In Vivo and In Situ Activated Aggregation-Induced ... - ACS Publications

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Biological and Medical Applications of Materials and Interfaces

In vivo and in situ activated aggregation-induced emission probes for sensitive tumor imaging using tetraphenylethenefunctionalized trimethincyanines-encapsulated liposomes Xianghan Zhang, Bo Wang, Yuqiong Xia, Sumei Zhao, Zuhong Tian, Pengbo Ning, and Zhongliang Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07727 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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ACS Applied Materials & Interfaces

In Vivo and In Situ Activated Aggregation-Induced Emission Probes for Sensitive Tumor Imaging Using TetraphenyletheneFunctionalized Trimethincyanines-Encapsulated Liposomes Xianghan Zhang†, Bo Wang†, Yuqiong Xia†, Sumei Zhao†, Zuhong Tian‡, Pengbo Ning† and Zhongliang Wang†* †

Engineering Research Center of Molecular-imaging and Neuro-imaging of ministry of

education, School of Life Science and Technology, Xidian University, Xi’an, Shaanxi 710026, China. ‡

Institute of Digestive Diseases, Xijing Hospital, Fourth Military Medical University, Xi’an,

Shaanxi 710032, China. Dr. Zhongliang Wang * Corresponding author

Tel.: +86-29-91891070 Fax: +86-29-91891070 E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT The design and exploration of fluorescent probes with high-sensitivity and low-background are essential for non-invasive optical molecular imaging. The in vivo and in situ activated AIE (aggregation-induced emission) probes were found to be ideal for achieving higher signal-tobackground ratios for tumor detections. We herein developed novel tetraphenyletheneencapsulated liposomes (TPE-LPs) constructed by loading TPE-trimethincyanine into liposomes for the first time, and the probes were applied to tumor bioimaging in vivo. TPE-functionalized trimethincyanines were synthesized with a new and efficient one-pot reaction. In TPE-LPs, TPE -functionalized bicarboxylic acids benzoindole trimethinecyanine (TPE-BICOOH) fluorophores were found to be well dispersed in lipid bilayers (with non-restricted rotation) during the blood circulation, and then aggregated (with restriction of intramolecular rotation) upon liposome rupture in the tumor tissue, achieving a low-background and high-target signal for tumor imaging. The in situ activated AIE probes not only had great accumulation at the tumor site after intravenous injection in 4T1 tumor-bearing mice but also demonstrated excellent signal-tobackground ratios, as well as low cytotoxicity and excellent biocompatibility. The proposed strategy is believed to be a simple and powerful tool for the sensitive detection of tumors.

KEYWORDS: cyanine; fluorescence imaging; aggregation-induced emission; liposome; tumor imaging

1. INTRODUCTION Non-invasive optical molecular imaging is one of the most promising methods for the early detection of tumors, especially when applied as a surgical navigation system during open


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surgical procedures, which is expected to improve cancer treatment outcome and further increase patient survival.1, 2 At present, such imaging for accurate detection of cancer is still limited by low detection sensitivities; thus the design and exploration of fluorescent probes with a hightarget and low-background signal are essential.3-7 However, traditional fluorescent probes, such as cyanine dyes tend to aggregate in aqueous solution with fluorescence emission aggregationcaused quenching (ACQ),8, 9 and the strong background signals will interfere with the imaging sensitivities. Recently, Tang and his coworkers developed aggregation-induced emission (AIE) fluorophores, which could overcome the shortcomings of traditional fluorescent probes, 10, 11 and showed good bioimaging efficiency of tumors.12-15 Among the various types of AIE materials, tetraphenylethene (TPE) is widely applied owing to its great fluorescence enhancement, restriction of the intramolecular rotation (RIR) process upon aggregation or in solid state, as well as its simple chemical modification.13, 16-18 Traditional TPE probes show signal enhancement only when the solvent is changed from an organic solvent (a good solvent such as chloroform, tetrahydrofuran or acetonitrile) to water (a poor solvent). Thus, to develop TPE bioimaging probes, TPE fluorophores were necessary to be assembled into self-aggregation nanoparticles16, 19-21 or polymer-encapsulated nanoparticles (e.g., BSA,22 DSPE-PEG23-25). TPE self-assembled nanoparticles show efficient cell imaging and excellent in vivo imaging by intratumoral injection. Targeted imaging of polymer-assembled nanoparticles 20-22 only depends on accumulation of the nanoparticles at the tumor sites through intravenous injection, which may result in a low signal-to-background ratio (SBR) and high blood background owing to their blood circulation in an always-ON state and the long clearance time.5, 26


