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Facile Preparation of Fluorescent Nanoparticles with Tunable Exciplex Emission and Their Application to Targeted Cellular Imaging Lu Liu, Ronghua Liu, Xiaoyu Wang, Qianling Cui, Chuang Yao, Shuxian Zhu, and Lidong Li ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00116 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 18, 2018

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ACS Applied Bio Materials

Facile Preparation of Fluorescent Nanoparticles with Tunable Exciplex Emission and Their Application to Targeted Cellular Imaging Lu Liu,#,† Ronghua Liu,#,† Xiaoyu Wang,*,† Qianling Cui,† Chuang Yao,§ Shuxian Zhu,† and Lidong Li*,†,‡ †

State Key Laboratory for Advanced Metals and Materials, School of Materials Science and

Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China. ‡

State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024,

P. R. China. §

Key Laboratory of Extraordinary Bond Engineering and Advanced Materials Technology

(EBEAM) Chongqing, Yangtze Normal University, Chongqing 408100, P. R. China.

KEYWORDS: fluorescence, nanoparticle, donor–acceptor system, exciplex, targeted cellular imaging ABSTRACT Fluorescent nanoparticles with tunable emission show good potential for using in biological imaging. Exciplex emission usually appears with a large red shift from the normal emission peak. The integration of exciplex emission into nanoparticles offers a rational strategy to designing fluorescent nanoparticles with tunable emission. In this work, we doped electron acceptors into the electron donor poly(N-vinylcarbazole) (PVK) to develop novel fluorescent 1

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nanoparticles with a conveniently modulated PVK emission. Through careful design of the molecular structures of the electron acceptors, we demonstrated that controlled donor– acceptor spatial stacking and electron transitions could regulate the exciplex emission of the PVK/acceptor nanoparticles. Thus, the structurally controlled exciplex formation allowed for the preparation of multi-colored fluorescent nanoparticles. Moreover, further modifications with the cyclic peptide RGD showed little disruption to the structure of the PVK/acceptor nanoparticles and the corresponding exciplex emission. Hence, the nanoparticles showed the ability to be used for targeted cellular imaging. On the basis of the RGD-integrin αvβ3 (ligand–receptor) interaction, the nanoparticles were effectively endocytosed by target cancer cells. We anticipate that this research could provide a new strategy for the fabrication of fluorescent nanoparticles with tunable emission, leading to useful materials for fluorescent imaging.

INTRODUCTION Fluorescence technologies are widely used in chemical sensing and biological imaging.1-5 The major growth of fluorescence technology can be attributed to the rapid development of various imaging materials with excellent optical performances.6-8 Owing to their small size and ability to be taken up by cells, fluorescent nanomaterials are regarded as attractively prospective imaging agents.9-11 To date, numerous fluorescent nanomaterials have been investigated for application to fluorescent imaging, such as small molecule nanoparticles12,13, polymer nanoparticles14-17, quantum dots18,19, metal nanostructures20,21, and composite nanoparticles22,23. Among these, semiconducting polymer nanoparticles with π-electron 2


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systems are attractive materials because of their unique optical and electrical properties.24,25 Moreover, the absence of heavy metals in these kinds of nanoparticles avoids heavy metal ion-induced toxicity to organisms, which is an important property for biological applications.26,27 However, regulating fluorescence emission properties of semiconducting polymer nanoparticles to meet the demands of certain applications remains challenging. The optical properties of semiconducting polymer nanoparticles are mainly determined by their molecular structures, which can be changed to tune their emission properties28,29. However, such structural changes are complex and time-consuming. A more straightforward method is to encapsulate energy acceptors within semiconducting polymer nanoparticles to ensure Förster resonance energy transfer and multicolor emission.30-32 However, it is difficult to obtain a single emission that is completely different from the native emission. In this respect, encapsulating electron acceptors to form exciplexes is a promising way to tune and change the fluorescent emission of semiconducting polymer nanoparticles. Exciplexes, generated from the association of excited molecules and other ground-state molecules, show a large red shift of their emission compared with the normal molecular emission.33-35 Semiconducting polymers are electronically active materials.36 The electron donor (D)–acceptor (A) system formed in a semiconducting polymer matrix can generate exciplexes upon photoexcitation.37 More importantly, manipulation of the D–A stacking in aggregates can control the formation of exciplexes38 and successfully circumvent π-stacking induced fluorescence quenching. In this study, we examined tuning of the fluorescence emission of poly(N-vinylcarbazole) (PVK) nanoparticles with three synthetic electron acceptors. PVK is a typical 3



