Microfluidics-Prepared Uniform Conjugated Polymer Nanoparticles for

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Biological and Medical Applications of Materials and Interfaces

Microfluidics Prepared Uniform Conjugated Polymer Nanoparticles for Photo-triggered Immune Microenvironment Modulation and Cancer Therapy Zhe Wang, Bing Guo, Eshu Middha, Zemin Huang, Qinglian Hu, Zhengwei Fu, and Bin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22579 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Applied Materials & Interfaces

Microfluidics

Prepared

Uniform

Conjugated

Polymer Nanoparticles for Photo-triggered Immune Microenvironment Modulation and Cancer Therapy Zhe Wang,a‡ Bing Guo,b‡ Eshu Middha,b Zemin Huang,a Qinglian Hu,*a Zhengwei Fu,*a Bin Liu*b

aCollege

of Biotechnology and Bioengineering, Zhejiang University of Technology,

Hangzhou 310032, China

b

Department of Chemical and Bio-Molecular Engineering, National University of

Singapore, 117585, Singapore

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Corresponding Author Dr. Qinglian Hu. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China. Email: [email protected]; Prof. Zhengwei Fu. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China. Email: [email protected] Prof. Bin Liu. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117585. Email address: [email protected]

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡Z. Wang and B. Guo contributed equally.



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ABSTRACT

Photothermal therapy (PTT) has shown great promise to spatiotemporally ablate cancer cells and further understanding of the immune system response to PTT treatment would contributes to improvement in therapeutic outcomes. Herein, we utilize microfluidic technology to prepare biocompatible conjugated polymer nanoparticles (CP NPs) as PTT agents and assess the immune response triggered by CP-based PTT treatment in

vitro and in vivo. Through careful control of the anti-solvent, CP NPs with uniform diameter of 52 nm were obtained. The c-RGD functionalized CP NPs exhibit high photothermal conversion efficiency, inducing effective cancer cell death under 808 nm laser illumination. Using macrophage cells as the model, CP NPs demonstrate effective activation of pro-inflammatory immune response. Furthermore, in tumor-bearing mice model, a single round of CP NPs assisted PTT could efficiently induce anti-tumor immunity activation and ultimately inhibit tumor growth. The study provides detailed understanding of both microfluidic technology for CP NPs fabrication and photothermaltriggered anti-tumor immune responses.


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KEYWORDS: Immune tumor microenvironment, conjugated polymer nanoparticles, photothermal therapy, cancer, microfluidics



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Introduction

Photothermal therapy (PTT) as a promising non-invasive cancer therapy has advantages in spatiotemporal controllability and negligible drug resistance.1-3 The PTT efficacy is strongly dependent on the properties of photothermal agents. As compared to conventional inorganic photothermal agents which have long-term safety concerns, the emerging organic PTT agents like small molecules and conjugated polymers (CPs) have advantages in good biocompatibility.4-8For small organic molecules, they often suffer from low photostability, leading to suboptimal PTT efficacy.7-9 CPs can be facilely designed via molecular engineering to achieve certain electrical and optical properties for optimal PTT performance, such as excellent photostability, long wavelength absorption and favorable non-radiative decay for exciton energy dissipation.12-14Most recently, we and others have reported a series of CP based biocompatible PTT agents, which showed excellent PTT performance due to their strong NIR absorbance, high photothermal conversion capability and excellent photostability.15-21


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To convert CPs into nanoparticles (NPs) with suitable size and water dispersibility for biological applications, NP formulation methods including in-situ polymerization, emulsion and solvent evaporation, self-assembly and nanoprecipitation are generally used.22 Among all the methods, nanoprecipitation usually facilitates NP fabrication with small sizes of less than 100 nm, but this method generally requires CP to be dissolve well in water miscible organic solvents like THF, acetone and methanol.23 However, many rigid conjugated polymers cannot dissolve well in the above mentioned organic solvents, but they are soluble in some water-immiscible solvents like chloroform and 1,4-dichlorobenzene. This leads to difficulties in the fabrication of small uniform NPs through nanoprecipitation. Most recently, microfluidic systems have been extended to the encapsulation of drugs/dyes into polymer matrix through nanoprecipitation, forming uniformly distributed NPs with tunable particle size due to efficient mixing.24-26 They have shown significant potential superiority over conventional NP preparartion methods, and therefore, microfluidics would be as an advanced technology to synthesize CP NPs with desirable sizes.27



