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Biological and Medical Applications of Materials and Interfaces

Brightness Enhancement of Near Infrared Semiconducting Polymer Dots for In Vivo Whole-Body Cell Tracking in Deep Organs Zhe Zhang, Ye Yuan, Zhihe Liu, Haobin Chen, Dandan Chen, Xiaofeng Fang, Jie Zheng, Weiping Qin, and Changfeng Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08735 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 26, 2018

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ACS Applied Materials & Interfaces

Brightness

Enhancement

Semiconducting

Polymer

of Dots

Near-Infrared for

in

Vivo

Whole-body Cell Tracking in Deep Organs Zhe Zhang, † Ye Yuan, † Zhihe Liu, † Haobin Chen, † Dandan Chen, ‡ Xiaofeng Fang, ‡ Jie Zheng, †

†

Weiping Qin, † Changfeng Wu *, ‡ State Key Laboratory of Integrated Optoelectronics, College of Electronic Science and

Engineering, Jilin University, Changchun, Jilin 130012, China ‡

Department of Biomedical Engineering, Southern University of Science and Technology,

Shenzhen, Guangdong 518055, China

ACS Paragon Plus Environment 1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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KEYWORDS: Near-infrared probe, semiconducting polymer, nanoparticle, cell tracking, fluorescence imaging

ABSTRACT: In vivo visualization of cell migration and engraftment in small animals provides crucial information in biomedical studies. Semiconducting polymer dots (Pdots) are emerging as superior probes for biological imaging. However, in vivo whole-body fluorescence imaging is largely constrained by the limited brightness of Pdots in near infrared (NIR) region. Here, we describe the brightness enhancement of NIR fluorescent Pdots for in vivo whole-body cell tracking in deep organs. We first synthesize semiconducting polymers with strong absorption in orange to far-red region. By molecular doping, the weak broad-band fluorescence of the Pdots was significantly narrowed and enhanced by one order of magnitude enhancement, yielding bright narrow-band NIR emission with a quantum yield of ~0.21. Under an excitation of far-red light (676 nm), trace amount of Pdots (~2 µg) in the stomach can be clearly detected in whole body fluorescence imaging of live mice. The Pdots coated with a cell penetrating peptide are able to brightly label cancer cells with minimal cytotoxicity. In vivo cell tracking in live mice indicated that the entrapment and migration of the tail-vein administered cells (~400,000) were clearly visualized in real time. These Pdots with deep-red excitation and bright NIR emission are promising for in vivo whole-body fluorescence imaging.


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1. Introduction Fluorescence imaging an important imaging modality in many areas of biomedicine.1 The ability to noninvasively visualize the biological processes in living animals can provide critical information for fundamental research and clinical applications.2 Non-hazardous optical radiation is used in fluorescence imaging as compared to the imaging methods such as X-ray computed tomography (CT) and positron emission computed tomography (PET). However, both the excitation and fluorescence are severely affected by the scattering and absorption of biological tissue.3 Therefore, the limited photon penetration depth has constrained the widespread applications of the fluorescence imaging, especially when the target is deep-seated in the animal models. The performance of in vivo fluorescence imaging is largely dependent on the optical properties of fluorescent probes.4-6 Plenty of fluorescent probes have been exploited for biomedical imaging in last decades. Pu et al. recently discovered afterglow imaging based on semiconducting polymer dots, which can eliminate the tissue autofluorescence for in vivo imaging.7 Dai and coworkers demonstrated superior fluorescence probes in the second NIR window for brain tumor imaging.8-9 Wang and coworkers reported the synthesis of inorganic quantum dots and achieved the real-time tracking of in vivo immigration dynamics and distribution of protein nanocages.10 As highly useful fluorophores by gene transfection, fluorescent proteins play important role in biological imaging.11 Verkhusha and his group demonstrated NIR fluorescent proteins for in vivo animal imaging.12 Despite these progress, bright NIR probes are still highly desirable for in vivo fluorescence imaging of deep-seated organs.13-15 Semiconducting polymer dots (Pdots) has shown exciting applications in biomedical imaging because of their high brightness and intriguing energy-transfer properties.16,17 Various



