Multifunctional polymer nanoparticles: ultra bright near-infrared fluorescence and strong magnetization and their biological applications

Abstract

Multifunctional polymer nanoparticles with great promise for biomedical applications have attracted many attentions. Using an aggregation-enhanced emission (AEE) molecule DPPBPA and magnetic Fe₃O₄ as the core, and biocompatible polymer Pluronic F-127 as the encapsulation matrix, multifunctional polymer NPs Fe₃O₄/DPPBPA@F-127 were fabricated by self-assembly procedures. These NPs have bright near-infrared fluorescence λ_ex at 654 nm with a high fluorescence quantum yield of 18.3%, and strong magnetism, and superparamagnetism. With good monodispersity and biocompatibility, the NPs not only can show effective MRI ability, but also can stain in cytoplasm with a strong near-infrared fluorescent signal, as well as little toxicity to living cells, which show a very good prospect in the field of biological applications.

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Introduction

Multifunctional nanoparticles (NPs) with unique magnetic, optical, catalytic, and electrical properties hold great promise for biomedical applications such as drug delivery carriers,^1,2 diagnostic analysis,³ magnetic resonance imaging,⁴ bioseparation,⁵ and fluorescent labeling,⁶ and have attracted much attention in the past few decades.^7–11 The combination of magnetic and fluorescent capabilities enables the design of new multifunctional NPs probes in multimodal optical/magnetic resonance imaging.^9–11 Because of the optical transparence and biological compatibility, polymers have become favorable encapsulation matrixes. Furthermore, the encapsulation can protect dye molecules from the external perturbation and prevent the magnetic nanoclusters from agglomerating into large chunks.¹²

To prepare fluorescent magnetic NPs for imaging, the luminescent QDs and organic dyes were both chosen in the past studies. However, QDs are less chemically stable, potentially toxic, and show fluorescence intermittence,¹³ and most commercially organic dyes typically exhibit rapid photobleaching and a low fluorescence quantum yield in the NPs due to the notorious aggregation-caused quenching (ACQ) effect. Therefore, the luminescent materials with high photo-stability, low optical background and bright light emission are required.¹⁴ Recently, opposite to the ACQ effect, a novel class of organic fluorophores^15–19 with aggregation-induced emission (AIE) characteristic has been developed. These fluorophores almost have no emission in their dilute solutions, but have high fluorescence quantum yields in the aggregate state when the intramolecular rotations are restricted.¹⁵ These AIE fluorophores opened a new gate to fabricate high brightness NPs by encapsulating large amount of dyes.

Due to the superparamagnetism, nontoxicity, and biodegradability, magnetic iron oxide NPs have become famous magnetic materials.²⁰ However, as we know, when the fluorescent material cones in contact with iron oxide, most of the fluorescence is quenched.^21,22 For the AIE characters, the NPs fabricated by combining AIE dyes and iron oxide may overcome this problem. In order to get both high fluorescence quantum yields and strong magnetism in the aggregate state, Tang and co-workers succeeded in synthesizing silica NPs with both efficient fluorescence and strong magnetization by using AIE dyes and iron oxide firstly.¹² And then, some other AIE fluorescent magnetic NPs have been developed.^23–26 However, most of these dyes are blue or green emitters,^15,27–29 whose fluorescence is still quenched to some extent by the magnetic materials, especially iron oxide, and the intensity of magnetism is always weak, which are not ideal for imaging applications. Because of the low photodamage, deep tissue penetration and minimal auto-fluorescence from biological substrates, the fluorophores with intense emission in the far red/near-infrared (FR/NIR) (650–900 nm) region have attracted much interest.^30–32 Up to now, the investigations about AIE dyes applied in near-infrared emission bioimaging are just emerged,^32–36 and the reports on NPs with both bright FR/NIR fluorescence and strong magnetism are quite limited.²⁶

