Specific Targeting, Imaging, and Ablation of Tumor-Associated

Apr 22, 2019 - ... AIE luminogens (AIEgens) are a series of molecules with propeller ..... Geissmann, F.; Manz, G. Z.; Jung, S.; Sieweke, M. H.; Merad...
1 downloads 0 Views 632KB Size
Subscriber access provided by IDAHO STATE UNIV

Article

Specific Targeting, Imaging and Ablation of Tumor-Associated Macrophages by Theranostic Mannose-AIEgen Conjugates Xiaoying Gao, Duo Mao, Xingang Zuo, Fang Hu, Jie Cao, Peng Zhang, Jingzhi Sun, Jianzhao Liu, Bin Liu, and Ben Zhong Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01053 • Publication Date (Web): 22 Apr 2019 Downloaded from http://pubs.acs.org on April 22, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 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

Analytical Chemistry

Specific Targeting, Imaging and Ablation of Tumor-Associated Macrophages by Theranostic Mannose-AIEgen Conjugates Xiaoying Gao,†,║,# Duo Mao,║,# Xingang Zuo,† Fang Hu,║ Jie Cao,† Peng Zhang,† Jing Zhi Sun,† Jianzhao Liu,*,† Bin Liu,*,║ and Ben Zhong Tang*,†,§,‡ MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Zheda Road 38, Hangzhou 310027, China





Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585, Singapore Department of Chemistry, Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

§

SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou, 510640, China



ABSTRACT: Tumor associated macrophages (TAMs) that exist in tumor microenvironment promote tumor progression, and have been suggested as a promising therapeutic target for cancer therapy in pre-clinical studies. Development of theranostic systems capable of specific targeting, imaging, and ablation of TAMs will offer clinical benefits. Here we constructed a theranostic probe, namely TPE-Man, by attaching mannose moieties to a red-emissive and AIE (aggregationinduced emission)-active photosensitizer. TPE-Man can specifically recognize mannose receptor that is overexpressed on TAMs by the sugar-receptor interaction, and enables to fluorescently visualize the mannose receptor-positive TAMs in a high contrast. The histologic study of mouse tumor sections further verifies TPE-Man’s excellent targeting specificity comparable with the commercial mannose receptor antibody. TAMs can be effectively eradicated upon exposure to white light irradiation via photodynamic therapy effect. To our knowledge, this is the first small molecular theranostic probe for TAMs which revealed combined advantages of low cost, high targeting specificity, fluorescent light-up imaging, and efficient photodynamic ablation.

Tumor cells exist in complex microenvironment which contains blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix.1 Among the immune cells residing in the tumor site, macrophages are particularly abundant and are present at all stages of tumor progression.1 Unlike other cells, macrophages are highly heterogeneous and could be polarized by local microenvironment stimuli from M0 stage to different subtypes, namely M1 and M2, showing totally opposite functions.2-3 The classically activated M1 macrophages enhance immune responses and inhibit tumor progression,45 however, M2 macrophages suppress antitumor immune responses and promote tumor development and angiogenesis, and are alternatively called tumor associated macrophages (TAMs).6 There is an increasing biological

evidence to suggest TAMs as a promising therapeutic target for cancer therapy.7 Given these facts, specific recognition and ablation of TAMs might be an appreciable way to increase cancer treatment efficiency.7 To achieve the recognition of TAMs, biochemical methods for the diagnosis of TAMs, including qRT-PCR (quantitative real time polymerase chain reaction), western blotting, and immunofluorescence staining of biomarkers, have been applied.8-9 These biological technologies often rely on time-consuming procedures, expensive apparatus and biological reagents, limiting their use for rapid in vivo detection. The ablation of TAMs mostly utilizes chemodrugs, and the performance of chemodrugs is often restricted by the drug resistance problem. Besides, recent studies also have revealed that TAMs are initially associated with the chemoresistance of cancer cells.10-11

ACS Paragon Plus Environment

Analytical Chemistry 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

Scheme 1. Illustration of the TPE-Man probe for specific recognition of TAMs and its photodynamic ablation effect. HO O HO

