Engineered cells-assisted photoactive nanoparticles delivery for

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Biological and Medical Applications of Materials and Interfaces

Engineered cells-assisted photoactive nanoparticles delivery for imageguided synergistic photodynamic/photothermal therapy of cancer Peng Wang, Wenbo Wu, Ruifang Gao, Han Zhu, Juan Wang, Renle Du, Xiaoyu Li, Chenglan Zhang, Shaokui Cao, and Rong Xiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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ACS Applied Materials & Interfaces

Engineered cells-assisted photoactive nanoparticles delivery for image-guided synergistic photodynamic/photothermal therapy of cancer Peng Wang†, Wenbo Wu‡, Ruifang Gao†, Han Zhu¶, Juan Wang†, Renle Du†, Xiaoyu Li†, Chenglan Zhang†, Shaokui Cao§, Rong Xiang†* †Department

of Immunology, Medical School of Nankai University, Nankai

UniversityTianjin, China, 300071. ‡Department

of Chemical and Biomolecular Engineering, National University of

Singapore, Singapore 117585 ¶Key

Laboraory of Synthetic and Biological Colloids Ministry of Education School of

Chemical and Material Engineering, Jiangnan University, Wuxi, China, 214122 §School

of Materials Science and Engineering, Zhengzhou University, Zhengzhou,

China, 450001 *Email:

[email protected]

Keywords: photo-activated therapy, immune cell-mediated drug delivery, cell surface engineering, metabolic labeling, synergistic therapy of cancer.

ABSTRACT Photo-activated therapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a spatiotemporally precise, controllable and noninvasive method for tumor therapy and has therefore attracted increasing attention in recent years. However, it is still a challenge to obtain highly efficient therapeutic photoactive agents (PAAs) and deliver them into tumor, especially the core of solid tumors. Here, we have developed a newly engineered monocyte (MNCs)-based PAA system that realizes

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precise and highly efficient tumor diagnosis and therapy. Firstly, a near infrared (NIR) emissive PAA molecule with both strong singlet oxygen (1O2) production and high photothermal conversion efficiency was precisely designed for realizing simultaneous PDT and PTT of tumor, and was further fabricated to form PAA nanoparticle (NPs). After loading the PAAs NPs into MNCs, the MNCs were then decorated with cyclic Arg-Gly-Asp (cRGD) groups through metabolic labeling method to further improve their ability of targeting and homing into the deep regions of tumors. By using this strategy, we have achieved highly efficient solid tumor ablation results both in vitro and in vivo, indicating that our strategy has a promising prospect for solid tumor therapy.

INTRODUCTION Photo-activated therapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is considered as a promising approach for solid tumor ablation due to its high selectivity, low bio-toxicity and non-invasive properties.1, 2 Under light irradiation, photoactive agents (PAAs) within tumor can generate singlet oxygen (1O2) with strong oxidizing features (for PDT) or heat (for PTT) that can induce severe tumor tissue necrosis.3-6 To date, single phototherapy strategy often suffers from poor treatment outcome and high risk of recurrence. Combination therapy of PDT and PTT have been considered as a highly desirable strategy to address these problems. Conventional strategies often integrate PDT agent and PTT agent into one system, 7-11 of which the therapeutic performance largely relies on the loading capacity of two agents. In addition, two different excitation wavelengths are usually needed to produce 1O2 and heat, respectively, leading to longer treatment time and more systemic side effects to patients.12, 13 More importantly, the 1O2 generation efficiency of PS could be much lower than before due to the energy transfer from PS to PTT agent, which directly affects the PDT efficacy in combination therapy strategy. In this regard, there is an urgent need for a single molecule to achieve both PDT and PTT simultaneously under one single excitation wavelength. The other critical problem that largely affects the therapeutic outcome of PAAs is their target specificity of tumor. Polymer encapsulated drug nanoparticles (NPs) have shown preferential accumulation in solid tumor tissue through the enhanced permeability and retention (EPR) effects or specific interaction between ligands and receptors.14


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However, the heterogeneous distribution of the tumor vessels and receptors makes most tumor regions inaccessible to NPs, leading to unstable therapeutic outcomes in preclinical trials.15, 16 To achieve better tumor-targeting effect, immune cell-mediated drug delivery is receiving more attention due to their natural capability of recognizing and recruitment to tumor tissues.17, 18 In addition, immune cells can protect nano-drugs from clearance, and carry them into the deep regions of tumors. Engineered cytotoxic T lymphocytes have been demonstrated to have not only surprising efficacy against hematologic malignancies, but also high drug-carrying efficiency with functionalized cell surfaces.19 However, their tumor infiltration and cytotoxicity could be impaired during treatment of solid tumor due to the complicated tumor microenvironment.20 Therefore, other types of immune cells, e.g. neutrophils, monocytes and macrophages, which have better ability of phagocytosis and migration to solid tumor tissues, have drawn growing research interests.17 However, it is still challenging to achieve specific enrichment of carrier cells in tumors due to uncontrollable cell behaviors and low cell survival rates in vivo. Cell surface engineering by decorating cells with bioactive molecules has been applied to the field of cell therapy and tissue engineering.21 Natural functional groups existing on the surface of cells are the most popular sites for biomolecular conjugation, but most of them are prevalently existing in the biological system with no specificity, causing a low conjugation efficiency and the off-target effect. Alternatively, metabolic labeling shows great potentials for cell surface engineering due to its high sensitivity and specificity.22, 23 To date, most studies in this field are mainly focusing on decorating tumor cells with functionalized sugars for better tumor-specific targeting in vivo.24-27 However, few examples of metabolic labeling of immune cells have been reported so far.