Here, we present a simple strategy to fabricate multifunctional polymer NPs Fe₃O₄/DPPBPA@F-127, by using oleic acid coated magnetic Fe₃O₄ (OA-Fe₃O₄) and an AEE molecule (2Z,2′Z)-3,3′-(2,5-di(piperidin-1-yl)-1,4-phenylene)bis(2-phenylacrylonitrile) (DPPBPA) as the core, and biocompatible polymer Pluronic F-127 as the encapsulation matrix. During the nanoparticle formation, OA-Fe₃O₄ and DPPBPA tended to entangled with the hydrophobic domains of Pluronic F-127 to afford the hydrophobic interiors, while the hydrophilic domains of Pluronic F-127 extended into the aqueous phase, which makes the NPs stable in the aqueous suspension. With very little fluorescence quenching and magnetic intensity reducing, these NPs possess bright near-infrared fluorescence (λ_ex = 654 nm, Φ_f = 18.3%), strong magnetism (M_s = 35.66 emu g⁻¹) and superparamagnetism (M_r = 0.3186 emu g⁻¹, H_ci = 6.377 G), which show that the goals to prepare nanostructured materials with high near-infrared fluorescence efficiency and magnetic susceptibility have been achieved. And these NPs are expected to facilitate their biological applications.

Results and discussion

Synthesis of DPPBPA and oleic acid coated Fe₃O₄ (OA-Fe₃O₄)

The synthetic route to DPPBPA is shown in Scheme S1.† (2Z,2′Z)-3,3′-(2,5-Dibromo-1,4-phenylene)bis(2-phenylacrylonitrile) (2) was synthesized by coupling 2,5-dibromobenzene-1,4-dicarbaldehyde (1) and benzyl cyanide through Knoevenagel reaction.³⁷ Under typical Suzuki reaction conditions, the target molecule was derived from connect piperidine to the 2,5-positions of compound 2.³⁸ All compounds were characterized by standard spectroscopic methods, from which satisfactory analysis data corresponding to their molecular structures were obtained. The molecular structure and purity were confirmed by ¹H NMR and MS (Fig. S1 and S2 in the ESI†), which all gave satisfactory spectroscopic data. The final product gives a [M + H]⁺ peak at m/z 498 (calcd: 498.66) in the GC-MS spectra for DPPBPA, confirming the formation of the expected adducts.

After the synthesis of AEE dye DPPBPA, we then worked on the preparation of magnetic particles. Among various magnetic materials, the Fe₃O₄ particles become a good candidate, since its biocompatibility and wide biological applications such as magnetic resonance imaging, enzyme and protein immobilization. The Fe₃O₄ particles were prepared by a coprecipitation method with oleic acid as stabilizer.³⁹ Ferric chloride hexahydrate and ferrous chloride tetrahydrate was dissolved in deionized (DI) water with the ratio of 2 : 1. Potassium oleate and ammonium hydroxide was added into the solution at 80 °C. The OA-Fe₃O₄ magnetic NPs were centrifuged by supercentrifuge.¹⁰ The oleic acid coated iron oxide retained the characteristic X-ray diffraction pattern of Fe₃O₄ at 2θ of 30.2, 35.5, 43.2, 53.3, 57.1, and 62.8 as listed in ASTM XRD standard card (19-0629) (as shown in Fig. S3,† green line). A transmission electron microscopy (TEM) image showed a mean size of OA-Fe₃O₄ particles of 10 nm in diameter (Fig. 2A), which indicates that these particles are monodisperse. The magnetic NPs were dispersed in THF at 10 mg mL⁻¹ for further study.

Fig. 1 Photographs of water solutions of Fe₃O₄/DPPBPA@F-127 taken under (A)–(C) normal room lighting and (B)–(D) UV illumination in the absence ((A) and (B)) and presence ((C) and (D)) of external magnetic field from a bar magnet.

Fig. 2 TEM image of OA-Fe₃O₄ (A) and Fe₃O₄/DPPBPA@F-127 (B).