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To overcome these disadvantages, some good water-soluble TPE fluorophores with lower background were developed by directly conjugating TPE fluorophores to hydrophilic groups (such as sulfonic acids27) or hydrophilic recognition ligands (such as FFGYSA,28 T7,29 R4,30 AP2H31). However, these relatively larger ligands and fluorophores may also have mutual interactions due to steric hindrance effects,32 resulting in weak signal AIE amplification. Further, TPE-type in situ activated AIE probes were developed using a solubility change strategy with enzyme-cleavable water-soluble groups such as phosphate33, β–galactopyranoside,34 glutamic acid,35 or hydrophilic peptides (GFLGD3-cRGD,36 or DVEDEE-Ac,37 DVED-cRGD38,

39

,

KFPE40, CPPs41). In the presence of specific enzymes, the corresponding hydrophilic groups are cleaved and TPE compounds with poor water solubility are generated, resulting in an activated AIE effect. The in situ activated AIE probes were found to be ideal for achieving a higher SBR, as well as for eliminating the false-positive signals in vivo. However, the research on in situ activated AIE probes has thus far been limited to the in vitro levels and few of these probes have realized in vivo applications. In addition, the modification of enzyme-cleavable groups not only increases the burden of organic synthesis and purification but also enhances the complexity toward clinical transformation.10, 13, 42 To achieve TPE-type in situ activated AIE and enhance the tumor signal, liposomes,43, 44 approved by the U.S. Food and Drug Administration (FDA), are considered to be excellent nanocarriers through loading hydrophilic probes in the interior and/or loading hydrophobic probes in the bilayer while maintaining hydrophobic monomers41. Inspired by their unique structure, we hypothesized that a low background of TPE fluorophores can be realized by simply dispersing hydrophobic TPE monomers into the bilayer of liposomes to subsequently achieve signal amplification by in situ activated AIE probes once the liposomes are ruptured in water


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(Figure 1). In this work, we first designed and synthesized TPE-functionalized cyanines, and then prepared TPE-liposomes (TPE-LPs) by loading TPE-BICOOH dyes into the hydrophobic lipid bilayers of the liposomes. TPE-LPs were expected to have the following characteristics: 1) maintain the TPE-BICOOH dyes in a non-restricted molecular rotation state, leading to weak fluorescence emission in aqueous media; 2) prevent TPE-BICOOH from aggregation during the blood circulation to reduce background interference; and 3) rupture the liposomal membranes when the TPE-LPs are delivered to the tumor cells,45 so that the TPE-BICOOH dyes are released and aggregate into AIE nanoparticles, achieving a high level of target signal amplification.

Figure 1. Illustration of the mechanism of TPE-LPs in imaging tumor in vivo.

2. EXPERIMENTAL SECTION 2.1 Materials and Reagents. 4-(1,2,2-triphenylvinyl)benzaldehyde (TPE-CHO) was synthesized according to the literature procedure[1]. Common reagents and solvents were purchased from Sigma-Aldrich, Tokyo Chemical Industry or J&K Chemical. Commercially reagents were used as received without extra purification. DPPC (1,2-dipalmitoyl-sn-glycero-3-


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phosphocholine),

cholesterol

and

DSPE-PEG2000

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(1,2-Distearoyl-sn-Glycero-3-

