Facile Preparation of AIE-Active Fluorescent Nanoparticles through

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Facile Preparation of AIE-active Fluorescent Nanoparticles through Flash Nanoprecipitation Mingwei Wang, Nan Yang, Zhiqian Guo, Kaizhi Gu, Andong Shao, WeiHong Zhu, Yisheng Xu, Jie Wang, Robert K Prud'homme, and Xuhong Guo Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b00501 • Publication Date (Web): 13 Apr 2015 Downloaded from http://pubs.acs.org on April 21, 2015

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Industrial & Engineering Chemistry Research

Facile Preparation of AIE-active Fluorescent Nanoparticles through Flash Nanoprecipitation Mingwei Wang†, Nan Yang†, Zhiqian Guo*,†, Kaizhi Gu†, Andong Shao†, Weihong Zhu†, Yisheng Xu*,†, Jie Wang†, Robert K. Prud’homme‡, Xuhong Guo *,† †

State-Key Laboratory of Chemical Engineering, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China

‡

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA

ABSTRACT: Flash nanoprecipitation (FNP) is an easy scalable and fast processing method for preparing of nanoparticles (NPs) with a simply vortex equipment. By using the FNP method, fluorescent nanoparticles are prepared less than 1 second in a multi-inlet vortex mixer, in which hydrophobic aggregation-induced emission (AIE)-active dye of EDP is incorporated within the biocompatible block copolymer PEG-b-PCL for EDP-NPs assembly. The formulation parameters of stream velocity, dyes as well as loading and concentration in FNP are optimized. The sizes of NPs ranged from 20 to 60 nm with the ratio change of mixed solvents. As a control, an aggregation-caused quenching (ACQ) molecule of BDP was also synthesized for BDP-NPs. To take insight into the effect of polymer on the aggregation state of hydrophobic dyes, the preparation of EDP and BDP NPs without block copolymer was also investigated. Apparently, the sizes of NPs display large distributions without hydrophilic block copolymer as the engineering template, suggesting that the block of polymers play key roles in tuning the aggregation state of encapsulated dyes in the FNP processes. Moreover, the peak shifts of dye with different microenvironment also confirmed the successful encapsulation of fluorescent dye in the NP cores. Finally, by externally applied forces in the FNP method, the engineered assembly of fluorescent AIE-active NPs possessing a narrow size distribution with desirable fluorescence properties was obtained. These features provide the possibility to rapidly construct controllable AIE-active fluorescent NPs as biomedical tracers.

KEYWORDS: fluorescent dyes, aggregation-induced emission, flash nanoprecipitation, nanoparticles

1. INTRODUCTION Fluorescent nanomaterials that could provide dynamic information of imaging biomolecules have become indispensable tools in biological research and clinical diagnosis.1,2 In particular, fluorecent organic molecules with excellent flexibility in synthesis and chemical modification have tremendously advantages in biomedical application.3-5 However, most organic fluorophores suffer from their inherent fluorescence quenching at high concentrations or during aggregation. Since the new concept of aggregation-induced emission (AIE) originally reported by Tang et al, the fascinating merits that AIE-active molecules exhibit highly bright fluorescence in aggregation make them ideally suitable for biosensing and imaging in vivo. 6-10 However, to facile control AIE-active organic molecules and form aggregated nanomaterials with desired sizes and photophysical properties remains largely unexplored. In order to apply the AIE-active molecules in bioimaging with high performance, control of the

aggregation degree as well as the fluorescence is necessary.10-12 Traditionally, the degree of aggregation can be controlled by modulating the solubility in a mixture of good and poor solvent. For example, adding a poor solvent, commonly water, into aggregation-induced emission (AIE) in their dissolved soltuion induces a decrease in solubility and hence the aggregation induced emission can be achieved.13-17 However, the formed nanoparticles usually show a large size with broad particle size distribution which limits the applications of these materials especially in biomedical areas. One solution is to encapsulate the AIE-active molecules inside biocompatible fluorescencet nanoparticles (NPs) using amphiphilic block copolymers. However, the traditional method to prepare such NPs relies on slow self-assembly of amphiphilic copolymers which is a spontaneous, naturally occurring process. The process is dictated by thermodynamics, meaning that the process often takes long time to form stable NPs.18-19 In addition, the size