Nanoparticle preparation and characterization

As shown in Scheme 1, the multifunctional polymer NPs Fe₃O₄/DPPBPA@F-127 were fabricated by a self-assembly procedure, where magnetic OA-Fe₃O₄ and DPPBPA served as the core, and biocompatible polymer Pluronic F-127 served as the encapsulation matrix.⁴⁰ Pluronic F-127 is a copolymer consisting poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) blocks, PEO₁₀₀–PPO₆₅–PEO₁₀₀. The exterior PEO corona provides an antifouling character to prevent aggregation, protein adsorption, and recognition by the reticulo-endothelial system (RES) and the hydrophobic PPO core can be adapted to encapsulate the fluorophores. The self-assembling characteristics of F-127 have been extensively explored in the form of micelles. OA-Fe₃O₄, DPPBPA and Pluronic F-127 were dispersed in THF by a vigorous bath sonicator. The solution mixture was quickly added into DI water in a vigorous bath sonicator. The THF was removed by nitrogen stripping. The solution was concentrated by continuous nitrogen on a 90 °C hotplate followed by filtration through a 0.2 micron filter.⁴¹ During the formation procedure of the NPs, the OA-Fe₃O₄ and DPPBPA tended to entangle with the hydrophobic domains of Pluronic F-127 and afford the hydrophobic interiors, while the hydrophilic domains of Pluronic F-127 extend into the aqueous phase of the NPs.

Scheme 1 Schematic illustration of chemical formation of Fe₃O₄/DPPBPA@F-127.

In order to optimize the size and morphology of the NPs, many parameters should be taken into account. As the previous studies, the sizes of the polymer NPs are affected by the amount of polymer added. Under the condition shown in Table S1,† the NPs are formed with four different polymer amount (mass of polymer: 6 mg, 8 mg, 10 mg, 12 mg). The number average hydrodynamic diameters of the nanoparticle are around 90–105 nm, and the NPs possess the uniform size and monodisperse confirmed by dynamic light scattering (DLS), which show a narrow peak in the size distribution diagram, as shown in Fig. S4.† Notably, when the addition amount of polymer increase to 10 mg, the encapsulated NPs show the narrowest peak and maximum yield. Therefore, we choose the addition amount of polymer at 10 mg to further fabricate the multifunctional polymer NPs. Zeta potential analyses reveal that the Fe₃O₄/DPPBPA@F-127 NPs possess appreciable surface charges and hence good colloidal stability, which shows a negative charge of around −12.5 mV due to the protonation of oxygen atom in the Pluronic F-127. These negative charges on the surface will stabilize the NPs in aqueous medium by electrostatic repulsion (Fig. S5†). According to the TEM, the average diameter of the NPs is about 65 nm. This size is smaller than that of NPs obtained by DLS analysis due to the shrinkage of the outer hydrophilic chains of Fe₃O₄/DPPBPA@F-127, which often occur under the high vacuum in the TEM chamber.

Photophysical properties

The images in Fig. 1 show that the suspension of Fe₃O₄/DPPBPA@F-127 in water exhibit good dispersion in the solutions and can be attracted by a bar magnet. The intense near-infrared light was observed in the suspension solution of Fe₃O₄/DPPBPA@F-127 with and without magnet upon the UV illumination.¹² It indicates that both DPPBPA and OA-Fe₃O₄ were successfully encapsulated into the NPs. The fluorescence quantum yield (Φ_f) of Fe₃O₄/DPPBPA@F-127 NPs is surprisingly as high as 18.3%. Compared with our previous report, the fluorescence quantum yield of DPPBPA@F-127 NPs without Fe₃O₄ encapsulated is 20% (Table S2†).⁴² This means that there is almost no quenching of fluorescence when the Fe₃O₄ co-encapsulated together with DPPBPA.