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Pdots have recently been explored for applications such as super-resolution imaging,18 biosensing,19,20 in vivo tumor targeting,21-22 photoacoustic imaging,23-25 and phototherapy.26 One prominent advantage of Pdots is the widely tunable emission colors by modifying the chemical structures of π-conjugated polymers. Besides, the large absorption coefficients make semiconducting polymer a unique nanoparticle matrix.27-29 After doping with other functional fluorophores, the Pdots can effectively transfer excitation energy to the dopant molecules, resulting in red-shifted emissions or singlet oxygen generation.30-32 For instance, Chiu and coworkers obtained a NIR emission and large stokes shift in Pdots by the energy transfer from PFBT matrix to NIR775.27 However, to the best of our knowledge, all the visible-emissive Pdots doped with NIR dye exhibited red-shifted emissions, but reduced quantum yields as compared to the undoped Pdots.27-28,

33

Meanwhile, the pure undoped Pdot species in NIR region are

intrinsically weakly fluorescent or non-fluorescent. The Pdots with long wavelength absorption and high NIR emission quantum yields are rarely reported despite the salient Pdot species in visible wavelength region. Here, we describe the brightness enhancement of NIR Pdots for in vivo whole-body cell tracking in deep organs. First, we synthesize semiconducting polymers with strong absorption in orange and deep-red region. Although the pure Pdots have low quantum yields, we show that molecular doping can cause one order of magnitude enhancement of the Pdot brightness, yielding NIR narrow-band emission with a quantum yield up to ~0.21. Under an excitation of deep-red light (676 nm), trace amount of Pdots in deep-seated organs can be clearly detected in whole body fluorescence imaging. In vivo cell tracking in live mice indicated that the entrapment and migration of the tail-vein administered cells (~400,000) were clearly visualized in real time. 2. Results and Discussion


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2.1. Design and Synthesis of Far-Red-Absorbing Semiconducting Polymers Several factors are essential to realize fluorescence imaging of deep organs, including long wavelength absorption, NIR emission, and high quantum yield. The long wavelength absorption

Scheme 1. a) Chemical structures and synthetic route of semiconducting polymer PHIDT-DBT, PHIDT-HBT, and PHIDT-DFDBT. b) Schematic illustration of NIR775-doped PHIDT-DFDBT Pdots as fluorescent probes for whole-body optical imaging.



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and emission could be obtained from low-band-gap semiconducting polymers.34 In this regard, the donor-acceptor (D-A) structures usually generate a relatively low HOMO-LUMO energy gap because of the molecular orbital interactions.9,

23, 35-39

We attempted to synthesize the low

bandgap polymers by selecting strong donor and acceptor monomers. An electron-rich donor (HIDT) was employed as electron-donating moieties. The benzothiazole was used as electron acceptor owing to its strong electron affinity. Another two benzothiazole derivatives were also used to synthesize the polymers and investigate their structure-dependent optical properties. Three polymers were synthesized via palladium-catalyzed Stille coupling and the resulting polymers were abbreviated as PHIDT-DBT, PHIDT-HBT, and PHIDT-DFDBT, respectively. The general synthetic routes and their chemical structures were shown in Scheme 1. The successful synthesis of conjugated polymers was verified by nuclear magnetic resonance (NMR) spectroscopy (Figure S4-6) and gel permeation chromatography (GPC). GPC characterizations revealed that PHIDT-DBT polymer had a number average Mn of 4 kDa with a polydispersity index (PDI) of 2.59, PHIDT-HBT polymer had a Mn of 8 kDa with PDI of 3.76, and PHIDT-DFDBT polymer had a Mn of 9 kDa with PDI of 2.35, respectively.

2.2. Preparation and Characterizations of the Dye-Doped Pdots The PHIDT-DBT, PHIDT-HBT, and PHIDT-DFDBT polymers were used to prepare Pdots by the reprecipitation method as described in previous report.16 Figure 1 shows the dynamic light scattering (DLS) data of the Pdots, which indicate the as-prepared Pdots are well dispersed in water with an average hydrodynamic diameter of ~12 nm. The DLS results were in good agreement with the transmission electron microscopy (TEM) image of the Pdots (Figure 1b),


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which show well-dispersed nanoparticles with spherical morphology. Optical spectroscopy was used to characterize the absorption and fluorescence properties of the Pdots. As indicated in Figure 1c, all the three types of Pdots showed broad-band absorption because of their alternating donor-acceptor backbones. Notably, the absorption bands of the Pdots span the visible wavelength range with strong absorption in the red region (>600 nm), which is quite valuable for achieving deep photon penetration of in tissues. The Pdots exhibited fluorescence spectra in far-red and near infrared region with emission peaks centered on ~730 nm. Although the absorption and emission wavelengths of these Pdots are suitable for NIR fluorescent imaging, the quantum yields are quite low (1~2%), thus limiting their practical applications.