Phosphoethanolamine-N-[Methoxy (Polyethethylene glycol)-2000] (Ammonium salt)) were purchased from Avanti lipids. DAPI was purchased from Thermo Fisher Scientific. Cell Counting Kit-8 (CCK-8) assay was purchased from Dojindo Molecular Technologies, Inc. 2.2 Synthesis and Characterization of dyes and liposomes. The details of the synthesis and characterization of TPE-functionalized vinyl benzoindole dye (TPE-VS), TPE-functionalized vinyl monocarboxylic acid benzoindole dye (TPE-COOH), TPE-functionalized vinyl quinoline dye (TPE-VQ), TPE–functionalized bimethyl benzoindole trimethinecyanine (TPE-BIVS), TPE functionalized bicarboxylic acids benzoindole trimethinecyanine (TPE-BICOOH), liposomes (TPE-LPs) and giant unilamellar vesicles (TPE-BICOOH-GUVs) are provided in the Supporting Information. 2.3 In Vitro Fluorescence Imaging. 4T1 and SGC-7901 cells with 80% confluence were dissociated with 0.5% trypsin-EDTA and suspended in fresh medium, then they were plated on Bioclean coverslips in Millicell EZSLIDE well at 1 × 105 cells/well and incubated at 37°C in a 5% CO2 incubator overnight. On the next day, the cells were incubated with 5 µM TPE-VQ, TPE-BIVS, TPE-BICOOH or TPE-LPs at 37 °C for 3 h. The cells were washed three times with PBS (pH 7.4) to remove free compounds before imaging and fixed with 4% aqueous paraformaldehyde solution, and then counterstained for visualization of nuclei using 4’6Diamidino-2-phenylindole (DAPI, 10 µg/mL) for 5 min. After washing several times with PBS and drying at room temperature, the coverslips were mounted on slides using the anti-fading buffer, examined on confocal laser microscopy and presented at the same intensity scale for comparison. An Olympus FV 10i laser confocal fluorescent microscope was used for cell


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imaging experiments. A Leica DMi8 inverted fluorescent microscope was used for GUV imaging experiments. 2.4 Procedure for Cytotoxicity Study. For cell viability study, 4T1 cells were cultured in 96well plates at the density of 0.5~1 × 105cells per well and cultured at 37 °C for 24 h. Then 100 µL different probe concentrations of TPE-VQ, TPE-BIVS, TPE-BICOOH and TPE-LPs (0, 1.25, 2.5, 5, 10, 20 µM) were added to continue culturing for 24h under dark condition. Next, the old medium were removed and then washed thoroughly with PBS buffer. Then 10 µL Cell Counting Kit-8 (CCK-8) agent was added per well and cultured for another 3 hours followed by washing with PBS. Finally the absorbance in each well of the 96-well plates was measured at 450 nm with a multi-well plate reader (Promega glomax discover system). Cell viability was calculated using the following formula: Cell viability = (Meanabsorbance of test wells - Mean absorbance of medium control wells)/(Meanabsorbance of untreated wells - Mean absorbance of medium control well) × 100%. Each concentration was measure in triplicate and used in three independent experiments. 2.5 Flow Cytometry Analysis. Flow cytommetric analysis was carried out in BD Accuri C6 machine. 4T1 cells were plated on 12-well plates at the density of 1 × 106 cells per well for 24 h. The cell media were replaced by 100 µL different probe concentrations of TPE-VQ, TPE-BIVS, TPE-BICOOH and TPE-LPs (0, 1, 5, 10, 15, 20 µM). After culturing for 3 h and washing with PBS, the cells were digested with 0.5% trypsin-EDTA, harvested and quantitatively determined by flow cytometry. 2.6 Animals and Tumor Xenograft Model. Female athymicnude mice (6–8 weeks) weighted 20–25 g were supplied by the Animal Center of the Fourth Military Medical University (Xi’an, China). To develop the breast tumor model, 2 × 106 Murine breast cancer 4T1 cells were