O

HO

OH

N N N

N N N

O

O

O

OH

O

OH HO

OH

TPE-MAN NC

CN

OH HO O HO HO

N

N N

N O

N

O

OH

N

O

HO

OH

OH

TPE-GAL

NC

CN

3O 2 1O 2

PDT Ablation Imaging

Tumor-Associated Macrophages

It is apparent that chemo-therapy is not a proper choice for the ablation of TAMs. In this regard, there is an urgent need to develop alternative methods for the recognition and ablation of TAMs. Photodynamic therapy (PDT) uses photosensitizers to achieve its therapy purpose and holds potential to circumvent the drug resistance problem. Photosensitizers are activated with light illumination and generate reactive oxygen species (ROS), which kills the malignant cells.12-13 Most photosensitizers exhibit photoluminescent property, which enables to track special cells or cellular process with high resolution in living systems. In principle, photosensitizers can be designed to possess dual-functions of imaging and ablation of malignant cells at the same time. However, Traditional photosensitizers like porphyrins tend to form aggregates through π-π stacking, and suffer from aggregation-caused fluorescence quenching (ACQ) issue.14 The aggregation results in not only quenched fluorescence but also reduced ROS generation ability.15 To address these issues, many efforts have been made to circumvent the aggregation of ACQ photosensitizers,16 but few effective results could be obtained. Recently, fluorophores with aggregation-induced emission (AIE) characteristics have attracted intense research

attention in many fields.17-22 In sharp contrast to ACQ luminogens, AIE luminogens (AIEgens) are a series of molecules with propeller shape, which are non-emissive when molecularly dissolved, but become intensely emissive when forming aggregates, owing to the restriction of intramolecular motions (RIM).23-24 The AIEgens often have larger Stokes shifts compared to ACQ luminogens and can be easily designed as light-up probes for imaging with high signal-to-noise ratio. Meanwhile, AIEgens provide better photo-bleaching resistance than ACQ luminogens due to their aggregate formation or higher working concentration. Besides, AIE photosensitizers are expected to exhibit reserved ROS generation ability in aggregate state and have been extensively applied in image-guided therapies recently.25-31 In this work, we designed and synthesized a costeffective theranostic probe, namely TPE-Man, for applications in TAM targeting, photo-imaging and ablation (Scheme 1). TPE-Man consists of a red-emissive AIE photosensitizer core and two flanking TAM-targeting αmannosides. TPE-Man shows excellent targeting specificity towards TAMs, owing to the favored interactions between mannose and CD206 which is over-expressed on the membrane of TAMs. The specificity is confirmed by

ACS Paragon Plus Environment

Page 2 of 9

Page 3 of 9 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

Analytical Chemistry flow cytometry analysis and confocal laser scanning microscope (CLSM) imaging. Furthermore, TPE-Man was applied in histological study and showed significant signal overlap with commercial antibody immunostaining, suggesting a potential in disease diagnosis. Upon exposure to visible light radiation, the internalized TPE-Man generated ROS to kill TAMs efficiently. Taken together, we successfully developed an AIE-active theranostic probe capable of specific targeting, imaging, and ablation of TAMs on its own.

EXPERIMENTAL SECTION

Cell Culture. The macrophages were isolated from Sprague-Dawley rats. 10 mL DMEM was injected into the enterocoelia of each rat, followed with massage for 3 min and non-disturbance for 7 min. The medium was then extracted, and the released cells were collected by centrifugation at 1000 rpm/min. Next, 3×105 cells were incubated in a well of 24 well culture plate containing 1 mL Dulbecco’s Modified Eagle Medium (DMEM) containing penicillin (100 U/mL), 10% heat-inactivated fetal bovine serum (FBS), and streptomycin (100 μg/mL) and were maintained in humidified incubator at 37 oC under 5% CO2 environment. The cells were incubated with 50 ng/mL macrophage colony-stimulating factor (M-CSF) to obtain M0 macrophages, and M0 macrophages were incubated with 40 ng/mL interleukin 4 (IL-4) for 72 h to obtain M2 macrophages (Scheme 2). Cell Imaging. Both M0 macrophages and TAMs were incubated with 10 μM TPE-Man and 10 μM TPE-Gal at 4 o C for 1 h. After that, the medium was removed and the cells were rinsed with phosphate buffer saline (PBS). Then, the cells were imagined by confocal laser scanning microscope. The excitation wavelength was 405 nm, and the emission filter was 580-680 nm. Flowcytometry. Both M0 macrophages and TAMs were incubated with 10 μM TPE-Man at 4 oC for 1 h. After that, the medium was removed and the cells were rinsed with PBS for three times. Cells were removed from plate and diluted with 330 μL PBS buffer. The fluorescence intensity was measured by flow cytometry (Becton and Dickinson, BD FACSCalibur ROW, USA).