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electron-donating polymer containing carbazole units.39 By designing the molecular structure of the electron acceptors, we successfully modulated the D–A stacking and exciplexes formation in PVK nanoparticles. Hence the fluorescence emission of the PVK/acceptor nanoparticles could be modulated by controlling the exciplex emission. A cyclic peptide of arginine-glycine-(aspartic acid)-(D-tyrosine)-cysteine (RGD for short) was also covalently conjugated to these nanoparticles to enhance the targeting specificity and efficiency. The RGD modified PVK/acceptor nanoparticles showed excellent structural stability under physiological conditions as well as the capability to specially bind with αvβ3 receptor-positive cells, resulting in a high exciplex fluorescent signals. Our exploration of structural control of exciplex formation in nanoparticles provides a uniquely simple approach to achieving the desired emission characteristics. EXPERIMENTAL SECTION Materials and Measurements. 2,4,6-Tri(4-bromophenyl)-1,3,5-triazine

(compound

2,4,6-tris(4-(diphenylphosphanyl)phenyl)-1,3,5-triazine

(compound

1), 2)

and

2-(4-bromophenyl)-4,6-bis(4-(diphenylphosphanyl)phenyl)-1,3,5-triazine (compound 3) were synthesized according to literature procedures.40,41 The detailed synthesis processes of TPOTZ, BPOTZ and BPOTZ-OR were provided in the supporting information. PVK, PSMA (cumene terminated, Mw ≈ 1,700),

1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide

hydrochloride (EDC), N-hydroxysuccinimide (NHS) and bis(triphenylphosphine)palladium (II) dichloride [Pd(PPh3)2Cl2] and other mentioned reagents were purchased from Sigma-Aldrich and J&K Chemical. RGD was obtained from Hangzhou Chinese Peptide 4


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Company. Three cell lines MDA-MB-231, MCF-7 and L-02 were obtained from the Chinese Academy of Medical Sciences. The 13C NMR and 1H NMR spectra were operated at resonance frequencies of 100 and 400 MHz, respectively, on AC Bruker spectrometers. Mass spectra data were measured on a Bruker Daltonics BIFLEX III MALDI-TOF analyzer. Cyclic voltammetry was conducted on a CHI860D electrochemical analyzer. Absorption spectra were recorded with Hitachi U-3900H spectrophotometer. Fluorescence spectra and excitation spectra were recorded with Hitachi F-7000 fluorescence spectrometer. The morphology of the nanoparticles was observed with a scanning electron microscope (SEM, Carl Zeiss Jena, SUPRA 55 SAPPHIRE). The size measurents were conducted on a Nano ZS90 (Malvern Instrument Ltd., England). The Bruker Hyperion 2000 spectrometer was used for fourier transform infrared spectroscopy (FT-IR) analysis. Fluorescence lifetimes were measured by a Delta Flex spectrofluorometer with an excitation wavelength of 340 nm. Cellular images were recorded on Olympus 1X73 fluorescence microscope with mercury lamp as the light source for 375/28 nm excitation with a 100 ms exposure time. Cell viability was determined using a Spectra MAX 340PC plate reader. Preparation of PVK/acceptor NPs The reprecipitation method was used to prepare PVK/acceptor nanoparticles. Compound PVK, TPOTZ, BPOTZ, and BPOTZ-OR were dissolved in THF at a concentration of 2 mg/mL. PVK solution (100 µL) and acceptor (TPOTZ, BPOTZ or BPOTZ-OR) solution (100 µL) were mixed and injected into 2 mL of deionized water rapidly. The solution was under ultrasonication for 5 min and then heated at 70 °C to remove THF and condensed to 1 mL. 5