Scheme 1. a) The flow chart of the preparation of MTD NPs. b) Schematic illustration of the fabrication of MN@RMNCs. c) The therapeutic strategy of breast solid tumor by combined PDT and PTT. d) The proposed process of MN@RMNCs delivery into the tumor site: (1) MN@MNCs bind with the integrin αvβ3 receptors overexpressed on the surface of cells within tumor tissue, (2) MN@RMNCs move across blood vessel wall and transmigrate into the tumor tissues, (3) MN@RMNCs penetrate and accumulate in the deep regions of the tumors, (4) MTD NPs are released from dead MNCs, (5) MTD NPs are taken up by tumor cells and then produce highly efficient tumor ablation under laser irradiation.

In this study, we developed a newly engineered immune cell-mediated PAAs delivery system for highly efficient tumor imaging and simultaneous photodynamic and photothermal tumor therapy. The proposed strategy is shown in Scheme 1. Firstly, a new organic PAA MTD (Scheme 1a) with NIR emission was designed and synthesized to show bright near infrared (NIR) emission, high 1O2 generation and photothermal potentials simultaneously. MTD was further encapsulated into amphipathic polymer DSPE-PEG2000 to form MTD NPs to improve water dispersibility (Scheme 1a). MTD NPs can be largely loaded into MNCs through simple incubation and this assembly is referred to as MN@MNCs. Subsequently, the tumor targeting capability of MTD NPsloaded MNCs were further improved by covalent conjugation with cyclic RGD through “copper free” metabolic labeling technique, hereafter referred to as MN@RMNCs (Scheme 1b). As shown in Scheme 1c and 1d, after intravenous (i.v.) injection, a large


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amount of MN@RMNCs were penetrated and accumulated into deep areas of breast tumor due to specific binding of cRGD with integrin overexpressed on the endothelial cells and tumor cells.28-31 After the cells apoptosis, MTD NPs were gradually released and aggregated in the deep tumor area. After injection of this smart drug delivery system, the tumors could be precisely detected by fluorescence imaging in mice and the tumor growth was also significantly inhibited under 660 nm laser irradiation. RESULTS AND DISCUSSION For PAAs design, it has been shown that a small ΔES1-T1 value (the energy gap between the lowest singlet state (S1) and the lowest triplet state (T1)), which is generally achieved by the separation of HOMO and LUMO in organic molecules, is helpful in realizing effective 1O2 production, since it favors intersystem crossing process.32 However, the HOMO-LUMO separation can easily lead to short wavelength absorption,33 which is contradictory to the requirement of PTT agent in long wavelength absorption. To solve this problem, in this work, we focused on the intersystem crossing process32 from S1 state to the higher energy triplet state, such as T2, rather than T1 for 1O2 generation. In our design, diketopyrrolopyrrole (DPP) with good planar and conjugated structure was selected as the acceptor, since it has been confirmed as a good unit for constructing long wavelength absorption molecules.34,35 By linking one or two methoxy substituted triphenylamine groups to DPP, MTD and DTD (Figure 1a) can be yielded easily, whose synthetic route and structural identification data are presented in Scheme S1, and Figures S1-S2, respectively. As shown in Figure 1a, in both MTD and DTD, the HUMO is distributed everywhere, due to the planar and conjugated structure. As a consquence, both of MTD and DTD show very large ΔES1-T1 values of 0.863 eV and 0.910 eV. Fortunately, their extreamly low ΔES1-T2 values make the intersystem crossing process from S1 state to T2 state possible for 1O2 generation. Meanwhile, the good conjugated structure would endow MTD and DTD with good longwavelengh absorptions, making them potential candidates of good PTT agents. To endow MTD and DTD with good water dispersibility and biocompatibility in biological applications, the polymerencapsulated NPs were then prepared by co-precipitation method using amphiphilic



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DSPE-PEG2000 as the polymer encapsulation matrix. Transmission electron microscopy (TEM) images and dynamic laser scattering (DLS) results (Figure 1b and Figure S3a, b) indicated that the both MTD NPs and DTD NPs had spherical shape with uniform sizes of around 40 nm, which are suitable for in vivo systemic circulation. Both MTD NPs and DTD NPs had broad absorption in the range of 300-700 nm (Figure 1c), and showed bright and broad FR/NIR emission (650-950 nm, Figure 1d), which covers the tissue-penetrating NIR region. There are two donors in DTD, while only one in MTD. Therefore, the charge transfer from the donor to acceptor in MTD is lower than DTD. As a consequence, the absorption peak of MTD NPs is slight blue shifted to that of DTD NPs, and the brightness of MTD NPs is much higher than that of DTD NPs. Also because of this reason, MTD NPs exhibited better 1O2 production than DTD NPs measured

by

the

UV-vis

spectrum

change

of

9,10-anthracenediyl-

bis(methylene)dimalonic acid (ABDA) under 660 nm laser irradiation (Figure 1e). The 1O