RESULTS AND DISCUSSION

Design, Synthesis and Bio-sensitivity of TPE-Man. The target compound TPE-Man and TPE-Gal, were synthesized via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry as shown in Scheme S1. The α-mannoside could be specifically recognized by CD206, which is over-expressed on the surface of TAMs. For comparison, we prepared a control probe named TPE-Gal, which contained β-galactoside analogs. There is no receptor on the surface of TAMs that can recognize βgalactoside. To verify the feasibility of the probe design, we firstly examined the AIE properties of TPE-Man and TPE-Gal, and the results are shown in Figure S1. Both TPE-Man and TPE-Gal are soluble in DMSO (10 μM) with almost no

fluorescence. When the water fraction (fw, by volume) increases from 0% to 30%, the photoluminescence (PL) intensity stays almost the same. When fw continuously increases, its PL intensity increases sharply and reaches the maximum when fw is 60%. The quantum yield of TPEMan in DMSO is 0.92%, and grows to 13.45% when fw is 99%, which is the experimental condition used in the subsequent experiments. These data confirm that TPEMan and TPE-Gal show obvious AIE characteristics (Figure S2). We then studied TPE-Man and TPE-Gal’s stability to the pH in the range of 3 to 10. The results are shown in Figure S3. The PL intensities of TPE-Man and TPE-Gal in PBS buffer with different pH value show subtle fluctuations, indicating the two probes are quite stable under physiological pH range. With these AIE-active probes in hand, their biosensitivity was studied by using concanavalin A (Con A) and peanut agglutinin (PNA). Con A and PNA are plant lectins, which can specifically bind certain glycose through sugar-lectin interactions.32 Based on previous investigation, Con A is known to specifically bind mannose and PNA can bind galactose. From the results of their spectroscopic response towards lectins (size distributions of these aggregates are shown in Figure S3), as shown in Figure 1, it is found that the spectra of TPE-Man and TPE-Gal showed a dose dependent behavior towards Con A and PNA respectively. With the increasing of the lectin concentration, the emission peak at 650 nm increased gradually. A linear region could be found in the concentration range of 20-80 μM. Moreover, the specificity of TPE-Man and TPE-Gal is also studied. It was found that in the presence of some other proteins, including bovine serum albumin (BSA), wheat germ agglutinin (WGA), and soybean agglutinin (SBA), TPE-Man and TPE-Gal showed no obvious response towards the interfering species. It could be concluded from the results that TPE-Man and TPE-Gal have high selectivity towards Con A and PNA, respectively, indicating the interactions between sugar and lectins are highly selective. Specific Recognition of TAMs. As previous work reported33, mannose has been widely applied for targeting TAMs with overexpressed CD206. Herein, we induced TAMs from primary macrophages (M0 phenotypes) by IL4 (see Scheme 2). To confirm the cells we obtained are TAMs, qRT-PCR was conducted to analyze gene expression change of macrophage during polarization process. Both TNF-α and iNOSs are specific marker of M1 macrophages, while CD206 and Arginase are selectively expressed by TAMs, and could be viewed as the markers of TAMs. As shown in Figure 2A, the mRNA contents of TNF-α and iNOSs are almost at the same level in M0 macrophages and those treated with IL-4. However, M0 macrophages treated with IL-4 possess much higher expression level of CD206 and Arginase, indicating that polarized TAMs were successfully obtained from M0 macrophages.

ACS Paragon Plus Environment

Analytical Chemistry

2.

The

schematic

B

A 600 0

450

Con A concentration (μmol / L) 0 10 20 30 40 50

of

M0

macrophages

C

6 5

1

0 0

400 300

0

200

30

40

50

F

6

5

4

4

3

1 0

550

600

650

700

Wavelength (nm)

750

3 2

1 0

0 500

PNA Con A BSA WGA SBA

6

5

2

100

0

I/I0-1

50

E

PNA concentration (μmol / L) 0 10 20 30 40 50

20

I/I0-1

D

10

Con A concentration (μmol / L)

700 750

Wavelength (nm)

5

2

1

150

600 650

culture.

3

2

500 550

TAMs

4

3

300

0

and

4

I/I0-1

50

PL intensity (a.u.)

illustration

I/I0-1

Scheme

PL intensity (a.u.)