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of NPs is difficult to control, and generaly there is a wide size distribution of NPs. Recently, a new simple and generic method described as flash nanoprecipitation (FNP) was developed to produce NPs with a desired particle sizes.18-31 FNP is a relative rapid process to prepare NPs within only 1 second using a Multi-Inlet Vortex Mixer (MIVM) system (Scheme 1). In FNP, the organic solute and amphiphilic block copolymer are simultaneously dissolved in a water miscible organic solvent, and then the organic solvent mixes with anti-solvent (water) in the mixing chamber dramaticly, forming NPs with organic solute being encapsulated inside. FNP is directed or engineered assembly driven by externally applied forces. In the case of FNP, the rapid change in the solvent polarity controls the kinetics of particle assembly.18 The size and its distribution of NPs are easy to be well tuned by the alteration of solvent ratio and stream velocity while maintaining the good stability of NPs. Notably, this process is conveniently scaled-up.32 The multiple advantages enable the method to be ideal for facile control AIE-active organic molecules with high performacance photophysical properties. Scheme 1. Schematic Diagram of Multi-Inlet Vortex Mixer (MIVM) system and the Flash Nanoprecipitation (FNP)

In this work, we presented an AIE molecule of EDP to prepare NPs by FNP method using PEG-b-PCL as a biocompatible amphiphilic block copolymer. Meanwhile, as a control, an ACQ molecule of BDP with very similar structure to the AIE molecule was also encapsulated at the same conditions in order to understand the aggregation process by the FNP method. The NP formation processes were optimized by changing the solvent ratio and velocities. The size and fluorescence properties of the NPs were systematically investigated. Such work is anticipated to provide a simple, rapid, and robust approach for fluorescence modulation of AIE molecules and enable them to be practically used as biomarkers with ﬂexibility for imaging. 2. EXPERIMENTAL SECTION

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Materials. Poly(ethyleneglycol)-blockPoly(ε-caprolactone) (2k-b-2k, PEG-b-PCL) was purchased from Shanghai Yarebio Co. Tetrahydrofuran(THF) were in chemically pure grade and purchased from Shanghai Tianlian Fine Chemical Co., Ltd. Pure water was obtained by a Milli-Q water purification system and was used in all experiments. Other reagent and solvents were used as received without any further treatment. The EDP and BDP were synthesized according to our previous paper.

THF/DMSO for 24 h using a Spectra/Por®6 MWCO 10kDa membrane and stored at room temperature. 3

RESULTS AND DISCUSSION

Molecular Design. As well-known red-emitting chromophores, the conventional dicyanomethylene-4H-pyran (DCM) derivatives35 with typical donor-π-acceptor (D-π-A) type structures suffer from severe ACQ effect in most cases. Recently, we firstly reported the impressive quinoline-malononitrile (QM) as AIE-active red-emitter building block, by merely replacing the oxygen atom in 4-dicyano-methylenechromene moiety with N-ethyl group.33,34 That motivates us to further explore novel red-emitting AIE chromophores for bioimaging. Specifically, employed an impressive QM as AIE-active core, an AIE-active molecule of EDP was easily synthesized with the reference compound BDP (Scheme 2). As shown in Figure 1a and Figure S1 in the supporting information, with the volume fraction of water (fw) in solvent of water/THF up to 70%, EDP exhibited an obviously 60-fold fluorescence enhancement (I/I0) at 555 nm compared with that in pure THF. In contrast, DCM-based BDP dispalyed a totally opposite phenomenon with ACQ effect in Figure 1b and Figure S2. Although the fluorescence intensity of BDP at 570 nm increased initially from pure THF solution to 30 % water fraction, there was continuously to sharply decrease to zero. In fact, its maximum emission peak exhibited a red-shifted by 103 nm from 570 to 673 nm. It could be ascribed to the synergy effect of the polarity effect and the forming extended π–conjugated planar aggregates34 (Figure S3). In a word, the fluorescence quantum yield of BDP has a sharply decrease with respect to the isolated single molecule state in THF solution (Figure 1b).