2

generation efficiency of MTD NPs is also higher than that of Ce6 NPs, a widely

used PS at present. Meanwhile, To verify specific 1O2 production of MTD NPs, commercial 1O2 probe SOSG was used as an indicator. Under laser irradiation, the green fluorescence of oxidized SOSG gradually increased but was significant suppressed in the presence of sodium azide (NaN3), which is well known to selectively quench 1O2 (Figure S4a, b). Therefore, MTD NPs are selected for the next study. Under 660 nm laser irradiation, MTD NPs showed good photostability (Figure S5) and the temperature of NPs solution rapidly reached 60 °C within a short time of 300 s (Figure 1f), indicating that MTD NPs can act as both PDT agent and PTT agent simultaneously. Meanwhile, after incubating 4T1 cancer cells with different concentrations of MTD NPs for 12 h, the cell viability was not significantly affected (Figure 1g), suggesting good biocompatibility of MTD NPs under the dark condition. In contrast, upon 660 nm laser irradiation for 5 min, MTD NPs loaded 4T1 cells showed bright-green emission of DCFDA, indicating strong ROS generation within cells (Figure S4c, d). Through MTT assay, MTD NPs exhibited obvious toxicity to tumor cells, with a half-maximal inhibitory concentration (IC50) of 5 µg/mL, indicating their excellent capacity to kill tumor cells. However, the killing efficiency of MTD NPs was partly eliminated when


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4T1 cells were pre-incubated with NAC, a ROS scavenger, indicating obvious synergistic treatment outcome of PDT and PTT.

Figure 1. a) The chemical structures and HOMO-LUMO distribution, which was calculated by TDDFT, of MTD and DTD. b) The DLS size distribution of MTD NPs. The inset shows the TEM image of MTD NPs. The scale bar: 100 nm. c) The UV-vis absorption and PL spectra d) of MTD and DTD NPs, respectively. e) The measurement of 1O2 production of MTD, DTD and Ce6 NPs under 660 nm laser irradiation (0.5 W/cm2) by ABDA in water. f) The temperature of MTD NPs and PBS solutions as a function of laser irradiating time at the power intensity of 0.5 W/cm2. g) The cell viability of MTD NPs labeled 4T1 cancer cells treated with N-acetyl-L-cysteine (NAC，1 mM) or not, followed by exposure to 660 nm laser (0.5 W/cm2, 5 min) and dark condition. Data are plotted as means ± SEM (n = 3).

Currently, deep region of tumor is one of the most promising targets for solid tumor therapy in preclinical studies. Herein, MNCs were chosen as drug carrier due to their



outstanding migration capacity towards the deep region of tumor. In addition, to strengthen their tumor targeting effect, we armed cell membrane of MNCs with cRGD through metabolic labeling method. Specifically, cRGD peptide with thiol group and BCN-NHS were firstly coupled through click reaction to generate BCN-cRGD which was then purified by HPLC. BCN-cRGD was thoroughly characterized to confirm its structure and high purity. The structure, synthesis route and identification results of BCN-cRGD are presented in Scheme S2, Figure S6 a and b, respectively. Next, MNCs were isolated from healthy mice and characterized with specific marker F4/80 by flow cytometry analysis. The result showed that on day 5 of cultivation, the purity of monocytes reached 71.4%, then monocytes gradually differentiated into macrophages after 5 days. (Figure S7a). After incubated with acetylated N-azidoacetylmannosamine (AC4ManNAz) for 12 h, which can be incorporated into glycan with azide group on the cell membrane through metabolic glycoengineering,[9b] MNCs were subsequently treated with DBCO-Cy5 for 20 min. The confocal images showed that obvious red fluorescence can be clearly observed in the cell membrane (Figure 2a). In addition, the cell labeling rate for Cy5 was around 95%, indicating the high labeling efficiency of azide group (Figure S7b). Furthermore, the Cy5 was precisely localized on the membrane, demonstrating that azide groups are successfully incorporated into the membrane proteins through metabolism process (Figure 2b). Then, azide modified MNCs were incubated with BCN-cRGD to generate cRGD-MNCs (RMNCs) through click chemistry. The linkage efficacy was estimated by incubation with integrin αvβ3FITC. As shown in Figure 2c, strong green fluorescence could be observed on the membrane of MNCs. Additionally, flow cytometry measurement also showed that the amount of FITC positive cells significantly increased as compared with that of untreated MNCs (Figure S8a and b). These results together demonstrate successful cRGD modification on the cell membrane. To investigate the ability of MTD NPs retention in carrier cells, MNCs were incubated with MTD NPs (10 μg/mL) for 12 h and then subcultured for 1 and 7 days, respectively. As shown in Figure 2d and 2e, all of the MNCs showed strong red fluorescence and their MTD NPs uptake rate was nearly 99% at 1 day. After 7 days, obvious fluorescence signals were still observed in the cytoplasm of 95.8% MNCs. These results verify that MTD NPs can be internalized very well by MNCs with a long retention time and excellent stability in cells over time. Meanwhile, the release curve of MTD NPs


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demonstrate that MTD NPs can be largely released from MNCs in the process of carrier cell death (Figure S9). Subsequently, MN@RMNCs were successfully obtained through this two-step treatment. Fluorescence images constructed in the 3D view showed that after successive treatment, MNCs exhibited obvious localization of red and green fluorescence in the cytoplasm and on the membrane (Figure 2f), respectively, and the co-stained MNCs rate was 93% (Figure 2g), indicating a high assembly efficiency. Meanwhile, cell viability and migration assay also exhibited a good biocompatibility and negligible side effects on cell behavior in this method (Figure S10a and b).