To get indepth understanding of emission behavior of the Fe₃O₄/DPPBPA@F-127 NPs, we check the effect of both DPPBPA and OA-Fe₃O₄ loading amounts (Table 1). With enlarging the loading amount of DPPBPA from 0.5 mg to 1 mg, the fluorescence quantum yield can increase. On the contrary, the fluorescence quantum yield shows obvious decrease when adding more OA-Fe₃O₄ particles. Although the combination of luminescent dye and magnetic particles often quench the fluorescence, the high quantum yield of these NPs can be achieved, even that a low dye loading was used for the nanoparticle fabrication and relative high loading of OA-Fe₃O₄ in the polymer NPs. It is well known that the luminescent dyes with AIE or AEE active have the enhancement emission in their aggregated states, which originate from the restriction of intramolecular rotations (RIR). The properties of enhancement emission is mainly affected by the aggregate state and quite different from the traditional luminescence process, such as fluorescence resonance energy transfer (FRET)⁴³ or photoinduced electron transfer (PET),^44,45 which often accompanied by the electronic process in the excited state of luminescent dye. Therefore, we infer that the extraordinary emission properties of these NPs are probably caused by less interaction between the excited state of the DPPBPA and magnetic Fe₃O₄.

Table 1 The fluorescent quantum yield (Φ_f) and spin lattice relaxation time T₁ with different content of DPPBPA and Fe₃O₄

	DPPBPA	OA-Fe3O4	F-127	THF	H2O	Φf	1/T1 (s^−1)
1	0.5 mg	1 mg	10 mg	5 mL	15 mL	16.4%
2	1 mg	1 mg	10 mg	5 mL	15 mL	18.3%	0.24
3	0.5 mg	2 mg	10 mg	5 mL	15 mL	7%
4	1 mg	2 mg	10 mg	5 mL	15 mL	14%	0.30

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DPPBPA is a typical AEE active luminogen, which show weak near-infrared emission peaked at 674 nm in its THF solution, and strong emission located at 654 nm in the solid state with high quantum yield (Φ_f = 78%). The emission spectra of Fe₃O₄/DPPBPA@F-127 and DPPBPA@F-127 NPs are almost same and with the peak located at 654 nm under the same measurement conditions, which are similar with the emission spectrum in solid state (Fig. 3). The fluorescence quantum yield of Fe₃O₄/DPPBPA@F-127 shows slight decrease compared with that of DPPBPA@F-127. In addition, time resolved fluorescence spectra of Fe₃O₄/DPPBPA@F-127 and DPPBPA@F-127 NPs shown in Fig. S6† reveals the similar fluorescence lifetime τ_FL of both systems, 8.25 ns for Fe₃O₄/DPPBPA@F-127 and 8.63 ns for DPPBPA@F-127, respectively. Since Φ_f equals the product of τ_FL and radiative deactivation rate (k_r), k_r and non-radiative deactivation rate (k_nr) can be approximately estimated as listed in Table S2.† It shows obviously that the radiative deactivation process of excited state have not be affected when the magnetic OA-Fe₃O₄ particles added, which is in agreement with the observation of high solid state fluorescence quantum yield. Furthermore, the fluorescence is so stable that there was nearly no change in the spectrum, after put on shelves for several weeks without protection from light and air. The leaching issue is also researched. There was little change in the spectrum after continuous ultrasound in a long time (Fig. S7†), which suggest that only few dyes leak out from the nanoparticles.

Fig. 3 Normalized PL spectra of DPPBPA and NPs.

Magnetism

Owing to nanoscopic magnetic Fe₃O₄ contained into Fe₃O₄/DPPBPA@F-127 NPs, they are expected to be magnetically susceptible. Fig. 4 shows the plots of magnetization versus applied magnetic field at 300 K for OA-Fe₃O₄ and Fe₃O₄/DPPBPA@F-127. With the increasing of the magnetic field strength (maximum field = 18 000 G), the magnetization of Fe₃O₄/DPPBPA@F-127 swiftly increases and ultimately reaches a saturation magnetization (M_s) of 35.66 emu g⁻¹. There is no hysteresis and both remanence (M_r = 0.3186 emu g⁻¹) and coercivity (H_ci = 6.377 G) are nearly zero, which indicates that the NPs are superparamagnetic.⁴⁶ It should be noted that the saturation magnetization often reduce when the iron oxide was cladded in some matrix. Although the M_s value of Fe₃O₄/DPPBPA@F-127 is a little bit lower than that of OA-Fe₃O₄ (M_s = 55.629 emu g⁻¹), the magnetization of Fe₃O₄/DPPBPA@F-127 is already superior to those NPs reported previously.