33,34

Characterizations. Dynamic light scattering (DLS) was carried out at 25 oC with a NICOMP 380 ZLS instrument with durations of 180 s. The angle of measurement is fixed at 90°. For data processing, the number particle size distribution (PSD) was chosen for plotting in order to avoid a dominant scattering effect of large aggregates. Average and standard deviations were obtained from three duplicates. UV-vis absorption spectra of samples were recorded on a UV-2550 UV-vis spectrophotometer. Fluorescence spectra were recorded on a Varian Cary Eclipse fluorescence spectrophotometer excited at 445 nm wavelength for AIE-NPs and 465nm wavelength for ACQ-NPs respectively. Nanoparticle morphology was observed on a JEOL JEM-1400 TEM instrument with at an acceleration voltage of 100 kV, and a JEOL JEM-2100 TEM instrument with at an acceleration voltage of 200 kV. One drop of the nanoparticle solution was deposited on carbon-coated copper grid. The droplet was allowed to dry under ambient conditions. The 1H and 13C NMR spectra in CDCl3 were recorded on a Bruker AM 400 MHz instrument (relative to TMS). High resolution mass spectra (HRMS) measurements were performed using a Waters LCT Premier XE spectrometer. Preparation of NPs. The AIE-NPs were prepared by FNP through a MIVM system. EDP (1 mg) was added into a solution of PEG-b-PCL (2000-b-2000 g/mol) (50 mg) in THF/DMSO (49 mL / 1 mL). The organic solution was fed (stream 1), along with water (streams 2 - 4), into a MIVM (Scheme 1) system using two digitally controlled syringe pumps (Harvard Apparatus, PHD 2000) to yield NPs. NP suspensions were dialyzed against Milli-Q water (3 L Milli-Q water per 20 mL NP suspension) to remove remaining THF/DMSO for 24 h using a Spectra/Por®6 MWCO 10kDa membrane and stored at room temperature.

Scheme 2. Molecular Structure of EDP and BDP

The ACQ-NPs were prepared similar to AIE-NPs. BDP (1 mg) was added into a solution of PEG-b-PCL (2000-b-2000 g/mol) (50 mg) in THF/DMSO (49 mL / 1 mL). The organic solution was fed (stream 1), along with water (streams 2-4), into a MIVM (Scheme 1) using two digitally controlled syringe pumps (Harvard Apparatus, PHD 2000) to yield NPs. NP suspensions were dialyzed against Milli-Q water (3 L Milli-Q water per 20 mL NP suspension) to remove remaining



60 a AIE

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Figure 2. EDP (a) and BDP (c) incorporated NPs at concentrations of 9.2 μM by FNP; (b) free EDP (b) and BDP (d) solution at concentrations of 9.2 μM.

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Size Control and AIE Properties. The NP sizes by the FNP method can be tuned by altering four velocities of streams. Specifically, the water fraction in mixed solvents was firstly optimized. The four stream velocities and the ratios of H2O : THF conversion were also shown in Table 1. The velocity of stream 1 (organic solution containing PEG-b-PCL block copolymer and EDP or BDP) and stream 2 (water) were fixed at 12 mL/min. The velocity of stream 3 (water) and stream 4 (water) were varied from 12 to 84 mL/min. Not only the velocity was changed, but also the ratio of H2O : THF in the mixed solvent was increased from 3:1 to 15:1. As shown in Figure 3a and 3c, with the increasing fraction of water in the mixed water/organic solvent, the sizes of both EDP and BDP NPs were continuously grown, where the sizes of NPs ranged from initial 20 to 60 nm.

100

fw (vol%) Figure 1. Emission intensity change I/I0 of EDP (a) and BDP (b) in a water/THF mixture with different water fractions (fw); I0 and I are the emission intensities (a) at 555 nm (excitation at 445 nm) and (b) at 570 nm (excitation at 455 nm) in THF (fw = 0) and a THF/water mixture with a specific fw, respectively. Inset: fluorescent photos in THF/water mixtures with various fw values and in the solid state.

Preparation of NPs by FNP. FNP method was used to prepare AIE and ACQ incorporated NPs, as shown in Figure 2. EDP, the AIE-active molecule, is a hydrophobic molecule with poor solubility in water, indicated by a little turbid solution (Figure 2b) with a concentration of 9.2 μM in a mixture solvent of H2O/THF with DMSO as a cosolvent (9: 0.98: 0.02, v: v: v). In contrast, the EDP solution, encapsulated inside the PEG-b-PCL NPs by FNP method, is crystal clear without precipitates observed (pure water solution, after dialysis). The results suggested that water soluble NPs were successfully formed with hydrophoibc moieties inside the core. The similar results were also obtained for ACQ-NPs in Figure 2b and 2d. In addition, the solubility difference also clearly demonstrated by size and size distribution in Table S1

