 (Figure 4h), respectively, inferring that they might be applied in realization of all the visible emission region and special imaging of living cells.

Photostability and cytotoxicity

The photostability and cytotoxicity of the obtained nanoparticles were examined to assess their potential application in biology. As shown in Figure 5a, the nanoparticles in aqueous solutions retained the emission intensity of about 60–80% under continuous irradiation for 60 s, indicating good photostability. TPPh and TTPy nanoparticles exhibited negligible cytotoxicity recorded by an MTT assay (Figure 5b). OTPy and TOPhP nanoparticles showed small cell cytotoxicity in the concentration of <1.0 μM, which can meet the standard for the test in the cell imaging.

Figure 5
figure 5

(a) Photostability of DBPy, TTPy, OTPy and TOPhP nanoparticles upon irradiation at 380 and 455 nm. (b) Viability of HeLa cells in the presence of the obtained nanoparticles with an MTT assay.

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Cell imaging

In light of the above facts, the cellular imaging in vitro based on these nanoparticles was further investigated by using confocal laser scanning microscope where red fluorescence PI was selected as the nucleus-located dyes for TPPh, TTPy and OTPy nanoparticles and blue fluorescence DAPI was utilized for TOPhP nanoparticles. The results are depicted in Figure 6. TTPy nanoparticles concentrated on the membrane of HeLa cells, while the other three nanoparticles are mainly observed in the cytoplasms. This might result from the small zeta potential (−1.7 mV) of TTPy nanoparticle solution. These particles were prone to agglomerate, which was a disadvantage to penetrate into the membrane.58, 59 Accordingly, TPPh, OTPy and TOPhP nanoparticles having high potential values could be stained to cytoplasm areas owing to endocytosis. Clearly, the desired nanoparticles could selectively be stained in the special cellular position, which is beneficial for the location, diagnostic and prognostic bio-information for pathologists.

Figure 6
figure 6

Fluorescence images of HeLa cells treated with TPPh (a), TTPy (b), OTPy (c) and TOPhP (d) nanoparticles.

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In summary, we have synthesized and characterized a family of π-conjugated dendrimers containing branched twistacenes. Molecular structures can significantly affect the size and shape of the aggregation that fabricated through re-precipitation method. Interestingly, the formed nanoparticles kept strong blue, cyan, green and red luminescence in aqueous solution compared with their THF solution. As a new class of detection systems, the self-assembled multicolor nanoparticles could selectively flow into the membrane and cytoplasm of HeLa cells, being indicative of feasibility and efficiency in cell fluorescent imaging. Obviously, our systematic study of these molecules could provide more room to rational design and functionality of the larger dendrimers. Pursuing the nanoparticles with low cytotoxicity and high photostability based on all-carbon conjugated dendrons and potential application in biological processes are on the way.

scheme 1

Molecular structures of DBPy (6, black), DPyT (11, black), TPPh (blue), TTPy (12, cyan), OTPy (18, green) and TOPhP (red).