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subcutaneously implanted into the right upper limb per mouse. Animals received care in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals. All probes were dissolved in saline for injection. 2.7 In vivo Fluorescence Imaging. For in vivo imaging, when the diameter of tumor developed to 4-6 mm after a half week inoculation, the mice were randomly divided into two groups (n = 3) and intravenously injected with TPE-BICOOH or TPE-LPs respectively. These two probes were dissolved in saline for injection. The equivalent probes dose was kept at 20nmol per individual. Mice were anesthetized with 5% isoflurane then transferred to the IVIS imaging system (Perkinelmer IVIS Lumina III) with a 580 nm excitation wavelength and 620 nm filter to obtain fluorescence imaging at different time. For tissue distribution study, the mice were immediately sacrificed by breaking the neck at the 18th h after injection and the organs including heart, lung, liver, spleen, stomach, kidney and tumors were collected for the ex vivo imaging. Quantification of tumor fluorescence images was analyzed using the IVIS software.

3. RESULTS AND DISCUSSION The general synthesis methods for functional trimethincyanines mainly need to construct of cyanine chromophores firstly, and then introduce functional groups on the methine chain46, which suffer from long span of reaction time and tedious purification steps of intermediates. To construct AIE-founctionalized trimethincyanines, TPE was substituted to the conjugated chain for benzothiazole trimethincyanines with a new and robust one-pot approach (Table 1). TPEtrimethincyanines (II) can be easily synthesized from TPE-CHO and benzothiazole quaternary salts (A or B) without purifying the intermediate TPE-vinyl dyes (I).


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Table 1. Optimization of TPE-vinyl dyes (I) and TPE-trimethincyanines (II)

Entry

S.M.

Cat.

Solvent

Temp.

Yield (I)

Yield (II)

1

A

Pyridine

DMF

25°C

82%

−

2 3

A A

Et3N Piperidine

MeOH MeOH

25°C 25°C

57% 20%

11% 57%

4 5

A A

Et3N Piperidine

DMF DMF

25°C 25°C

38% 13%

52% 70%

6

A

Et3N

MeOH

60°C

16%

60%

7 8

B B

Pyridine Et3N

DMF MeOH

25°C 25°C

77% 68%

− −

9 10

B B

Piperidine Et3N

MeOH DMF

25°C 25°C

22% 43%

56% 46%

11 12

B B

Piperidine Et3N

DMF MeOH

25°C 60°C

15% 25%

67% 58%

Further, we investigated the effect of different catalysts, temperatures and solvents on the formation of TPE-vinyl dyes (I) and TPE-trimethincyanines (II). We found that improving the alkali strength and reaction temperature increased the overall possibility of the synthesis process for TPE-trimethincyanines, and replacing the solvent methanol with dimethylformamide (DMF) increased the yields of the product (II) under the same temperature and catalysts. The proposed one-pot reaction pathway for the TPE- trimethincyanines is shown in Scheme 1. The experimental phenomenon revealed that a two-step reaction pathway should be considered for the formation of TPE-trimethincyanines (II). First, benzothiazole quaternary salts (A or B) and piperidine produced the intermediate carbanion Da, and a subsequent nucleophilic addition reaction with TPE-CHO was conducted under an alkali environment to produce the TPE-vinyl


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dyes (I). Second, another benzothiazole intermediate carbanion Da could produce the conjugate intermediate Db, followed by a conjugate addition reaction on the methine chain of TPE-vinyl dyes (I) to give intermediate (II)a. Finally, subsequent proton transfer from (II)b to the highly alkaline piperidine gave TPE-trimethincyanines (II).

Scheme 1. Proposed reaction pathway for TPE-benzothiazole trimethincyanines.

To confirm the mechanism for the formation of TPE-trimethincyanines, we further explored the reaction of TPE-CHO with 4-methyl quinoline heterocycle quaternary salts (C) (see Table 1). Only the vinyl dyes TPE-VQ was obtained regardless of whether the reaction was catalyzed by Et3N or piperidine. This is because compound C cannot transform a stable conjugate intermediate compared with 2-methyl benzothiazole quaternary salts (Scheme 1, Db),47 which is consistent with the experimental result. In short, reasonable control of the reaction conditions for the reaction between TPE-CHO and heterocycle quaternary salts can selectively form the TPEvinyl dyes or TPE-trimethincyanines through simply controlling nucleophilic addition or the conjugate addition reaction. All of the above compounds were synthesized in the one-pot reaction with high yields, resulting in less by-products with a simple purification procedure. The structures were confirmed by 1H NMR, HRMS and HPLC (Figure S1, S2 and S3).