Fig. 4 Plots of magnetization versus applied magnetic field at 300 K for OA-Fe₃O₄ and Fe₃O₄/DPPBPA@F-127.

Next we explore the magnetic resonance imaging (MRI) ability of the NPs, due to the excellent magnetic characters of Fe₃O₄/DPPBPA@F-127. Normally, with the same imaging parameters, the higher concentration of the contrast agent is the larger change of water relaxation in tissue. The spin lattice relaxation time T₁ of Fe₃O₄/DPPBPA@F-127 NPs with different concentration of Fe₃O₄ loading in water was performed. From Table 1, we can know that 1/T₁ increases with increasing the concentration of Fe₃O₄ from 1 mg to 2 mg. Fig. 5 shows the comparison of the T₁ contrast MRI image of water without and with the presence of Fe₃O₄/DPPBPA@F-127 NPs carried out in vitro MRI experiments. It can be clearly observed that the different MRI contrast between map A and B, where the contrast for map A, which is made with pure water, is fairly poor, whereas that for map B, which contain the Fe₃O₄/DPPBPA@F-127 NPs, is much better. The contrast with the presence of Fe₃O₄/DPPBPA@F-127 is significantly enhanced, suggesting the effective MRI ability.

Fig. 5 MRI results of (A) water and (B) Fe₃O₄/DPPBPA@F-127.

Intracellular imaging

The cytotoxicity of Fe₃O₄/DPPBPA@F-127 in the MCF-7 cells was studied by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fig. S8† shows the cell viability after incubation with the NPs suspension at concentrations of 0, 5, 10, 20, 40, 60, 80, 100 ppm for 24 h, respectively. It was observed that cell viabilities for all the fluorogen concentrations within the tested periods of time were more than 95%, indicating that the Fe₃O₄/DPPBPA@F-127 NPs had low cytotoxicity or good biocompatibility. The low cytotoxicity makes the NPs promising for bioimaging applications and superior to QDs, which are well-known for their concentration-dependent cytotoxicity.

The in vitro cellular imaging of Fe₃O₄/DPPBPA@F-127 was performed by using confocal laser scanning microscopy (CLSM) with a 405 nm laser excitation and the fluorescent signals were collected at 620–680 nm to monitor the cell uptake behaviour to the NPs. Previous studies have showed that the FSNPs could selectively stain the cytoplasmic regions of the living cells. Phagocytosis may be the possible mechanism for intracellular uptake of the NPs. The NPs were enclosed by the cell membrane to form small vesicles, which were then internalized by the cells. The NPs were further processed in endosomes and lysosomes and are eventually released into the cytoplasm. As shown in Fig. 6, after incubating with 50 ppm of Fe₃O₄/DPPBPA@F-127 for 6 h at 37 °C in the culture medium, an intense red fluorescence was observed in the cellular cytoplasms, which suggested the intracellular uptake of the NPs. These results manifested that the Fe₃O₄/DPPBPA@F-127 NPs were effective FR/NIR fluorescent bioprobes for cellular imaging with a high fluorescence contrast.

Fig. 6 Confocal laser scanning microscopy images of MCF-7 cells after incubation with Fe₃O₄/DPPBPA@F-127 for 6 h at 37 °C. (A) Bright field image; (B) fluorescence image; (C) overlay of (A) and (B). AEE NPs concentration: 50 ppm. Scale bar for all images = 30 μm.