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Figure 1. a) Typical hydrodynamic diameter of the NIR775 doped PHIDT-DFDBT Pdots determined by dynamic light scattering. b) A representative TEM image of the NIR775 doped PHIDT-DFDBT Pdots. The scale bar represents 100 nm. c) Absorption spectra of the three types of Pdots in water. d) Fluorescence spectra of the three types of Pdots in water.

We demonstrate that a molecular doping strategy significantly enhances the NIR fluorescence brightness of the Pdots. As shown in our previous reports,30 efficient energy transfer could occur from semiconducting polymers to small dye molecules inside Pdots. Hydrophobic molecules such as NIR fluorophore,28 photosensitizer,40 or oxygen-sensitive dye,41


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can be doped into the Pdots, yielding red-shifted fluorescence, singlet oxygen generation, or oxygen sensing properties, respectively. In the prior studies, the semiconducting polymers used for dye doping were highly fluorescent and the doped Pdots typically had reduced quantum yields as compared to the undoped ones. Here we examine the optical properties of the newly-synthesized Pdots by molecular doping despite the low fluorescence quantum yields (1~2%) of the pristine Pdots. In fact, the doping approach takes advantages of the strong broad absorptions in the red wavelength region and generates red-shifted narrow fluorescence in NIR range, which are desirable for in vivo imaging. Table 1. Optical properties of PHIDT-Based Pdots in water and NIR775 dye in THF solvent

fluorescence probes

doping concentrations (w/w)

λmax abs (nm)a

λedge (nm)b

Egopt (eV) c

λmax em (nm)d

fwhm (nm)e

PHIDT-DBT

0

575

693

1.79

739

148

3×107

0.5 %

575

693

1.79

783

20

3×107

0

558

683

1.82

733

151

2×107

0.5 %

558

683

1.82

784

19

2×107

PHIDT-HBT

PHIDT-DFDBT

NIR775

ε(M-1cm-1, 560 nm)

7

0

558

681

1.82

720

137

2×10

0.5 %

558

681

1.82

779

18

2×107

772

780

Mn (kDa)f

Size (nm)g

Φ (%)h

15.4

17

1

17

10

18

5

19

14

16

2

18

21

15.7

18.9

7

42

a) Absorption maximum; b) λedge is absorption edge of the Pdots; the c) Eg = 1240/λedge; d) Emission maximum; e) Full width at half maximum; f) Number-average molecular weight; g) Hydrodynamic size of Pdots in water; h) Fluorescence quantum yield.

A fluorescent dye (NIR775) was chosen for preparing the dye-doped Pdots owing to the good spectral overlap between its absorption and the emission of the semiconducting polymers (Figure 2a). To optimize the doping concentration, we investigated the absorption and fluorescence properties of NIR775-doped Pdots at different doping concentration (Table S1). Figure 2b-2c show the absorption and fluorescence spectra of the doped Pdots as the doping



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concentration was increased from 0.2 wt% to 5.0 wt%. As can be seen, the polymer fluorescence was efficiently quenched by NIR775 even at low concentrations, which indicated the efficient energy transfer from the polymer to the dye. In addition, the intensity of NIR775 emission varied under different doping concentration. The NIR775 fluorescence intensity was increased at low doping concentrations (0.2 wt%-0.5 wt %), but decreased at high concentrations (1.0 wt%-5.0 wt %). In addition, the excited state lifetime of the Pdots exhibited apparent decrease after dye doping (Figure 2d), further confirming the energy transfer from the polymer to the dye. Based on the spectroscopic results, the optimal doping concentration was determined to be 0.5wt%, yielding the brightest fluorescence for the doped Pdots. Surprisingly, the NIR fluorescence of the NIR775-doped Pdots was significantly enhanced as compared to the pristine Pdots. All the three types of Pdots show significantly enhanced fluorescence narrowed emission bandwidth after dye-doping (Figure S9). The quantum yield of PHIDT-DFDBT-NIR775 Pdots was determined to be 21% in aqueous solution, which was 10 times than that of the PHIDT-DFDBT Pdots (Table 1). Such a great improvement was likely attributable to the hydrophobic environment and the steric hindrance of hexyl groups provided by PHIDT-DFDBT,43 which yielded strong fluorescence from the NIR dye. It is worth noting that the quantum yield of the dye in the Pdots was even higher than the free dye in an organic solvent. Combined with the large absorption cross section of the polymer and the high quantum yield of the NIR dye, the doped Pdots provided an unparalleled NIR fluorescence brightness under far-red light excitation.