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

Page 4 of 9

10

20

30

40

PNA concentration (μmol / L)

50

0

PNA Con A BSA WGA SBA

Figure 1. (A) TPE-Man and (D) TPE-Gal in the presence of increasing specific lectins (concentration from the bottom curve to the top curve: 0, 10, 20, 30, 40 and 50 μM). Plotting of lectin sensitivity of (B) TPE-Man and (E) TPE-Gal as a function of lectin concentration. Fluorescence response of (C) TPE-Man and (F) TPE-Gal in the presence of 50 μM of different proteins. Photoluminescence spectra were measured in PBS buffer (pH 7.4). Probe concentration: 1 μM; DMSO:H2O=1:99. Subsequently, the macrophages with different phenotypes were incubated with TPE-Man (10 μM) or TPE-Gal (10 μM) at 4 oC for 1 h, respectively. As shown in Figure 2B, both M0 macrophages and TAMs incubated with TPEGal show almost no fluorescence, and there is no obvious difference in terms of fluorescence intensity between M0 macrophages and TAMs. This is because no β-galactoside receptors exist on the surface of both M0 macrophages and TAMs, resulting in ineffective non-specific uptake of TPE-Gal into cells. In contrast, TPE-Man exhibits intense

and specific imaging effect towards TAMs rather than M0 macrophages because of mannose recognition-mediated endocytosis, which is in great consistence with the higher CD206 level in TAMs than M0 macrophages. These results indicate that the mannose group in TPEMan plays a major role in the recognition and labeling of TAMs. Thus, it could be concluded that TPE-Man is CD206-targeting.

ACS Paragon Plus Environment

Page 5 of 9

TAMs

B

M0

TPE-Man

A

C TAMs

85.3%

M0

9.1%

TPE-Gal

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

Analytical Chemistry

Control

Figure 2. (A) Relative content of mRNA level of TNF-α and iNos (M1 macrophage markers), CD206 and arginase (TAM markers) in TAMs over M0 macrophages by qRT-PCR analysis. (B) CLSM images of TPE-Man (10 μM) and TPE-Gal (10 μM) incubated with TAMs and M0 phenotype for 1 h, respectively. (C) Flow cytometry analysis of TAMs and M0 macrophages incubated with TPE-Man (10 μM) for 1 h at 4 oC. Excitation wavelength: 405 nm. Scale bar: 50 μm.

40 μM

0 μM

80 μM

A

B

C

D

E

F

TAMs

M0

Figure 3. CLSM images of the TPE-Man (10 μM) incubated with (A-C) TAMs and (D-F) M0 macrophages pretreated with increasing amount of free mannose (0, 40, 80 μM) for 1h, respectively. Excitation wavelength: 405 nm. Temperature: 4 oC. Scale bar: 50 μm. Comparison with Immunofluorescence Staining. Next, flow cytometry analysis was utilized to test TPEMan’s efficiency in the recognition of TAMs. Flow cytometry is a conventional method for quantitative analysis of a large amount of samples and offers complete information about the distribution of cells through the analysis of their physical properties, such as fluorescence intensity. As shown in Figure 2C, M0 macrophages and

TAMs were incubated with TPE-Man under the same condition. It was found from the results that TAMs exhibited more intense red fluorescence than M0 macrophages. The results showed that 85.3% of TAMs had positive response while the percentage for M0 is only 9.1%, which indicated the high efficiency of TPE-Man in specific recognition of TAMs.

ACS Paragon Plus Environment

Analytical Chemistry

A

B

1.5

UV PL

1.2

125

TPE-Man Alexa647

100

0.9 75

175 nm

0.6

50

0.3 0.0 300

0s

500

600

700

30 s

25

0

20

60 s

40

60

80

100

90 s

TPE-Man

C

400

Alexa647

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

Page 6 of 9

Figure 4. (A) Absorption and emission spectra of TPE-Man. Concentration: 10 μM. (B) Changes in PL intensity of different dyelabeled TAMs with an increase of irradiation time. (C) CLSM images of TAMs labeled with TPE-Man and Alexa647 for different periods of irradiation time, respectively. Excitation wavelength: 405 nm for TPE-Man and 647 nm for Alexa647; Emission filter: 550-700 nm for TPE-Man and 650-700 nm for Alexa647. Scale bar: 50 μm.