PVK/acceptor nanoparticles were obtained. Calculation Method The optimized geometries of TPOTZ, BPOTZ, and BPOTZ-OR ground states were calculated by the B3LYP/Def2-SVP level. Imaginary frequency was not found for the optimized structures.42 The dimers were constructed based on the relative optimized geometry and optimized by the BLYP/Def2-SVP level. The geometrical Counterpoise Correction43 was used to remove the artificial overbinding effects in these calculations. To improve the accuracy, atom-pairwise dispersion correction was also used.44 The whole calculations were executed on the ORCA 4.0.0 software.45 Preparation of PVK/BPOTZ-OR@RGD NPs First, 100 µL of PVK solution, 100 µL of BPOTZ-OR solution and 200 µL PSMA solution (2 mg/mL) were mixed and rapidly injected into 2 mL of deionized water. After 5 min of ultrasonication, the solution was heated at 70 °C to remove THF and condensed to 1 mL. Then, RGD was conjugated onto PVK/BPOTZ-OR nanoparticles by adding 300 µL of EDC (1 mg/mL) and 300 µL of NHS (1 mg/mL) to the PVK/BPOTZ-OR nanoparticle solution. After 1 h, 300 µL of RGD solution (2 mg/mL) was injected and the mixture was then kept stirring for 12 h. The mixture was dialyzed for 24h to purify PVK/BPOTZ-OR@RGD nanoparticles. The concentration of PVK/BPOTZ-OR@RGD nanoparticles was 1.8 mg/mL. Cellular Imaging Experiments. MDA-MB-231 cells were cultured with DMEM containing 10% fetal calf serum (FBS) (v/v) and then seeded on confocal dishes. The cells were incubated with 1 mL of culture medium containing 50 µL of PVK/BPOTZ-OR and PVK/BPOTZ-OR@RGD nanoparticles, 6


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respectively. The cells were incubated for 3 h and then washed three times with PBS. The fluorescent images of cells were observed with an Olympus 1X73 fluorescence microscope. For selective imaging, MCF-7 and L-02 cells were chosen as control groups and incubated with PVK/BPOTZ-OR@RGD nanoparticles, respectively. Cell viability assay MDA-MB-231, MCF-7, and L-02 cells were cultured in 96-well plates. After incubation with various concentrations of PVK/BPOTZ-OR@RGD nanoparticles (9−180 µg/mL) for 10 h,

the

medium

in

each

well

was

replaced

by

100

µL

of

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution. The cells were incubated for another 4 h. Then, the MTT was replaced by 100 µL of DMSO. By using a Spectra MAX 340PC plate reader, the absorbance of purple formazan at 570 nm was measured. RESULTS AND DISCUSSION Preparation and characterization of the PVK/acceptor nanoparticles The electron-rich polymer PVK38 was used as the electron-donating matrix in this work. Three electron acceptors TPOTZ, BPOTZ, and BPOTZ-OR were synthesized according to the synthetic routes shown in Figure S1. As shown in Figure 1a, the electron-withdrawing groups of 1,3,5-triazine and triphenylphosphine oxide were introduced into these molecules46 to produce excited-state interactions with PVK. We measured the lowest unoccupied molecular orbital (LUMO) by cyclic voltammetry (Figure S2a-S2c).47 From the onset of the absorption wavelength (λedge), the optical bandgap was calculated: Egopt = 1240/λedge (UV–vis absorption results are shown in Figure S2d). We then calculated the highest occupied 7



molecular orbital (HOMO) from the LUMO and optical bandgap: EHOMO = ELUMO–Egopt.47 As expected, the electron-withdrawing groups induced the LUMO of these molecules to be low lying at approximately –3.2 eV (Figure 1b). The electron affinity was also improved, which can lead to charge-transfer (CT) interactions within the electron donor polymer PVK.