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Figure 2. The characterization of TPE dyes and TPE-LPs. (A) Fluorescence emission spectra and plot of corresponding fluorescence intensity at 584 nm of TPE-BICOOH in methanol/H2O mixtures with different volume fractions of water (fw). fw = 0: ΦF = 4.7%; fw = 80%: ΦF = 54.8%. (B) Fluorescence images of GUV containing TPE-BICOOH; scale bar: 25 µm; Leica DMi8 with maxima exposure time and gain; Colorless small particles are sucrose and salt crystals. (C) DLS analysis of TPE-LPs in PBS. (D) Fluorescence emission spectra of TPE-BICOOH in methanol, TPE-LPs in PBS and TPE-LPs-R in PBS containing 0.05% Triton X100 to rupture membranes (ΦF of TPE-LPs and TPE-LPs-R in PBS are 4.4% and 50.5%, respectively). Dye concentration: 10 µM. λex: 530 nm. Photos were taken under 365 nm UV light.

Next, the fluorescent emission of TPE-cyanines in the monomer and aggregate state were investigated. As shown in Figure 2A and Figure S4, TPE-BIVS and TPE-BICOOH showed very weak fluorescence in a good solvent of methanol, and exhibited significant enhancement in the emission intensity upon increasing the water fraction (fw) due to the AIE effect in poorer solvents, demonstrating an 18-fold enhancement at an fw of 80%. On the other hand, the fluorescent intensities and AIE enhancements of TPE-vinyl dyes (I) were weaker than TPEtrimethincyanines (II) (Figure S5). This indicated that introducing TPE groups to the conjugate chain on trimethincyanines provided an excellent AIE effect in aqueous solution.


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It was well-known that liposomes can load hydrophilic molecules in the hydrophilic interior and can load hydrophobic molecules in the bilayer.48 To demonstrate that TPE-BICOOH dyes localized in the hydrophobic bilayer of liposomes instead of the hydrophilic interior, giant unilamellar vesicles (GUVs, cell-sized liposomes) containing TPE-BICOOH were prepared using a natural swelling method. As shown in Figure 2B, the ring structure of the fluorescent image proved that the hydrophobic TPE-BICOOH dyes are indeed loaded into the bilayer and not the interior. Indeed, TPE-LPs greatly improved the solubility of TPE-BICOOH dyes in PBS, and had a well-defined spherical shape with a size of 118±4 nm and a relatively narrow distribution (polydisperisty index (PDI): 0.16±0.02, Figures 2C and Figure S6), but induced almost no change in the fluorescence intensity as TPE-BICOOH dyes in methanol solvent (Figure 2D). It is because that TPE-BICOOH dyes were evenly distributed within hydrophobic lipid bilayer of the liposomes as monomers, where the dyes remained rotational freedom of their phenyl rotors. The zeta potential was −1.42 ± 0.53 mV, showing that the TPE-LPs had an almost neutral charge, and will therefore have longer blood circulation retention. Otherwise, the Zeta potential of TPE-BICOOH aggregates in water was -34.23±1.63 mV, and the size distribution was non-uniformed with a PDI of 0.5±0.05 (Figure S7). Further, to investigate the stability of TPE-LPs in vivo, the diameter of TPE-LPs was monitored in the medium with 10% FBS. As shown in Figure S8, there wasn’t significant change of size over a week, which indicated TPELPs were stable in the medium with 10% FBS. To investigate the activated AIE effect of TPE-LPs, we compared the fluorescence and morphology of TPE-LPs and ruptured TPE-LPs (TPE-LPs-R, Figure S9). Encouragingly, the fluorescence was up to 20-fold higher through rupturing the membrane of TPE-LPs in PBS by the addition of Triton X-100 (Figure 2D and Figure S10). Correspondingly, the fluorescence


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quantum yields of TPE-BICOOH in methanol, TPE-LPs and TPE-LPs-R in PBS were determined to be 4.7%, 4.4% and 50.5%, respectively. This indicated the superior photo-physical properties of TPE-LPs, demonstrating their potential as in situ activated AIE probes for in vivo bioimaging application.