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Figure 2. a) Spectral overlap between the normalized fluorescence spectrum of PHIDT-DFDBT and absorption spectrum of NIR 775. b) Absorption spectra of NIR775-doped PHIDT-DFDBTPdots by varying dye concentrations. c) Fluorescence spectra of NIR775-doped PHIDT-DFDBT Pdots by varying dye concentrations. d) Fluorescence decay curves of NIR775-doped PHIDT-DFDBT- Pdots by varying dye concentrations.

2.3. In Vitro Cellular Studies



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Cellular labeling studies were performed with NIR775-doped PHIDT-DFDBT Pdots because of its highest fluorescence quantum yield. The Pdots can be easily internalized by endocytosis, which provides an effective approach for using the Pdots in cellular tracking.44 In addition, Pdots shows a good stability under physiological environments.28 We choose human breast cancer (MCF-7) cells for the cell labeling. A cell penetrating peptide octa-arginine (R8) was employed to modify the carboxyl Pdots to enhance labeling brightness as described previously.32 The cell viability of MCF-7 cells was measured with a colorimetric MTT assay. As seen in Figure 3a, cell cytotoxicity was not observed even at high concentrations of Pdots (100 µg/mL), indicating the biocompatible feature of the doped Pdots. We use flow cytometry to characterize the cell labeling brightness. With increasing the incubation time at the same Pdots concentration (40 µg/mL), the cell labeling brightness was increased in a time-dependent manner (Figure S10). Apparently, cell penetrating peptide R8 greatly enhanced the endocytosis of the Pdots, leading to an enhancement of labeling brightness by about 100 times as compared to the Pdots without R8 peptide (Figure 3b). Flow cytometry was also used to examine the in vitro cell tracking capability of the NIR775-doped Pdots. After 4-hour incubation with R8-Pdots (40 µg/mL), MCF-7 cells brightness was monitored at the various time point (Figure 3c). The freshly labeled cells yielded the brightest fluorescence signal. Although the fluorescence intensity was much weaker after 13 days of incubation, it can still be clearly identified by flow cytometry. Fluorescence imaging of the cells indicates that the Pdots was mainly distributed in the cytoplasm (Figure 3d). The bright fluorescence and the long-term cell tracking capability make the Pdots a valuable fluorescent probe for in vivo cell tracking studies.


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Figure 3. a) Cellular toxicity study of MCF-7 cells incubated with NIR775-doped PHIDT-DFDBT Pdots at different concentrations. b) Brightness comparison of MCF-7 cells labeled with R8-Pdots and carboxyl Pdots by flow cytometry. c) Long-term cell tracking of MCF-7 cells labeled with R8-Pdots. d) Fluorescence imaging of MCF-7 cells labeled with R8-Pdots (red). The cells were also stained by Hoechst 33258 nuclear dye (blue). The scale bar represents 20 µm.

2.4. In Vivo Animal Studies of the NIR775-doped Pdots



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The combination of red excitation and NIR fluorescence of the doped Pdots results in a large penetration depth for in vivo studies. We designed an experiment to examine the penetration depth by using chicken breast to mimic the biological tissue. Two glass capillaries were filled with 20 µg/mL NIR775-doped Pdots and pristine Pdots, respectively. The fresh chicken breast was placed over the capillaries layer by layer to examine the light penetration. As shown in Figure 4a, the two capillaries were clearly observed when the thickness of the chicken breast was ~2.1 mm. However, the NIR775-doped Pdots showed a signal to noise ratio that is apparently higher than the pristine Pdots. The fluorescence of NIR775-doped Pdots was still clearly identified through the chicken breast of 5.0 mm thickness, while the pristine Pdots were hardly detectable, confirming the large penetration depth by using the doped Pdots. As further shown in Figure 4b, the signal to noise ratios of dye-doped Pdots was significantly improved because of the enhanced fluorescence quantum yields by the dye doping.


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Figure 4. a) Penetration depth studies of NIR775-doped Pdots (top capillary) and pristine PHIDT-DFDBT Pdots (bottom capillary). b) Signal to noise ratio of the Pdots relative to the tissue autofluorescence at different tissue depths. c) In vivo fluorescence images of the mice that were orally administered with different amounts of NIR775-doped Pdots. The images were collected 5 minutes after the oral administration. d) SNRs of the Pdots in the stomach of the mice with different amounts of Pdots orally administered.