Figure 1. (a) Molecular structures of PVK, TPOTZ, BPOTZ, and BPOTZ-OR. (b) HOMO and LUMO energy levels for PVK, TPOTZ, BPOTZ, and BPOTZ-OR. Owing to the hydrophobic properties of the molecules, PVK/acceptor nanoparticles were formed in the PVK and acceptors (1:1 mass ratio) via nanoprecipitation. PVK nanoparticles were prepared in the same manner. SEM images in Figure 2 show the spherical morphology of the PVK, PVK/TPOTZ, PVK/BPOTZ and PVK/BPOTZ-OR nanoparticles, with average diameters of 45 ± 3, 52 ± 5, 54 ± 4, and 53 ± 4 nm, respectively. Moreover, dynamic light scattering confirmed their good uniformity with polydispersity indexes of 0.16 ± 0.01 for PVK, 0.19 ± 0.01 for PVK/TPOTZ, 0.22 ± 0.01 for PVK/BPOTZ, and 0.20 ± 0.01 for PVK/BPOTZ-OR (Figure S3).

8


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Figure 2. SEM images of (a) PVK, (b) PVK/TPOTZ, (c) PVK/BPOTZ, and (d) PVK/BPOTZ-OR nanoparticles, respectively. The optical properties of these nanoparticles were investigated. The strong absorption peaks of PVK at 290, 330, and 342 nm were due to the π–π* transitions of the carbazole moiety48 (Figure S4a). Compared with the strong emission at 373 nm of PVK in THF solution, the two emission peaks of the PVK nanoparticles located at 375 and 402 nm resulted from the intra-chain face-to-face overlap of carbazole groups49 (Figure 3a). After TPOTZ, BPOTZ, and BPOTZ-OR were embedded into PVK matrices to form nanoparticles, new emission features appeared at approximately 500 nm for TPOPZ, BPOTZ, and BPOTZ-OR. These features were different from the original emission peaks at 372, 465, and 450 nm for TPOTZ, BPOTZ and BPOTZ-OR, respectively (Figure S4b). The absolute fluorescence quantum yields of PVK, PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR 9



nanoparticles were measured to be 6.8%, 9.1%, 2.7% and 2.0%, respectively. Consequently, a change of the bright fluorescence from blue/cyan to green could be directly observed under irradiation by UV light (Figure 3b).

Figure 3. (a) Fluorescent emission spectra of PVK, PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR nanoparticles. Excitation wavelength was 330 nm. (b) Photographs of PVK, PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR nanoparticles under irradiation at 254 nm. [PVK] = 0.05 mg/mL, [PVK/TPOTZ] = 0.1 mg/mL, [PVK/BPOTZ] = 0.1 mg/mL and [PVK/BPOTZ-OR] = 0.1 mg/mL. We inferred that the new emission originated from exciplexes based on the D–A system in PVK/acceptor nanoparticles. In the formed nanoparticles, the electron-rich PVK and electron-deficient acceptors were forced into proximity, which promoted the CT interaction between them. The excited PVK molecules could associate with the ground-state acceptor molecules and thus led to formation of exciplexes. The corresponding photon energy of the exciplex emission was related to the energy difference between the HOMO donor and LUMO acceptor: hvem ~ LUMOdonor–HOMOacceptor–C, where C represents the electron–hole Coulombic binding energy in the exciplex.47 The emission peak at 500 nm was evaluated to be 2.5 eV. These results are consistent with the differences between the LUMO of the three 10


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acceptors (TPOTZ, BPOTZ, and BPOTZ-OR) and the HOMO of PVK50. Thus, the new emissions could be attributed to exciplex emission. We then measured the fluorescence excitation spectra of the new emission bands from the PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR nanoparticles. As shown in Figure 4a, the excitation spectra of the nanoparticles were similar. The peaks at 295, 330, and 342 nm matched with the absorption bands of PVK, as shown in Figure S4a. This result confirms that these new emission peaks featured the same excitation pathway as PVK, and ruled out a ground-state CT interaction. These features clearly suggested the unique characteristics of exciplexes.51 Furthermore, we prepared TPOTZ, BPOTZ, and BPOTZ-OR nanoparticles with diameters of 80 ± 7, 72 ± 3, and 76 ± 5 nm, respectively (Figure S5). After mixing with the PVK nanoparticles, no new emission peaks appeared in the binary nanoparticle solutions (Figure 4b). Thus, depending on the relative proximity of PVK and the acceptors in the nanoparticles, a D–A system formed and red-shifted emission from exciplexes was observed.