As expected, we observed distinctly fluorescence changes for the preparation of AIE-active dye incorporated EDP-NPs and ACQ dye incorporated BDP-NPs (Figure 3b and 3d). For the preparation of BDP-NPs, the fluorescent intensity at 570 nm decreased concomitantly with the increasing ratio of H2O : THF from 3:1 to 15:1 in Figure 3d. That can be attributed to the accumulation of BDP in the NPs hydrophobic cores to generate the quenching effect. However, for EDP-NPs, there was a dramatically fluorescence increase at 544 nm when the ratio of H2O : THF in the mixed solvent was increased from 3 : 1 to 9 : 1 (Figure 3b). It can be attributed that, by the FNP method, more EDP molecules were encapsulated into the hydrophobic moiety of the polymer then aggregated to result in an AIE-active fluorescence


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enhancement. While the ratios of H2O : THF in the mixed solvent were over 9 : 1, there was a slight fluorescence decrease of EDP NPs. It may be interpreted as the formation of amorphous agglomerates, which has specific stacking structure and prevents the π–conjugation length. 33,34

Figure 4, with the stream velocity ratio changes from 1 to 3, (such as the stream velocity ratio of 2 represents that stream 1: stream 2: stream 3: stream 4 = 24: 24: 96: 96 mL/min), the size of NPs was remained ca. 50 nm, and there was a 2-fold fluorescence enhancement at 570 nm. Clearly, with the increasement of stream velocity, there were a significantly fluorescence enchantment of EDP-NPs while its sizes remained 50-60 nm (Figure 5). This continuous fluorescence enhancement can be ascribed that EDP molecules have higher kinetic energy at higher velocity to collide each other and then aggregate more efficiently during the FNP process.

60

a

Fluorescent Intensity (a.u.)

Average Diameter (nm)

The effect of velocity with the same ratio of H2O : THF on the EDP-NPs size and fluorescent property was also investigated in Figure 4. Herein, the ratio of H2O : THF in the mixed solvents (9 : 1) and the fixed four stream velocity (stream 1: stream 2: stream 3: stream 4 = 12: 12: 48: 48 mL/min, EDP concentration at 9.2 μM) was chosen as a control because the highest fluorescent intensity of NPs was observed. As shown in

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Figure 3. Size diameter (a) and fluorescent intensity (b) at 570 nm (excitation at 445 nm) of EDP-NPs with the changes of H2O : THF ratio in mixed solvents; the size (c) and fluorescent intensity (d) at 544 nm (excitation at 462 nm) of BDP-NPs with the changes of H2O : THF ratio in mixed solvents. The data point indicated by the red arrow in (a) was re-measured after 24 hours and the size is ~ 39 nm with a PDI of 0.426.

Table.1 Four Stream Velocity and The Ratio of H2O : THF in Mixed Solventsa Water : THF ratio 3:1

Stream 1 THF

Stream 2 Water

12

Stream 3 Water

Stream 4 Water

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The unit of stream velocity is mL/min Figure 5．．HRTEM (a) and TEM (b) images of EDP-NPs by the FNP method. The NP sizes are around 50nm.

a

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Particle Preparation Eﬀects. In FNP, the hydrophobic block of the amphiphilic block copolymers and the hydrophobic organic dyes are encapsulated in the core of the nanoparticles.36 (Scheme 1) In the preparation process of NPs by FNP, hydrophilic block of polymer forms a corona, then sterically stabilizing particles so that the aggregation of encapsulated dye could be tuned by preventing further aggregation. In our case, to better understand of the aggregation state of hydrophobic dye incorporated NPs, the control samples without block copolymer by FNP were also investigated at the same experimental conditions. As shown in Figure S4, the fluorescent spectra of free EDP nanoparticles with PEG-b-PCL copolymer by FNP become more broadly with subtle shifts in contrast to EDP-NPs in the absence of polymers. Meanwhile, as shown in Figure S5 and Table S2, the particles of BDP without copolymers coating displayed red shift in emission spectra while there was a blue shift as the systems in the presence of polymer. Emission intensity of BDP-NPs at 570 nm was lower than free BDP solution after FNP process indicating that limited BDP molecules aggregate in the core of NPs. It was suggested that the dye BDP become more insoluble during the FNP process as the water content was increased. Correspondingly, the fluorescence quenching effect of NPs become more obvious as the H2O: THF ratio was continuously increased. In addition, the experiment of EDP-NPs was re-measured after 24 hours and its size still remained about 39 nm with a PDI of 0.426.