To further prove the receptor-targeting ability of TPEMan, a competition assay was carried out by using free mannose. As shown in Figure 3, with the amount of free mannose incubated with TAMs increasing, a prominent decrease in fluorescence intensity could be observed. When TAMs were not treated with mannose, intense red fluorescence could be detected, while when 40 μM of mannoses were added, only weak fluorescence left. Further, fluorescence was hardly seen when 80 μM of mannose was used. However, when mannose-pretreated M0 was incubated with TPE-Man, no obvious fluorescence could be seen. These results indicate that the preincubation of mannose can saturate the receptors and suppress the internalization of TPE-Man, proving that TAM imaging is achieved by specific CD206-targeting effect. Immunofluorescence staining is a technique which utilizes the specific binding of fluorophore-labeled antibody to antigen in the tissues. This technique is widely applied in the biological research and clinical medicine field as a gold standard. Here, we compared the photo-physical property between the fluorophore used in conventional antibody immunostaining and our AIE-active molecular probe. According to the UV absorption and PL spectra (Figure 4A), TPE-Man showed an absorption maximum around 425 nm and emitted at 600 nm, showing a large Stokes shift of 175 nm. In contrast, commercial Alexa647conjugated antibody immunostaining only displayed a small Stokes shift of 20 nm (Figure S4). The large stokes shift of TPE-Man could help significantly avoid selfabsorption issue. Photo-stability is another key criterion

to evaluate imaging reagent. In the photo-bleaching experiment, CD206-positive TAMs were stained with TPEMan and commercial Alexa647-labeled antibody, respectively. Continuous laser scanning of the stained cells was carried out and its PL intensity at each scan was recorded. Compared with Alexa647, TPE-Man showed more resistance to laser irradiation (Figure 4B and 4C). The fluorescence of Alexa647 faded rapidly, while TPE-Man’s PL intensity stayed almost the same. It could be concluded that TPE-Man has superior photo-physical properties to commercial Alexa647 used in immunostaining. Encouraged by excellent performance of TPE-Man in specific cell imaging test and its excellent photo-physical property, we next tried to stain solid tumor tissue sections by TPE-Man. The results were compared with traditional immunofluorescence staining to evaluate if TPE-Man could also work at tissue level. As shown in Figure 5, The CLSM images of tumor sections indicated TAMs can be easily and precisely labeled by TPE-Man probe. Compared with the TAM-targeting commercial Alexa488-labelled antibody, the overlap coefficient reaches 89.7% based on MATLAB calculation. These data demonstrated that TPEMan could be potentially applied in clinical tissue sections test, and its specificity towards CD206 behaved similar to that of commercial antibody. Apart from this, TPE-Man-staining has advantages of short procedures and easy operation, while immunofluorescence staining requires time-consuming procedures and costly antibody.

ACS Paragon Plus Environment

Page 7 of 9

TPE-MAN

Alexa anti CD206

merge

Enlarged Views

DAPI

Figure 5. Representative immunofluorescence images of tumor sections stained with TPE-Man (4 μM, red channel) and Alexa488 anti CD206 (2.5 μg/mL, green channel), respectively. Scale bars: 25 μm (top row), 10 μm (bottom row).

A 125

Cell viability (%)

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

Analytical Chemistry

B

2 μM

D

100 μM

Light Dark

100 75 50 25 0

C

0

2

5

10

20

50 100

Concentration (µM) 20 μM

Figure 6. (A) Viability of TAMs preincubated with different concentrations of TPE-MAN in the dark (black bar) or with white light irradiation (400−800 nm) (red bar) for 8 min at the power density of 100 mW/cm2 (B-D) Selected laser scanning confocal microscopy images of TAMs stained with Fluorescein Diacetate (FDA, green fluorescence, live cells) and Propidium Iodide (PI, red fluorescence, dead cells) at 2, 20 and 100 μM of TPE-Man. Scale bar: 50 μm.

Photodynamic Therapy. We further studied the photodynamic cell ablation efficiency of TPE-Man. As a kind of appropriate and gentle approach for cancer therapy, PDT can be activated by visible light to generate shortlived but highly toxic reactive oxygen species (ROS), which could effectively circumvent cancer cells’ drug resistance caused by chemo-therapy, and has been clinically approved by authorities for cancer therapy with minimal invasion and high spatiotemporal precision. The TPEMan showed increased 1O2 production efficiency with the extent of probe aggregation and irradiation time (Figure S5).