A small amount of NIR775-doped Pdots was orally administered to ICR mice to examine the penetration depth of the Pdots by fluorescence imaging. As shown in Figure 4c, the fluorescence of the Pdots was clearly visible in the whole-body imaging, identifying the location of the stomach of the live mice. The Pdots would enter the intestine and be discharged in about



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48 hours post oral administration.45 We reduced the amount of Pdots orally administered and a quantity of 2 µg Pdots in the stomach was detected by the fluorescence imaging. The SNRs of the imaging results were presented in Figure 4d, indicating SNR of ~1.5 for the 2 µg Pdots in the stomach in the whole-body small animal imaging. Finally, we investigated in vivo cell tracking capability of the NIR775-doped Pdots in live mice. First, MCF-7 cells were labeled by incubation with 40 µg/mL R8-coated Pdots for 4 hours, the cells were then administered to the ICR mice via the tail vein. Three groups of ICR mice were administered with 0.4, 0.7 and 1.0 million MCF-7 cells, respectively. The fluorescence images were collected every another day. As shown in Figure 5, an intense fluorescence signal was observed in the lung of the mice 10 min post injection of the label cells. From day 1 to day 7, the fluorescence signal from the lung was gradually decreased, while the fluorescence from the liver and intestine was increased. These imaging results were consistent with the in vivo cell tracking results of other cells.32 When the Pdot-labeled cells (0.7 and 0.4 million, respectively) were tail-vein administered, fluorescence signal was clearly observed from the lung and liver (Figure S11-S12). In addition, we examined the fluorescence imaging of the tissue sections (Figure S13). It appears that the fluorescence pattern in the lung is different from that in the liver. There is a possibility that a small fraction of the Pdots is excreted from the cancer cells. The Pdots eventually accumulate in the liver. The major route of excretion from the body is through the liver, into bile and into feces (Figure S14). Figure 5b summarized the fluorescence signal of lung relative to body background in the three groups of mice, which indicated similar time-dependent fluorescence intensities from the lungs. Organs were dissected from the mice on the seventh day, and their ex vivo imaging (Figure 5c) confirmed that the fluorescence was dominantly detected from the lungs and livers of the mice. The bright cell labeling and long-term


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tracking of cells in live mice by whole-body imaging revealed the promising applications of the NIR-doped Pdots in biomedical studies.

Figure 5.

In vivo whole-body cell tracking in deep organs. a) In vivo fluorescence imaging

of the mouse intravenously injected with 1.0 million Pdot-labeled MCF-7 cells at designated time points. b) Fluorescence signal to noise ratio of the lung after the injection of Pdot-labeled cells (1.0 million, 0.7 million, and 0.4 million, respectively). c) Quantitative fluorescence analysis of the Pdot-labeled cells in the heart (H), kidney (Ki), spleen (Sp), liver (Li), and lung (Lu) 7 days post the injection. Inset shows the fluorescence image of the heart, kidney, spleen, liver, and lung from injected mice.



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3. Conclusion We synthesized a series of semiconducting polymers that exhibited long wavelength absorption and NIR emission. The dye doping strategy maintained the high light-harvesting capability of the polymer and more importantly improved emission quantum yields (779 nm). In one case, the quantum yield was increased to 21% after the Pdots were doped with 0.5 wt% fluorescent dye NIR775. The dye-doped Pdots exhibited superior properties for both in vitro and in vivo cell imaging and tracking as compared to the pristine Pdots. Trace amount of Pdots (2 µg) in the stomach of live mice resulted in a clear and strong fluorescence signal in whole body fluorescence imaging. In vivo tracking studies of MCF-7 cells labeled with the doped Pdots indicated that the entrapment of the tail-vein administered cells in the lung and gradual migration to the liver of the mice, which were clearly visualized in real time. The effective red excitation and enhanced NIR fluorescence of the NIR775-doped Pdots are promising for in vivo whole-body fluorescence imaging.

ASSOCIATED CONTENT Supporting Information: 1H-NMR (CDCl3) spectra. Typical hydrodynamic diameter and TEM image of Pdots. Zeta potential distribution of Pdots. Absorption and fluorescence spectra of NIR775-doped Pdots at different doping concentrations (w/w), Flow cytometry of labeled MCF-7 cells, Representative in vivo fluorescence images. Fluorescence imaging of the frozen tissue sections. Fluorescence imaging of excretion. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author: E-mail address: [email protected] Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENT C. Wu acknowledges financial support from the National Natural Science Foundation of China (Grant No. 61335001; Grant No. 81771930), and Shenzhen Science and Technology Innovation Commission (Grant No. JCYJ20170307110157501).

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