Conclusions

In summary, we succeeded in developing a simple strategy to fabricate multifunctional polymer NPs Fe₃O₄/DPPBPA@F-127, by using oleic acid coated magnetic Fe₃O₄ and an AEE molecule DPPBPA as the core, and biocompatible polymer Pluronic F-127 as the encapsulation matrix. The structure, morphology, and property of the NPs are characterized and investigated. The fluorescent magnetic NPs could be successfully dispersed in aqueous solution and showed significantly near-infrared emission (654 nm) with high fluorescence quantum yields (18.3%). The high quantum yield of the fluorescent magnetic NPs are attributed to the restricted intramolecular rotation of the DPPBPA molecules in the spatially confined hydrophobic core environment of the NPs, which prevents direct interactions of DPPBPA molecules. The NPs are magnetically susceptible with zero remanence and coercivity, suggesting that they are good superparamagnets with high magnetization, which show effective MRI ability. The Fe₃O₄/DPPBPA@F-127 NPs show less cytotoxicity in aqueous solution, suggesting NPs are biocompatible nanocarriers. Due to its high brightness, low cytotoxicity and excellent stability, the obtained NPs have been successfully utilized in in vivo fluorescence imaging with high fluorescence contrast. We anticipate that this strategy of multifunctional NPs will inspire the development of a novel biological nanomaterials with high efficient fluorescence and strong magnetic susceptibility.

Experimental section

Materials

All reagents and starting materials are commercially available and were used without further purification, unless otherwise noted. 1,4-Dibromo-2,5-dimethylbenzene was purchased from Aladdin (Shanghai, China). Benzyl cyanide was purchased from J&K Scientific Ltd (Beijing China). Pluronic F-127 and MTT were purchased from Sigma-Aldrich (St. Louis, USA). Deionized water (18.2 MΩ cm resistivity) from a Milli-Q water system was used throughout the experiments before being used as solvents.

Instrumentations

Mass spectra were recorded on an Agilent 1100 LC-MS system. ¹H NMR spectra were recorded on Bruker AVANVE 500 MHz spectrometer or Varian 300 MHz with tetramethylsilane as the internal standard. UV-vis absorption spectra were recorded using a Shimadzu UV-3600 UV-vis spectrophotometer. Solid state PL efficiencies were measured using an integrating sphere (C-701, Labsphere Inc.) with a 365 nm Ocean Optics LLS-LED as the excitation source, and the laser was introduced into the sphere through the optical fiber. Photoluminescence spectra were collected on a Shimadzu RF-5301PC spectrophotometer. DLS and zeta potential measurement was performed using a Malvern Zetasizer Nano ZS size analyser at room temperature. Transmission electron microscopy (TEM) images were obtained using a transmission electron microscope (TEM, JEM-2100F). The cellular imaging was performed on an Olympus IX71 microscope with mercury lamp as the excitation source.

Measurements of fluorescence quantum yields. Fluorescence quantum yields for the solutions and suspensions were obtained by comparing to the fluorescence spectrum of rhodamine in ethanol (absorbance value < 0.1, excitation wavelength: 365 nm, PL efficiencies Φ_r = 69%) with corrections of refractive index differences using eqn.⁴⁷
where Φ_r and Φ_s are the fluorescence quantum yields of standards and the samples, respectively. A_r and A_s are the absorbances of the standards and the measured samples at the excitation wavelength, respectively. I_r and I_s are the integrated emission intensities of standards and the samples. n_r and n_s are the refractive indices of the corresponding solvents of the solutions, respectively.

Measurements of the leaching issue. The leakage degrees of the dye in the matrix were investigated using fluorescence spectra. After continuous vigorous sonicating, Fe₃O₄/DPPBPA@F-127 NPs were separated by magnet from 1 mL aqueous solution and then dispersed in 1 mL water by sonicator. After that, the fluorescence was measured.