TAMs were incubated with TPE-Man at various concentrations for 1 h, respectively. The 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the viability of TPE-Manstained TAMs under dark condition was around 100%, indicating negligible dark toxicity of TPE-Man. In contrast, after exposed to white light irradiation, cell viability of TAMs gradually decreases with increase of concentration of TPE-Man (Figure 6A). In addition, live/dead staining was conducted to further test the PDT efficiency of TPE-Man. As shown in Figure 6B-6D, it was obvious that when TAMs were incubated with lower concentration of TPE-Man, majority of TAMs were labeled with green fluorescence, indicating few cells died. When the concentration of TPE-Man increased, the number of cells with red fluorescence gradually increased, suggesting TAMs could be effectively killed by PDT. Therefore, TPE-Man has good biocompatibility and significant therapeutic effect on TAMs upon light irradiation.

CONCLUSIONS To sum up, we have developed an AIE theranostic probe, TPE-Man, by conjugating mannose with a redemissive AIE-active photosensitizer. Mannose moiety in the probe offers binding specificity to CD206 on TAMs via sugar-protein recognition interactions. Upon white light irradiation, the AIE photosensitizer can generate ROS efficiently within TAMs to induce cell death. The TPEMan’s combinatorial roles of TAM recognition, imaging, and ablation make itself a promising theranostic reagent. The high fluorescence staining contrast between TAM and M0 macrophage by TPE-Man potentially becomes a criterion for rapid disease detection in clinical uses.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxx.

ACS Paragon Plus Environment

Analytical Chemistry 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

Experimental details of TPE-Man and TPE-Gal synthesis (Scheme S1); the photophysical properties of the probes (Figures S1-S4); 1O2 production efficiency of TPE-Man (Figure S5); cell incubation and imaging procedures; Characterization data of the probes (Figures S6-S15).

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] *[email protected]

Author Contributions # X. Gao and D. Mao contributed equally. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Science Foundation of China (91853110, 51573158, 21788102), the key project of Ministry of Science and Technology of China (2013CB834704), the Innovation and Technology Commission (ITC-CNERC-14SC01), and the Research Grants Council of Hong Kong (16301614, 16305015, 16308016, N_HKUST604/14 and A_HKUST605/16). J. L. thanks the Thousand Young Talents Plan of China, Hundred Talents Program of Zhejiang University, the Fundamental Research Funds for the Central Universities, and Dabeinong Funds for Discipline Development and Talent Training in Zhejiang University. B. Z. T. is also grateful for the support from the Science and Technology Plan of Shenzhen (JCYJ201602229205601482), B. L. thanks the National University of Singapore (R279-000-482-133) for financial support.

REFERENCES (1) Geissmann, F.; Manz, G. Z.; Jung, S.; Sieweke, M. H.; Merad, M.; Ley, K. Science 2014, 327, 656-661. (2) Murray, P. J.; Wynn, T. A. Nat. Rev. Immunol. 2011, 11, 723. (3) Gordon, S.; Taylor, P. R. Nat. Rev. Immunol. 2005, 5, 953. (4) Sutterwala, F. S.; Noel, G. J.; Salgame, P.; Mosser, D. M. J. Exp. Med. 1998, 188, 217-222. (5) Mosser, D. M.; Edwards, J. P. Nat. Rev. Immunol. 2008, 8, 958. (6) Kawanishi, N.; Yano, H.; Yokogawa, Y.; Suzuki, K. Exerc. Immunol. Rev. 2010, 16, 105-118. (7) Casazza, A.; Laoui, D.; Wenes, M.; Rizzolio, S.; Bassani, N.; Mambretti, M.; Deschoemaeker, S.; Ginderachter, J. A. V.; Tamagnone, L.; Mazzone, M. Cancer Cell, 2013, 24, 695-709. (8) Madsen, D. H.; Leonard, D.; Masedunskas, A.; Moyer, A.; Jurgensen, H. J.; Peters, D. E.; Amornphimoltham, P.; Selvaraj, A.; Yamada, S. S.; Brenner, D. A.; Burgdorf, S.; Engelholm, L. H.; Behrendt, N.; Holmbeck, K.; Weigert, R.; Bugge, T. H. J. Cell Biol. 2013, 202, 951-966. (9) Song, M.; Liu, T.; Shi, C.; Zhang, X.; Chen, X. ACS Nano 2016, 10, 633-647. (10) Yang, C.; He, L.; He, P.; Liu, Y.; Wang, W.; He, Y.; Du, Y.; Gao, F. Med. Oncol. 2015, 32, 352. (11) Jinushi, M.; Chiba, S.; Yoshiyama, H.; Masutomi, K.; Kinoshita, I.; Dosaka-Akita, H.; Yagita, H.; Takaoka, A.; Tahara, H. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 12425-12430.