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b 1000 800 600 400 200 0

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Stream Velocity Ratio Figure 4. Size diameter (a) and (b) fluorescent intensity at 570 nm (excitation at 445 nm) of EDP-NPs with the change of stream velocity ratio. The ratio of stream velocities (1, 1.5, 2.0, 2.5, 3.0) represent stream 1: stream 2: stream 3: stream 4 as 12: 12: 48: 48; 18: 18: 72: 72; 24: 24: 96: 96; 30: 30: 120: 120; 36: 36: 144: 144 mL/min, respectively.

Notably, the sizes could be well controlled with the block copolymer by FNP. As shown in Table 2, for the preparation of AIE EDP-NPs with the block copolymer, the NPs range in size from 30 to 55 nm as the ratio of the mixed solvent changes. However, the sizes of NPs for bare EBP-NPs display an extraordinary large size range from 50-350 nm without the help of polymer coating. Similar results also were found for the preparation of ACQ BDP-NPs in Table S3. All these data confirmed that the hydrophilic block of polymers played key roles in tuning the aggregation of encapsulated dyes37 in the FNP processes. Hence, in the


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1

H NMR, 13C NMR and HRMS spectra of EDP and BDP, absorption and emission spectra of EDP and BDP in mixed THF-H2O solution. Fluorescence changes of DT with various parameters in the formation NPs by FNP. This material is available free of charge via the Internet at http://pubs.acs.org/

generation of fluorescent NPs, especially for AIE-active molecules with desired sizes and photophysical properties, the FNP method certainly have unique advantages as a rapid, simply and scalable method. Table 2. Size distribution of EDP-NPs in the absence and presence of copolymer by FNP Only EDP a EDP-NPs a without Water : THF &PEG-b-PCL polymer 3:1 27 ± 3 155 ± 36 5:1

a

32 ± 1

AUTHOR INFORMATION Corresponding Author Fax: (+86) 21–6425–2758; E-mail: [email protected]; [email protected]; [email protected]

68 ± 4

7:1

45 ± 5

232 ± 50

9:1

49 ± 3

328 ± 46

11 : 1

46 ± 2

54 ± 2

13 : 1

52 ± 4

248 ± 94

15 : 1

55 ± 5

116 ± 18

ACKNOWLEDGMENT This work was supported by National 973 Program (2013CB733700), and Distinguished Young Scholars (21325625), NSFC/China, Oriental Scholarship, Shanghai Pujiang Program (13PJD010), Fok Ying Tong Education Foundation (142014), the Open Project of State Key Laboratory of Chemical Engineering (SKL-ChE-14C01), Fundamental Research Funds for the Central Universities (222201313010, 222201314029).

The size unit is nm.

4. Conclusions Novel AIE-active NPs have been successfully constructed by flash nanoprecipitation using a multi-inlet vortex mixer. The effect of formulation parameters in FNP on stream velocity, dyes as well as loading and concentration were investigated. The optimum solvents for AIE-active NPs was a mixed solvent (H2O: THF = 9: 1, v/v) and the concentration of EDP was determined at 9.2 μM. By using the optimized conditions in FNP, the AIE-active EDP-NPs sizes can be well tuned from 20 to 60 nm with desirable strong fluorescence properties. In addition, electron microscopy studies the nanoparticle structure by FNP were also performed. With the increasing stream velocity, there was significantly fluorescence enchantment of EDP-NPs while its size remained unchanged. Meanwhile, as a control, the ACQ dye of BDP has been constructed as NPs to better certify the encapsulation of fluorescent substance in FNP. Moreover, the difference of fluorescent substance aggregation state between in NP cores and in solution was demonstrated. Definitely, preparation AIE-active NPs by FNP has unique advantages in narrower size distribution and tuning fluroescence properties. Particularly, the method is easy scalable and fast processing with a simple vortex equipment. We believed that encapsulating AIE-active fluorophores in NPs by FNP would be a novel and promising way to prepare bio-imaging nanomaterials.

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Facile Preparation of AIE-active Fluorescent Nanoparitcles through Flash Nanoprecipitation Mingwei Wang†, Nan Yang†, Zhiqian Guo*,†, Kaizhi Gu†, Andong Shao†, Weihong Zhu†, Yisheng Xu*,†, Jie Wang†, Robert K. Prud’homme‡, Xuhong Guo *,† †

State-Key Laboratory of Chemical Engineering, Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China

‡

Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, USA


Facile Preparation of AIE-Active Fluorescent Nanoparticles through

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