Figure 3. Confocal images of 4T1 and 7901 cells stained with TPE-VQ, TPE-BIVS, TPE-BICOOH and TPELPs (5 µM) for 3 hours. The masked blue color represents fluorescence from the nuclei of cells stained by DAPI (λex = 359 nm, λem = 461 nm) and the red color represents fluorescence by dyes (λex = 559 nm, λem = 570620 nm); Scale bar: 30 µm.

To evaluate the in vitro cellular imaging of the probes, confocal laser-scanning microscopy (Figure 3) and flow cytometry (Figures 4A and 4B) were performed. Different intensities of red fluorescence originated from the AIE nanoparticles in the cytoplasm were observed for the 4T1 and 7901 cell lines. The red fluorescence signals TPE dyes upon incubation with cells decreased in the following order: TPE-LPs > TPE-BICOOH > TPE-BIVS >> TPE-VQ, and the order is consistent with their fluorescence intensities in solution. It is worth noting that the brightest


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fluorescence was derived from the cells incubated with TPE-LPs, suggesting that the liposomeencapsulating TPE-BICOOH can efficiently internalize into cancer cells and result in efficient AIE aggregation. Further, the cytotoxicity of the probes was evaluated with the CCK-8 assay. As shown in Figure 4C and Figure S11, the cellular viability of 4T1 cells remained above 95% after incubation with TPE-LPs (20 µM TPE-BICOOH) over 24 h. The cells still exhibited good proliferative ability after being incubated with TPE-BICOOH and TPE-LPs for 48 h and 72 h. The cytotoxicity assay also confirmed their good biocompatiblity with negligible toxicity.

Figure 4. (A) The quantitative analysis of fluorescence intensity by flow cytometry upon incubating the 4T1 cells with different concentrations of TPE-VQ, TPE-BIVS, TPE-BICOOH and TPE-LPs for 3 hours. (B) Average fluorescence intensity of TPE dyes in plot (A) versus concentration. (C) Cell viability of 4T1 cells after incubation with different concentrations (1.25, 2.5, 5, 10, 20 µM) of TPE-VQ, TPE-BIVS, TPE-BICOOH and TPE-LPs for 24 h and further incubation with CCK-8 for 3 hours.

Encouraged by the living cell imaging results, in vivo tumor imaging of TPE-LPs was thus performed and compared with that of TPE-BICOOH dyes. A tumor implantation mouse model was established using female athymic nude mice that were subcutaneously inoculated with 4T1 cancer cells. After tail intravenous injection of TPE-LPs and TPE-BICOOH, respectively, the


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fluorescence distribution in mice was immediately monitored at different time points (Figure 5A). The fluorescence was clearly observed in mice as early as 30 min, which was indicative of the rapid distribution of TPE fluorophores via the blood circulation. To quantitatively characterize the fluorescence signal, the region-of-interest (ROI) was measured. As shown in Figure 5B, the ROI in the tumor reached a maximum at 2 h post-injection (HPI) of TPE-LPs, and then gradually decreased over time. However, the ROI in the tumor did not change at 24 HPI for the TPEBICOOH-treated mice.

Figure 5. (A) In vivo imaging for 4T1 tumor-bearing mice after intravenous injection of TPE-LPs or TPEBICOOH (at 20 nmol of TPE-COOH per mouse), the white and black circles represented tumor tissue. (B) Fluorescence signal intensity in tumor tissue over time. (C) Signal-to-background ratios (SBR) over time. SBR = (tumor ROI - mean background ROI)/(muscle ROI - mean background ROI). **p

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