Synthesis of 2,5-dibromobenzene-1,4-dicarbaldehyde (1)

2,5-Dibromobenzene-1,4-dicarbaldehyde (1) was synthesized according to the procedure shown in Scheme S1.† All chemicals were purchased commercially, and used without further purification. 4 g of 1,4-dibromo-2,5-dimethylbenzene was dissolved in 20 mL of acetic acid and 40 mL of acetic anhydride at 0 °C. 14 mL of sulfuric acid was added dropwise into the solution, which was stirred for another 10 min. CrO₃ was grinded into powders, and then added to the mixture in portions. The resulting mixture was stirred vigorously for another 5 h at the temperature under 10 °C.³⁸ The greenish slurry was poured into ice water and filtered. The solid was washed with water and microscale cold methanol. The white compound would be get. The diacetate was then hydrolyzed by refluxing with a mixture of 20 mL of water, 20 mL of ethyl alcohol, and 2 mL of sulfuric acid for 5 h. After the mixture cooled, the pale yellow product was separated by filtration. The crude product was purified by recrystallization from chloroform. Yellow crystal will be get (1.83 g, 40%). ¹H NMR δ_H (500 MHz, CDCl₃) 10.38 (2H, s), 8.19 (2H, s).

Synthesis of (2Z,2′Z)-3,3′-(2,5-dibromo-1,4-phenylene)bis(2-phenylacrylonitrile) (2)

The mixture of benzylcyanide (201 mg, 1.5 mmol) and compound 1 (659 mg, 3 mmol) in tert-butyl alcohol (10 mL) was stirred at 46 °C for 30 min. Then, potassium tert-butoxide (1 M solution in tetrahydrofuran, 0.25 mL) and tetrabutylammonium hydroxide (TBAH, 1 M solution in methanol, 0.25 mL) were slowly added, and stirred for 60 minutes.³⁷ The resulting precipitate was filtered and purified by column chromatography using dichloromethane. Compound 2 (348 mg) was obtained in yield of 70% by evaporated the solvent. ¹H NMR: δ_H (500 MHz, CDCl₃) 8.39 (2H, s), 7.79 (2H, s), 7.77 (4H, dd, J 7.9, 1.2), 7.56–7.43 (6H, m).

Synthesis of (2Z,2′Z)-3,3′-(2,5-di(piperidin-1-yl)-1,4-phenylene)bis(2-phenylacrylonitrile) (DPPBPA)

A vial tube (25 mL) equipped with a magnetic stirring bar was charged with Pd₂(dba)₃ (2.06 mg, 22.5 μmol) and Ruphos (42 mg, 90 μmol). The vial tube was then capped with a rubber septum, evacuated for 5 min and charged with nitrogen. The evacuation–purge operation was repeated twice. Toluene (10 mL) was added to the vial at room temperature under a nitrogen atmosphere and the resulting mixture was stirred at room temperature for 5 min. To the solution were added compound 2 (0.147 g, 0.3 mmol), piperidine (0.297 mL, 3 mmol), and K₃PO₄ (0.87 g, 3 mmol).⁴⁸ The mixture was stirred at 100 °C for 15 h and then allowed to cool to room temperature. The reaction mixture was passed through a short pad of neutral alumina (activated level I). The filtrate was concentrated by evaporation. Then the crude product was purified by silica gel chromatography (hexane/CH₂Cl₂ = 1 : 1) to give a red solid. Then, the pure product was obtained by sublimation in the yield of 15%. ¹H NMR: δ_H (500 MHz, CDCl₃) 8.06 (2H, s), 7.93 (2H, s), 7.78 (4H, d, J 7.6), 7.51 (4H, t, J 7.6), 7.43 (2H, t, J 7.4), 3.06–2.93 (8H, m), 1.80–1.71 (8H, m), 1.62 (4H, s), GC-MS: C₃₄H₃₄N₄, calcd 498.66; found 498.66.