Figure 2. a) Fluorescent image of MNCs pretreated with AC4ManNAz for 12 h, followed by incubation with DBCO-Cy5 for 20 min. b) Fluorescent image of Cy5 (red color) labeled MNCs costained with membrane tracker CellMask Green (green color). c) Fluorescent image of MNCs pretreated with AC4ManNAz for 12 h, followed by successively incubated with BCN-cRGD and integrin αvβ3-FITC (green color). The scale bar: 20 μm. d) Confocal images of MN@MNCs cultured in vitro for 1 day and 7 days after MTD NPs uptake. The scale bar: 50 μm. e) Flow cytometry histograms of MTD NPs labelled MNCs cultured in vitro for 1 day and 7 days. f) The sectional confocal image of MNCs (left panel) incubated with MTD NPs (red color) and subsequently pretreated with AC4ManNAz, followed by successively incubated with BCN-cRGD and integrin αvβ3-FITC (green color). The right panel shows 3D image of the cells. g) Flow cytometry analysis of FITC and MTD NPs labeled MNCs.

Before in vivo application, the potential toxicity of MN@RMNCs in healthy mice was firstly tested. As shown in Figure S11a, after i.v. injection with MN@RMNCs, MTD NPs and saline, respectively, neither death nor obvious difference in body weight was



observed among different treatments over a span of 15 days. In addition, blood biochemistry analysis and histochemistry analysis also showed almost no changes in liver and kidney function, indicating negligible in vivo toxicity of the delivery system (Figure S11b-i). Then the in vivo tumor targeting ability of MN@RMNCs was evaluated. To precisely track the biodistribution and behavior of MN@RMNCs in vivo, Luc/GFP double positive MNCs were isolated from FVB-luc-GFP transgenic mice. After i.v. injection of MN@RMNCs, MN@MNCs or MTD NPs into 4T1 tumorbearing mice, respectively, time-dependent distribution of them in the mice were then monitored at designated time intervals. As shown in Figure 3a, b and Figure S12a, b, on day 0 post-injection, bioluminescent signals of mice can only be detected in the liver and spleen in MN@RMNCs and MN@MNCs groups, and both of them gradually decreased in the following days. In contrast, bioluminescence signals in the tumor continuously increased and reached the highest level on day 4 post-injection in both groups. More importantly, MN@RMNCs exhibited more intense and longer half-life of bioluminescence signal in 4T1 tumor-bearing mice as compared to MN@MNCs. These results verify better tumor targeting of MN@RMNCs due to the surface modification of targeting groups and more NPs accumulation of tumor. On the other hand, the fluorescent signals of the tumor and reticuloendothelial system (RES) organs (liver and spleen) of mice in MN@RMNCs and MN@MNCs groups gradually increase over time, and the highest signal was achieved on day 4 post different treatments. The tumor of the mouse treated with MN@RMNCs exhibits a more intense signal with longer half-life than that of the mouse treated with MN@MNCs. Quantitative analysis of the collected tissue showed MN@RMNCs treated tumor had 1.7-fold higher fluorescence intensity than that treated with MN@MNCs and 2.3-fold higher than that treated with MTD NPs (Figure 3c, d and Figure S13), indicating more effective MTD NPs accumulation in tumor tissue assisted by RMNCs. To examine if MTD NPs could be released from carrier cells, designated tumor tissues in all groups were collected for histological analysis. As shown in Figure 3e and Figure S14, on day 4 post-injection, there were more GFP+ cells in MN@RMNCs treated tumor than those in MN@MNCs treated ones, which is consistent with our previous in


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vivo imaging results. In addition, most of the red fluorescent dots were precisely colocalized with these GFP+ cells in both groups, indicating all MTD NPs in the tumor are derived from MN@RMNCs and have migrated into the tumor instead of free MTD NPs in blood circulation. On day 6, the number of GFP+ cells obviously reduced in both groups. Besides, a large number of MTD NPs were widely distributed in the tumor tissues. These results demonstrate that MTD NPs can be delivered and then released into the tumor by engineered MNCs. Similarly, by means of RMNCs, MTD NPs in MN@RMNCs group had the furthest distribution after release from the blood vessels among three groups (Figure 3f). Furthermore, MN@RMNCs delivery system could cause more accumulation of MTD NPs in the deep region of the tumors than the other two administrations (Figure S15). Taken together, these results suggest that MN@RMNCs delivery system significantly enhances tumor-targeting efficacy of MTD NPs and increases accumulation of MTD NPs at tumor sites lack of blood vessels. Furthermore, the migration capacity of MNCs is helpful for deeper tissue infiltration of NPs.



Figure 3. a) The time-dependent bioluminescence and fluorescence images of the mice i.v. administrated with MN@RMNCs at indicated time points. Yellow circles indicate tumor regions. b) Bioluminescence signal intensities of the tumors with different treatments. Data are plotted as means ± SEM (n=3). *p < 0.05 MN@RMNCs group versus MN@MNCs group. c) Ex vivo fluorescence images of the whole major organs and tumors harvested from the tumor-bearing mice on day 4 after injection of MN@RMNCs. Statistical results of the fluorescence intensities are shown in d). Data are plotted as means ± SEM (n=3). *p < 0.05, MN@RMNCs versus MN@MNCs or MTD NPs group, respectively. e) Representative fluorescence images of tumor sections of MN@RMNCs treated mice on day 4 and day 6, respectively. Middle picture shows the magnified view of square area in the left picture. Green color: GFP, red color: MTD NPs. f) Representative fluorescence images of tumor sections stained with CD31 from mice with different treatments. Dash line indicate NPs aggregates regions. Green color: CD31, red color: MTD NPs. The scale bar: 50 μm.