(12) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380-387. (13) Price, M.; Reiners, J. J.; Santiago, A. M.; Kessel, D. Photochem. Photobiol. 2009, 85, 1177. (14) Sekkat, N.; Bergh, H. V. D.; Nyokong, T.; Lange, N. Molecules 2012, 17, 98-144. (15) Park, S. Y.; Baik, H. J.; Oh, Y. T.; Oh, K. T.; Youn, Y. S.; Lee, E. S. Angew. Chem. Int. Ed. 2011, 50, 1644-1647. (16) DeRosa, M.; Crutchley, R. Coord. Chem. Rev. 2002, 351, 233234. (17) Gao, X.; Feng, G.; Manghnani, P. N.; Hu, F.; Jiang, N.; Liu, J.; Liu, B.; Sun, J. Z.; Tang, B. Z. Chem. Commun. 2017, 53, 1653. (18) Gao, X.; Cao, J.; Song, Y.; Shu, X.; Liu, J.; Sun, J. Z.; Liu, B.; Tang, B. Z. RSC. Adv. 2018, 8, 10975. (19) Qian, J.; Tang. B. Z. Chem 2017, 3, 56-91. (20) Feng, G.; Liu, B. Small 2016, 12, 6528-6535. (21) Gao, M.; Li, S.; Lin, Y.; Geng, Y.; Ling, X.; Wang, L.; Qin, A.; Tang, B. Z. ACS Sens. 2016, 1, 179-184. (22) Hu, F.; Liu, B. Org. Biomol. Chem. 2016, 14, 9931. (23) Mei, J.; Hong, Y.; Lam, J. W. Y.; Qin, A.; Tang, Y.; Tang, B. Z. Adv. Mater. 2014, 26, 5429-5479. (24) Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z. Chem. Rev. 2015, 115, 11718-11940. (25) Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D. Anal. Chem. 2014, 86, 7987-7995. (26) Gu, B.; Wu, W.; Xu, G.; Feng, G.; Yin, F.; Chong, P. H. J.; Qu, J.; Yong, K. –T.; Liu, B. Adv. Mater. 2017, 29, 1700548. (27) Wu, W.; Mao, D.; Hu, F.; Xu, S.; Chen, C.; Zhang, C. –J.; Cheng, X.; Yuan, Y.; Ding, D.; Kong, D.; Liu, B. Adv. Mater. 2017, 29, 1700548. (28) Wang, D.; Su, H.; Kowk, R. T. K.; Hu, X.; Zou, H.; Luo, Q.; Lee, M. M. S.; Xu, W.; Lam, J. W. Y.; Tang, B. Z. Chem. Sci. 2018, 9, 3685. (29) Yuan, Y.; Zhang, C. –J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Angew. Chem. Int. Ed. 2015, 54, 1780-1786. (30) Yuan, Y.; Zhang, C. –J.; Kwok, R. T. K.; Xu, S.; Zhang, R.; Wu, J.; Tang, B. Z.; Liu, B. Adv. Funct. Mater. 2015, 25, 6586-6595. (31) Gu, K.; Qiu, W.; Guo, Z.; Yan, C.; Zhu, S.; Yao, D.; Shi, P.; Tian, H.; Zhu, W. -H. Chem. Sci. 2019, 10, 398. (32) He, X. –P.; Zhu, B. –W.; Zang, Y.; Li, J.; Chen, G. –R.; Tian, H.; Long, Y. –T. Chem. Sci. 2015, 6, 1996. (33) Locke, L. W.; Mayo, M. W.; Yoo, A. D.; Williams, M. B.; Berr, S. S. Biomaterials 2012, 33, 7785-7793.

ACS Paragon Plus Environment

Page 8 of 9

Page 9 of 9 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

Analytical Chemistry

For Table of Contents Use Only

9 ACS Paragon Plus Environment