Figure 4. (a) Excitation spectra of PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR nanoparticles monitoring emission at 500 nm. (b) Fluorescent emission spectra of a mixture of PVK nanoparticles with TPOTZ, BPOTZ, and BPOTZ-OR nanoparticles. Excitation wavelength was 330 nm. [PVK/TPOTZ] = 0.1 mg/mL, [PVK/BPOTZ] = 0.1 mg/mL, 11



[PVK/BPOTZ-OR] = 0.1 mg/mL, [PVK] = 0.05 mg/mL, [TPOTZ] = 0.05 mg/mL, [BPOTZ] = 0.05 mg/mL, and [BPOTZ-OR] = 0.05 mg/mL. Notably, only exciplex emission was presented in the PVK/BPOTZ-OR nanoparticles. Figure 3b shows that the fluorescence color of the PVK/BPOTZ-OR nanoparticles was green, and different from the blue fluorescence of the PVK nanoparticles and the cyan fluorescence of the other nanoparticles. By comparing these nanoparticles, the modulated emission depended only on the nanostructure of the acceptor molecule in the formed nanoparticles. Thus, we simulated face-to-face stacking of the three acceptors. The π−π interactions could be expressed by the overlap of 1,3,5-triazine cores in the two molecules.52 As shown in Figure 5 and Figure S6, the two TPOTZ molecules overlapped well and two BPOTZ molecules showed a slight displacement. This arrangement resulted in a very strong π−π interaction. For BPOTZ-OR, there was a remarkable displacement between two molecules. The alkyl chains reduced the π−π interaction of BPOTZ-OR. Thus, the weakened π−π interactions between the two neighboring BPOTZ-OR molecules facilitated the formation of a D−A system for PVK and BPOTZ-OR. These factors might account for the exciplex emission from the PVK/BPOTZ-OR. Moreover, considerable emission quenching occurred in TPOTZ and BPOTZ nanoparticles, owing to the strong π−π stacking in the aggregates of these molecules (Figure S7a and S7b). However, an obvious blue-shifted emission band and 40% intensity reduction were observed in the BPOTZ-OR nanoparticles (Figure S7c). This result confirmed that the spatial restrictions imposed by the alkyl chains reduced the extent of the BPOTZ-OR π−π stacking in the aggregates.53 This conclusion is consistent with the results shown in Figure 5. 12


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Figure 5. Top view of the face-to-face π−π-stacking of TPOTZ, BPOTZ, and BPOTZ-OR, respectively. Exciplex emission competes with the corresponding monomer emission. Based on the above results, we infer that the exciton to exciplex emission of PVK could be well modulated through control of the stacking of PVK and the acceptor molecules. As shown in Scheme 1, the PVK nanoparticles emitted their own blue fluorescence. After encapsulation of TPOTZ or BPOTZ within the PVK nanoparticles, these acceptors tended to show strong π−π stacking. Only part of the acceptor molecules were in contact with PVK to generate intermolecular electron transfer complexes that showed exciplex emission. Thus, the exciplex emission was weak. The cyan emission of the nanoparticles was dominated by the internal emission of PVK. However, owing to the relatively weak π−π stacking of BPOTZ-OR, more molecules were in proximity to PVK and formed strong D−A systems, which gave rise to strong exciplex emission.