Encouraged by the excellent in vivo drug delivery effects, we expected a better therapeutic efficacy of photo-activated therapy against tumors that could be achieved by deeper MTD NPs delivery assisted by RMNCs. Similar to the cell tracking strategy as discussed above, a 4T1 tumor-bearing mouse model was i.v. injected with MN@RMNCs, MTD NPs and saline for three consecutive days, respectively.


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According to the in vivo fluorescence imaging, MTD NPs accumulation achieved to the maximum on the 5th day after the last injection (Figure S16). Therefore, the site of tumor was precisely localized and then photo-activated therapy of the tumors was conducted. (Figure 4a). As shown in Figure 4b, 4c and S17, 4T1 tumor-bearing mice were irradiated by the 660 nm laser with the power of 0.5 W/cm2 for 10 min, which caused the rise of tumor temperature to ~66 °C within 3 min. This data is much higher than that of mice treated with MTD NPs alone and saline, indicating stronger photothermal therapeutic efficiency of tumor. After laser treatment, the tumor sizes were continuously monitored for 14 days. As shown in Figure 4d, the tumors of mice in saline group exhibited very rapid growth, while the tumor growth was modestly delayed when the mice were injected with MTD NPs alone. In contrast, the tumor growth of mice treated with MN@RMNCs was significantly inhibited and tumor recurrence was prevented as well, indicating the remarkable therapeutic effect of MN@RMNCs. In addition, through a higher dose injection of MN@RMNCs, tumor tumor growth can be totally inhibited by combination therapy of PDT and PTT, indicating tumor killing ability of MN@RMNCs in a dose-dependent manner (Figure S18). Furthermore, compared with the other groups, the survival rate of mice treated with MN@RMNCs was obviously increased. (Figure S19). Taken together, these results demonstrate that MTD NPs can significantly inhibit the growth of tumor and with the help of MN@RMNCs delivery system the tumoricidal efficacy of MTD NPs can be further improved. To further analyze the therapeutic outcome, the mice in three groups were sacrificed and the tumor tissues were collected and sliced for histological analysis on day 14 after laser treatment. As seen in Figure 4e, hematoxylin and eosin (H&E) staining results displayed no obvious abnormalities and lesions in tumor sections from saline injected mice. Modest necrosis and nucleus dissociation could be observed in tumor sections of MTD NPs treated mice. In comparison, both surface and deep tumor sections from mice treated with MN@RMNCs exhibited severe nucleus dissociation and inflammatory cell infiltration, suggesting serious tissue deterioration and immune reactions within tumor. Similarly, the TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining results



showed that cell apoptosis in both surface and deep tumor sections was significantly increased in MN@RMNCs group as compared with those in other control groups (Figure S20). These data confirm that MN@RMNCs have excellent therapeutic outcome, which is in well accordance with previous observation of tumor growth curve and survival rate.

Figure 4. a) Scheme of the experimental design. b) IR thermal images of 4T1 tumor bearing mice receiving different treatments under 660 nm laser irradiation (0.5 W/cm2). c) The tumor temperature changes of 4T1 tumor bearing mice receiving different treatments under 660 nm laser irradiation. Data are plotted as means ± SEM (n=3). d) Tumor size measurement of mice receiving diffeent treatments at different time points after 660 nm laser irradiation treatment. Data are plotted as means ± SEM (n=10). *p < 0.05, **p < 0.01. e) H&E staining for tumors (surface and deep regions) on day 14 after PDT and PTT in MN@RMNCs, MTD NPs or saline groups. The scale bar: 100 μm.

CONCLUSIONS In conclusion, we report a novel diagnosis and therapeutic strategy for tumor based on engineered immune cell-assisted PAAs delivery. MTD molecule was firstly designed and synthesized as a new PAA molecule, which exhibit intense NIR fluorescence


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emission, strong 1O2 and photothermal generation. For better water dispersibility and biocompatibility, MTD was further encapsulated into DSPE-PEG2000 to form MTD NPs. Through a series of cell experiments, MTD NPs have been considered as an ideal antitumor PAAs. Subsequently, surface engineered MNCs were developed by metabolic glycoengineering technique to confer themselves with excellent ability of tumor targeting and migration. Through two-step treatment, the MN@RMNCs were obtained, which led to a deep and wide distribution of MTD NPs within tumor tissue after i.v. administration. Finally, MN@RMNCs were successfully applied in vivo and got a precise tumor imaging and highly effective tumor ablation upon laser irradiation treatment. Considering the unique performance of simultaneous diagnosis, PDT and PTT of tumor, we anticipate that our system will have many applications in further translational research.