Synthesis of oleic acid coated Fe₃O₄

A solution of 0.01 mol ferric chloride hexahydrate and 0.005 mol ferrous chloride tetrahydrate in 100 mL of de-ionized (DI) water in a 3-neck round bottom flask was stirred by mechanical stirrer at 80 °C with nitrogen bubbling for 30 min. 0.01 mol of potassium oleate was added, and the mixture was stirred for another 30 min. 35 mL of 4% ammonium hydroxide was added to the mixture. The reaction system turned to black immediately. The reaction continued at 80 °C under nitrogen bubbling for 30 min. The black liquid was centrifuged at 60 000 rpm for 20 min at 20 °C to separate unreacted oleic acid from magnetic NPs. The magnetic NPs were dispersed in THF at 10 mg mL⁻¹ for further application.

Preparation of Fe₃O₄/DPPBPA@F-127

In a typical preparation, the fluorescent DPPBPA was first dissolved in tetrahydrofuran (THF) to make a 0.5 mg mL⁻¹ stock solution. OA-Fe₃O₄ (1 mg, 2 mg), DPPBPA (0.5 mg, 1 mg) and Pluronic F-127 (6 mg, 8 mg, 10 mg, 12 mg) with different contentions were dispersed in 5 mL THF by a vigorous bath sonicator. The solution mixture was quickly added to DI water, and the THF was removed by nitrogen stripping in a vigorous bath sonicator. The solution was concentrated by continuous nitrogen on a 90 °C hotplate followed by filtration through a 0.2 micron filter. During nanoparticle formation, the OA-Fe₃O₄ and DPPBPA tended to entangled with the hydrophobic domains of Pluronic F-127, to afford the hydrophobic interiors, while the hydrophilic domains of Pluronic F-127 extend into the aqueous phase of the NPs.

Cell culture

MCF-7 breast cancer cells were provided by American Type Culture Collection (ATCC). The cells were cultured in DMEM medium containing 10% heat-inactivated FBS (Invitrogen), 100 U mL⁻¹ penicillin, and 100 μg mL⁻¹ streptomycin (Thermo Scientific) and maintained in a humidified incubator at 37 °C with 5% CO₂. Before experiment, the cells were precultured until confluence was reached. MCF-7 cells were seeded in a 96-well flat-bottomed microplate (10 000 cells per well) and cultured in 100 μL growth medium at 37 °C and 5% CO₂ for 24 h. Cell culture medium in each well was then replaced by 100 μL cell growth medium, containing Fe₃O₄/DPPBPA@F-127 with concentrations ranging from 0 to 100 ppm. After incubation for 20 h, 20 μL of MTT (5 mg mL⁻¹ in PBS solution) was added to each well, and cells were incubated further for 4 h at 37 °C. The growth medium was removed gently, and 150 μL of DMSO was then added to each well, sitting at room temperature overnight to dissolve the formazan crystals completely. The absorbance at the wavelength of 570 nm was measured by Multiskan EX (Thermo Electron Corporation), and each data point represents a mean + SD from triplicate wells.

Percentage cell viability = (average Abs value of experimental group − average Abs value of blank group)/(average Abs value of control group − average Abs value of blank group) × 100%.

Percentage cell cytotoxicity = [1 − (average Abs value of experimental group − average Abs value of blank group)/(average Abs value of control group − average Abs value of blank group)] × 100%.

Percentage cell cytotoxicity = (1 − percentage cell viability) × 100%.

Cell imaging

To study the cellular uptake, MCF-7 cells were added to 6-well plate, treated with various concentrations Fe₃O₄/DPPBPA@F-127 (50 ppm) for 12 h. The cells were washed with PBS before observations on fluorescence microscopy. The cellular imaging was performed on a Fluorescence microscope. Cellular uptake of the NPs was investigated by using flow cytometer (FACS Calibur, Becton and Dickinson Company) with excitation and emission wavelengths of 460 nm and FLA-3. In total, 10 000 cells were analyzed per sample.

Acknowledgements

This work was supported by (2013CB834701), the Natural Science Foundation of China (No. 51373063, 51573068, 21221063) and Program for Chang Jiang Scholars and Innovative Research Team in University (No. IRT101713018).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07520g

‡ These authors contributed equally to this work and should be considered co-first authors.

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