13



Scheme 1. Schematic diagram of PVK, PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR nanoparticles structures. Application to targeted cellular imaging The PVK/BPOTZ-OR nanoparticles showed a large Stokes shift of 170 nm and red shifted emission at 500 nm, which suggested good potential for applications to bioimaging. To fabricate PVK/BPOTZ-OR nanoparticles for targeted cellular imaging, we modified the PVK/BPOTZ-OR nanoparticles with a cyclic peptide RGD. The poly(styrene-co-maleic anhydride) (PSMA) coating presented carboxyl groups for bioconjugation with RGD through the typical carbodiimide mediated coupling reaction (Scheme 2).

Scheme 2. Schematic diagram of RGD modified PVK/BPOTZ-OR nanoparticle preparation. By comparing the FT-IR spectra in Figure S8, the enhanced peaks at 1630 cm−1 (amide I) and 1325 cm−1 (amide III) were attributed to the amide bond being modified by RGD. These 14


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results indicate the successful preparation of PVK/BPOTZ-OR@RGD nanoparticles. The SEM image in Figure S9 shows that the PVK/BPOTZ-OR@RGD nanoparticles maintained their spherical morphology with a uniform size of 84 ± 3 nm. Compared with PVK/BPOTZ-OR nanoparticles, the increased size was attributed to the modified RGD. The optical properties of PVK/BPOTZ-OR@RGD nanoparticles were investigated further. As shown in Figure 6a, the PVK/BPOTZ-OR@RGD nanoparticles showed only exciplex emission. The fluorescence lifetimes of these nanoparticles were also investigated (Figure 6b). The calculated average lifetimes were 48 and 46 ns for PVK/BPOTZ-OR@RGD and PVK/BPOTZ-OR nanoparticles, respectively. There was almost no change between these. These results confirmed that the RGD modification did not affect the CT interaction between PVK and BPOTZ-OR. The D–A system in these nanoparticles was stable and maintained exciplex emission. Notably, these nanoparticles possessed a longer lifetime than that of the PVK nanoparticles (6.2 ns). These long-lived exciplexes are typical of transitions involving molecular interactions.54 These results confirmed the formation of exciplexes through interactions of PVK and BPOTZ-OR. The stability of PVK/BPOTZ-OR@RGD nanoparticles in biological solutions also need to be investigated for their application to cellular imaging. As shown in Figure S10, the hydrodynamic diameters of PVK/BPOTZ-OR@RGD nanoparticles had little effect in both PBS (pH 7.4) and cell culture. These results indicated that the PVK/BPOTZ-OR@RGD nanoparticles possess good structural stability in different solutions, which promotes exciplex emission and is beneficial for their application to cellular imaging.

15



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Figure 6. (a) Fluorescence spectra of PVK/BPOTZ-OR and PVK/BPOTZ-OR@RGD nanoparticles. Excitation wavelength was 330 nm. (b) Fluorescence intensity decay of PVK/BPOTZ-OR, PVK/BPOTZ-OR@RGD and PVK nanoparticles, respectively. Owing to the recognition of RGD to integrin αvβ355, the targeting capability of PVK/BPOTZ-OR@RGD nanoparticles for the integrin-positive cells was evaluated in vitro. The human breast cancer cells MDA-MB-231 were selected as integrin-positive cancer cells because of their overexpression of integrin αvβ3. The human breast cancer cells MCF-7 and human liver cells L-02 were selected as a control group owing to their low expression of integrin αvβ3.56,57 As shown in Figure 7, a clear green fluorescence signal was detected in MDA-MB-231 cells after incubation with PVK/BPOTZ-OR@RGD nanoparticles for 3 hours. Conversely, PVK/BPOTZ-OR without RGD modification cannot be endocytosed by the MDA-MB-231 cells in 3h (Figure S11). It confirmed the modification of RGD on the PVK/BPOTZ-OR

nanoparticles.