EXPERIMENTAL SECTION Materials：

Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under dry nitrogen immediately prior to use. Compound S436 and S637 were prepared by the same procedure reported in the literature, according to the synthetic route presented in Scheme S1. All the other materials for organic synthesis were purchased from SigmaAldrich. DSPE-PEG2000 were purchased from Avanti Polar Lipids, Inc. All antibodies were purchased from Abcam. CellMaskTM Green Stain Plasma Membrane were purchased from Thermo Fisher Scientific, Inc. Instrument: 1H

and 13C NMR spectra were measured on a Bruker Avance 400 spectrometer using

tetramethylsilane (TMS; δ = 0 ppm) as internal standard. UV-vis and photoluminescence spectra were recorded using Shimadzu UV-1700 and Perkin-Elmer LS 55 spectrometer, respectively. Particle size and size distribution were determined by dynamic light scattering (DLS) with a particle size analyzer (90 Plus, Brookhaven Instruments Co., United States) at a fixed angle of 90° at room temperature. TEM images were obtained from a JEOL JEM-2010 transmission electron microscope with



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an accelerating voltage of 200 KV. A 0.1% TFA solution in H2O and acetonitrile was used as the eluent for high-performance liquid chromatography (HPLC) experiments (Agilent). Synthesis of compound MTD and DTD: To a solution of S6 (215.5 mg, 0.4 mmol) in 10 mL of dry THF at -78 oC, n-butyllithium (0.6 mL, 1 M in hexane, 0.6 mmol) was added dropwise. The mixture was stirred at 78 oC for 1 h. Then, tri-n-butyltin chloride (1.6 mL, 6 mmol) was added rapidly to the above solution and the mixture was stirred for 2 h. The resulting mixture was warmed up to room temperature and stirred for 24 h, then poured into water, and extracted with chloroform. The combined extract was washed with brine, dried over anhydrous Na2SO4. After the removal of solvents, compound S4 (384.3 mg, 1 mmol), palladium(II) chloride (5.0 mg) and toluene (20 mL) was added to the mixture, and the mixture was

then carefully degassed and charged with argon. Then the reaction mixture was stirred at 80 oC for 12 h. After the removal of solvents under reduced pressure, the crude product was purified by column chromatography on silica gel using nhexane/dichloromethane (2/1~1/2, v/v) as eluent to afford dark blue solid MTD and DTD simultaneously. MTD: 88.3 mg, 26.2% yield. 1H NMR (400 MHz, CDCl3, 298K)  (TMS, ppm): 8.95 (d, J = 4.0 Hz, 1H), 8.85 (d, J = 4.0 Hz, 1H), 7.58 (d, J = 7.2 Hz, 2H), 7.40-7.24 (m, 5H), 7.02 (d, J = 8.8 Hz, 4H), 6.84 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 3.98 (m, 4H), 3.71 (s, 6H), 1.86 (s, 2H), 1.32-1.10(m, 16H), 0.88-0.75 (m, 12H). 13C NMR (100 MHz, CDCl3, 298K)  (ppm): 161.9, 161.6, 156.5, 150.7, 149.5, 149.2, 140.4, 140.1, 139.0, 137.6, 136.4, 133.3, 129.2, 129.1, 128.7, 127.2, 127.1, 126.9, 126.1, 124.7, 124.4, 123.0, 119.7, 114.9, 108.4, 107.7, 55.5, 46.0, 39.3, 30.4, 28.6 23.7, 23.1, 14.1, 10.6. HRMS (ESI), calcd for m/z [M+H]+: 841.3942; found: m/z 841.3956. DTD: 132.6 mg, 28.9% yield. 1H NMR (400 MHz, CDCl3, 298K)  (TMS, ppm): 8.89 (d, J = 4.0 Hz, 2H), 7.36 (d, J = 7.6 Hz, 4H), 7.22 (d, J = 4.4 Hz, 2H), 7.02-6.98 (m, 8H), 6.82 (d, J = 8.8 Hz, 4H), 6.80-6.74 (m, 8H), 3.98 (t, J = 11.6 Hz, 4H), 3.71 (s, 12H), 1.86 (s, 2H), 1.32-1.10(m, 16H), 0.88-0.75 (m, 12H).

13C

NMR (100 MHz,

CDCl3, 298K)  (ppm): 161.7, 156.4, 150.1, 149.4, 140.1, 140.0, 139.5, 137.1, 127.4, ACS Paragon Plus Environment

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127.1, 127.1, 126.8, 125.5, 124.8, 122.9, 119.8, 114.9, 107.8, 55.5, 46.0, 39.2, 30.4, 28.5, 23.7, 23.1, 14.1, 10.6. HRMS (ESI), calcd for [M+H]+: 1131.5123; found: m/z 1131.5162. Synthesis of MTD NPs: The THF solution including 1 mg of MTD and 2 mg of DSPE-PEG2000-NH2 was poured into water with 10-fold dilution. The THF/water mixture was then sonicated for 2 min using a microtip ultrasound sonicator at 12 W output (XL2000, Misonix Incorporated, NY). After THF evaporation by stirring the obtained suspension in fume hood overnight, the NPs (8 mL, 0.1 mg/mL based on MTD) were obtained by filtration through a 0.2 μm syringe driven filter. Cell lines and cell culture: 4T1 murine breast cancer cells and 4T1 cells overexpressing luciferase (4T1-Luc) were purchased from ATCC and PerkinElmer Inc., and maintained in our lab. The cells were cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS (GIBCO) and PS (10 U/mL penicillin and 10 mg/mL streptomycin). The cells were maintained in an atmosphere of 5% CO2 and 95% humidified air at 37 °C. The culture medium was changed every day. Isolation and culture of mouse bone marrow-derived MNCs: 6-8 weeks old female Balb/c mice were killed under anaesthesia. The bone marrow cells were extracted from marrow cavities of femurs and tibias of the mice. Then bonemarrow cell suspensions were filtered through a 70-μm nylon strainer in order to remove bone debris. Cells were harvested by centrifugation at 1000 rpm for 10 min. Following by using red blood cell lysis buffer (5.0 mL; Sigma Aldrich, USA) at 37 °C for 30 min, erythrocytes were depleted. After collection by centrifugation, cells were seeded into 6-well ultra-low-attachment surface plates containing 6 mL of RPMI-1640 medium (GIBCO) supplemented with 10% FBS (GIBCO), PS (10 U/mL penicillin and 10 mg/mL streptomycin), and 20 ng/mL macrophage colony-stimulating factor (MCSF, PEPROTECH) at a density of 106 cells/mL. Subsequently, cells were incubated at 37 °C for 5 days in a humidified atmosphere containing 5% CO2. During culture period, the culture medium was changed every other day. On day 3, 5 and 7 of



incubation, the non-adherent cells were harvested and then identified with marker F4/80 by flow cytometry analysis to confirm functional and phenotypic properties of MNCs.38 Compared with mature monocytes, which show intermediate expression of F4/80, macrophages are strong for the marker. However, juvenile monocyte progenitors cannot express F4/80. 38 In vitro cellular uptake assay and metabolic labeling with cRGD peptide: MNCs were seeded and cultured in 10 cm dish for 12 h. MTD NPs (final concentration, 10 μg/mL) were then added into medium and incubated with MNCs for 12 h. The fluorescence signals of MTD NPs within MNCs were captured by confocal laser scanning microscopy (CLSM, Leica TSC SP8, Germany) with excitation at 633 nm and signal collection from 650 nm to 750 nm. After MTD NPs uptake, MNCs were treated with AC4ManNAz (50 μM). After 12 h, the medium was then gently removed, and the cells were washed twice with complete medium. To assess the metabolic labeling efficiency, DBCO-Cy5 (0.5 mg/mL) was added into the medium and incubated for 20 min. After cells were washed twice, the cell nuclei were live stained with Hoechst 33342 for 5 min, and then the cells were imaged immediately by a confocal microscope (CLSM, Zeiss LSM 410, Jena, Germany). To conjugate the cRGD peptide through click reaction, Azide groups labeled MNCs were incubated with BCN-cRGD (1 mg/mL) for 20 min and washed twice with complete medium. Cell viability test: The 4T1 cancer cells were seeded in 96 well plates at density of 3000 cells in 200 μL per well for 12 h. Cells were incubated with different concentration MTD NPs for 12 h and then treated with or without NAC (1 nM) for 30 min, followed by 660 nm laser irridiation for 5 min. After 12 h, MTT (40 μL, 1 mg/mL) was added into medium for 3 h. The medium was removed, and DMSO (100 μL) was added into each well and gently shaken for 10 min at room temperature. The absorbance of MTT at 550 nm was measured by using a SpectraMax M5 Microplate Reader. Cell viability was measured by the ratio of the absorbance of the cells incubated with different concentration NPs to that of the cells incubated with normal culture medium.


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Animals and 4T1 mouse breast cancer models: The luciferase-GFP transgenic mice (6-8 weeks old), which constitutively express firefly luciferase and GFP protein, were purchased from Jackson Laboratory. Female Balb/c nude mice and Balb/c mice were obtained from Beijing Huafukang bioscience Co. INC. (Beijing, China). These mice were maintained under specific pathogen-free (SPF) condition in the Animal Center of Tianjin Medical University (Tianjin, China). All animal experiments were performed in accordance with the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals, and the study was approved by the Animal Ethics Committee of Nankai University. To establish mouse breast cancer models, 4T1 cells (5×104) suspended in 30 μL serum-free cell medium were subcutaneously injected into the second mammary fat pad (left) of each mouse. Tumors were grown until a single aspect reached ∼5 mm (approximately 11 days) before drug treatment. The retention study of NPs in carrier cells: The procedures were similar with cellular NPs uptake. Briefly, on day 5 after cell culture, primary MNCs were collected and seeded into a 6-well plate for 12 h. Then MNCs were incubated with MTD NPs (final concentration, 10 μg/mL) for 12 h and subsequently washed twice with PBS buffer, and the fresh medium containing 20 ng/mL M-CSF was added. Then the cells were subcultured for 7 days. The culture medium was changed every other day. At indicated time points, the cells were collected, then the ability of NPs retention in carrier cells was analyzed by flow cytometry (BD FACSCalibur) and confocal microscopy (CLSM, Leica SP8). The fluorescence intensities of particles in the carrier cells were detected for different time points by confocal microscopy with excitation at 633 nm and emission at 750 nm. At the same time, quantitative analysis of particles in the carrier cells was performed by flow cytometry. Ten thousand events were counted for each sample to plot the histogram. Transwell assay: The migration assay was performed using a transwell cell culture system (5 μm pore size, EMD Millipore Corporation). Briefly, 4T1 cells (5×104 cells/well) were seeded into a 24-well plate containing RPMI-1640 medium supplemented with FBS (10% v/v)