After

incubation

with

PVK/BPOTZ-OR@RGD

nanoparticles with other two cells, the MCF-7 cells showed very faint fluorescence. There was no fluorescent signal for the L-02 cells. This observation indicates that PVK/BPOTZ-OR@RGD nanoparticles were efficiently endocytosed by the MDA-MB-231 cells, which was reasonably attributed to endocytosis mediated by the RGD−αvβ3 integrin 16


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interaction. Such nanoparticles could be applied to target imaging of integrin-positive cells and to differentiate between the two breast cancer cells MDA-MB-231 and MCF-7. The cytotoxicity of PVK/BPOTZ-OR@RGD nanoparticles was investigated by the MTT assay to evaluate biocompatibility. As shown in Figure 8, the RGD modified nanoparticles displayed low toxicity to all of the three cell lines even at the high doses of PVK/BPOTZ-OR@RGD nanoparticles (180 µg/mL). More than 92% of the MDA-MB-231 cells remained alive. Meanwhile, the MCF-7 cells maintained 93% cell viability and the normal L-02 cells maintained more than 100% cell viability. Thus, the PVK/BPOTZ-OR@RGD nanoparticles showed good biocompatibility. More importantly, the nanoparticles were safe and non-toxic to normal L-02 cells, which is promising for applications to targeted cellular imaging.

Figure 7. Bright field, fluorescent and overlay images of MDA-MB-231, MCF-7, and L-02 cells after incubation with PVK/BPOTZ-OR@RGD nanoparticles for 3 h. The scale bar 17



represents 5 µm.

Figure 8. Cell viability of MDA-MB-231, MCF-7, and L-02 cells after incubation with PVK/BPOTZ-OR@RGD nanoparticles at a concentration of 0−180 µg/mL. CONCLUSION In this work, we provided a simple method to tune the emission of PVK nanoparticles by embedding electron acceptors and forming exciplexes. Through modulating the molecular structures of electron acceptors, we demonstrated that exciplex formation in the PVK/acceptor nanoparticles strongly depends on the optimized D–A stacking and efficient electron transition between them. Thus, the fluorescence color of the obtained PVK/acceptor nanoparticles changed from blue/cyan to green owing to the structurally induced exciplex formation. The exciplex formation in the PVK/acceptor nanoparticles was robust and showed good structural stability even after modification of RGD. On the basis of the recognition of RGD by integrin, the RGD modified nanoparticles were able to bind with integrin-positive cells and showed strong fluorescence signals. Furthermore, their good biocompatibility allowed us to specifically image cells in a safe manner. Therefore, it provides new insights into the fabrication of fluorescent nanoparticles with tunable emission for biological imaging. ASSOCIATED CONTENT 18


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Supporting Information.

Synthesis routes of TPOTZ, BPOTZ and BPOTZ-OR. Cyclic voltammograms of TPOTZ, BPOTZ and BPOTZ-OR. Normalized absorption spectra of PVK, TPOTZ, BPOTZ and BPOTZ-OR in THF solution. Hydrodynamic diameter distributions of PVK, PVK/TPOTZ, PVK/BPOTZ, and PVK/BPOTZ-OR NPs in aqueous dispersions. Normalized emission spectra of PVK, TPOTZ, BPOTZ and BPOTZ-OR in THF solution. Hydrodynamic diameter distributions of TPOTZ, BPOTZ and BPOTZ-OR NPs in aqueous dispersions, respectively. Side view of the face-to-face π−π-stacking of TPOTZ, BPOTZ, and BPOTZ-OR, respectively. Emission spectra of TPOTZ, BPOTZ and BPOTZ-OR NPs in water. FT-IR spectra of PVK/BPOTZ-OR@PSMA NPs and PVK/BPOTZ-OR@RGD NPs. SEM images of

PVK/BPOTZ-OR@RGD

NPs.

Hydrodynamic

diameter

distributions

of

PVK/BPOTZ-OR@RGD NPs in water, PBS and DMEM, respectively. Bright field, fluorescent and merged images of MDA-MB-231 cells after cultured with PVK/BPOTZ-OR nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

L.L. and R.L. contributed equally to this study.

Notes 19



The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51673022, 51703009), the State Key Laboratory of Fine Chemicals (KF1613), the State Key Laboratory for Advanced Metals and Materials (2018Z-18) and the Fundamental Research Funds for the Central Universities (FRF-TP-16-026A1).

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