at 37 °C for 24 h. Different payload-containing MNCs (3×105 cells/well) were added to the upper chamber of the transwell in 24-well plates, the upper chambers added FBS free medium. After incubation at 37 °C for 24 h, the medium in the upper and lower chamber was removed. Then the cells migrating to the lower membrane surface were stained with 2% crystal violet at room temperature overnight. The migratory cells were observed and counted by an optical microscope (Olympus IX71). In vivo tracking of MN@RMNCs within tumor-bearing mouse: On day 11 after tumor cells inoculation, Balb/c nude mice were randomly divided into 3 groups (3 mice per group). The mice in three groups were separately i.v. administrated with MN@RMNCs (5×106, 300 μL), MN@MNCs (5×106, 300 μL) and MTD NPs (0.5 mg/mL, based on MTD, 100 μL). MNCs involved in MN@RMNCs and MN@MNCs were extracted from the luciferase-GFP transgenic mice. Subsequently, at designated time points, D-luciferin (150 mg/kg) was intraperitoneally injected into the mice, then bioluminescence signals of the tumors were acquired using a Xenogen IVIS2000 system (Caliper, Waltham, MA, USA) to precisely monitor the in vivo distribution of implanted cells, which were quantified in units of maximum photons per second per square centimeter per steradian. In addition, fluorescent signals of mice were also acquired by in vivo Maestro animal imaging system (CRi, USA) at the same point. Furthermore, on the 4 days post-administration, the tumor-bearing mice in three groups were sacrificed, then the whole major organs and tumors were collected, subjected to ex vivo fluorescence imaging and quantified by the Maestro system. On day 4 and 6 after injection, tumors of mice in different groups were further collected for immunohistochemistry analysis, which were dissected and fixed in 4 % paraformaldehyde (PFA). Afterwards, the tissues were embedded in 30% sucrose/PBS overnight and embedded in Optimal Cutting Temperature (OCT) compound (TissueTek). The slices were subsequently immunostained with rat anti-CD31 (BD Biosciences) and rabbit anti-GFP (Abcam), respectively. Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen) and goat anti-rat IgG (Invitrogen) were used as the corresponding secondary antibodies. The nuclei were stained with DAPI containing mounting solution (Dapi Fluoromount G, Southern Biotech). The sections without


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incubation with primary antibodies were used as negative controls. The slices were finally imaged by CLSM. In vivo anti-tumor therapy: On day 11 after tumor inoculation, 30 mice were randomly divided into three groups (10 mice per group). For three consecutive days, all the tumor-bearing mice were separately i.v. injected with MN@RMNCs (5×106, 300 μL), MTD NPs (0.5 mg/mL, based on MTD, 100 μL) or saline (300 μL). The tumors of mice were subjected to 660 nm laser irradiation (0.5 W/cm2) for 10 min on day 5 after the last injection. The tumor size was measured by a caliper every other day, and the tumor volume was calculated as (tumor length) × (tumor width)2/2. Data related to natural death were recorded as a survival curve. Histological analysis: On day 14 after laser treatment, tumors of mice in different groups were further collected for histological observation, which were dissected and fixed in 4 % paraformaldehyde (PFA). Afterwards, the tissues were embedded into paraffin, sliced at a thickness of 5 µm, and stained with H&E. In addition, a series of immunohistochemistry analyses were performed to assess the therapeutic effect of different treatments. The tumors from mice in different groups were fixed in 4% paraformaldehyde for 2 h, incubated in 30% sucrose/PBS overnight and embedded in Optimal Cutting Temperature (OCT) compound (Tissue-Tek). The slices were subsequently conducted with apoptosis staining according to manual instruction of In Situ Cell Death Detection Kit (Roche Applied Science) and finally imaged by CLSM (Leica TSC SP8, Germany). In vivo toxicity evaluation: In order to test the potential in vivo toxicity of different therapeutics, the body weight of each mouse was recorded every three days after injection. The healthy female Balb/c mice were divided randomly into three groups (5 mice per group). On day 0, the mice were intravenously injected with 200 μL of 3 different therapeutics (MN@RMNCs (5×106, 300 μL), MTD NPs (0.5 mg/mL, based on MTD, 100 μL) or saline (300 μL)). On day 15, all the mice were killed under anaesthesia, then their blood was collected



for blood chemistry analyses. Four important hepatic indicators (i.e., ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase and ALB: albumin) and three indicators for kidney functions (i.e., CRE: creatinine, UA: Uric Acid and URE: urea) were chosen to display. Furthermore, major organs (i.e., hearts, livers, spleens, lungs and kidneys) were excised for histological observations. The organs were fixed in 4% paraformaldehyde fixative solution, which were then processed routinely into paraffin and sectioned at 5 μm thickness. Following the sections were stained with hematoxylin and eosin (H&E) and then imaged by an optical microscope (BX51, Olympus, Japan). Statistical analysis: Statistical analyses were performed using OriginPro 9.1. Quantitative data were expressed as means ± SEM. Statistical comparisons were made by ANOVA analysis and Student’s t-test. Statistical significance threshold was set at P value < 0.05.

SUPPORTING INFORMATION Materials and methods, Synthesis and structure identification data of MTD, DTD and BCN-cRGD, flowcytometry analysis of MNCs with different treatments, In vivo toxicity evaluation, In vivo tracking of MN@MNCs in tumor, In vivo tissue accumulation of MN@RMNCs, IR thermal images of 4T1 tumor-bearing mice receiving MTD NPs treatment

ACKNOWLEDGMENT This work is supported by grants from The National Natural Science Foundation of China (81470354) and Project of Science and Technology Assistance in Developing Countries (KY201501006).

REFERENCES


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