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During cancer therapy, PTT ablates cancer cells and as well triggers release of cancer antigens and endogenous adjuvants, which would further activate the stream of immune cells and ultimately inhibit cancer progress and metastasis.28-30 It has been demonstrated that PTT combined with other therapeutic methods such as chemotherapeutic drugs, immune-adjuvants and immune checkpoint molecules could induce strong immune response and greatly improve therapeutic outcomes.31-33 However, the “immune enhancement” strategy often results in rare objective responses and causes frequent immune-related adverse events in vivo.34 With great achievement in understanding the roles that tumor microenvironment (TME) plays on cancer therapy, modulation of TME to “normalized level” becomes the new direction of cancer immunotherapy. Tumor-associated macrophages (TAMs) are important components in TME, exhibiting anti-inflammatory and pro-tumor effects. 35 Therefore, strategies to reset the TME such as anti-PD therapy and TAM polarization are of enormous interest in cancer immunotherapy.36-37 To the best of our knowledge, people have rarely investigated the detailed contribution of PTT alone on TME modulation, especially on macrophage polarization. 8 ACS Paragon Plus Environment

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In this contribution, we first demonstrate the synthesis of small and uniform conjugated polymer NPs through microfluidic glass capillary mixer by using a modified nanoprecipitation process. We then study the relationship between NP morphology and formulation parameters such as anti-solvent composition and the Reynold number (no) for screening the optimal process parameters. To achieve tumor targeting, tumorhoming peptide (cyclic RGD peptide) was decorated on the NP surface to form P0RGD NPs. Subsequently, we confirmed the photo-triggered cancer cell killing capability of CP NPs in vitro and assessed whether PTT treatment was able to activate macrophages for the generation of pro-inflammatory cytokines. At last, the PTT effect on TME modulation and antitumor efficacy of CP NPs was systematically investigated in vivo (Scheme 1).



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Scheme 1. Schematic illustration of immune response activation induced by P0RGD NP assisted PTT treatment.

Experimental Section

Microfluidic glass capillary mixer for synthesis of CP NPs. The synthesis of NPs was done by using microfluidic glass capillary with two inlets, one for organic solvent (10% 10 ACS Paragon Plus Environment

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CHCl3 + 90% THF) with conjugated polymer P0 and lipid-polymer DSPE-PEG dissolved in it and the other for anti-solvent (100-X% water + X% MeOH), linked with a syringe driven by a programmable syringe pump. The value of X is varied from 0 to 75% during experiments. The total flow rate of water and organic solvent was varied from 1 mL/min to 15 mL/min (Re from 21.2 to 318) by maintaining outer (anti-solvent) to inner (solvent) ratio as 10. P0 and DSPE-PEG were dissolved into solvent with a total concentration of 1.5 mg/mL and a DSPE-PEG to P0 mass ratio of 2. The synthesized NPs were characterized by using dynamic light scattering (DLS) at a 90-degree scattering (ZetaSizer Nano-ZS, Malvern Instruments, UK) after solvent evaporation. Each measurement was repeated 3 times. Decoration of c-RGD on NP surface. After microfluidic process, 10 mL of P0 NPs suspension (0.05 mg/mL) was stirred vigorously for 2 days to remove the organic solvent. For NP functionalization, thiolatedcyclo(Arg-Gly-Asp-D-Phe-Lys(mpa)) peptide (c-RGD) (0.5 mg) was added and was further shaken overnight at room temperature. Following the reaction, nanoparticles were dialyzed against pure water using a membrane (molecular cutoff = 8 KDa) for 3 days to remove the unreacted peptide. 11 ACS Paragon Plus Environment


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Cell Culture. Mouse-breast 4T1 cancer cells and RAW264.7 cells were received from Zhejiang University. The cells were pre-cultured in RPMI 1640 medium, which was supplemented with 10% FBS, 100 U/mL penicilins and 100 µg/mL streptomycin, at 37 oC

in a humidified atmosphere containing 5% carbon dioxide (CO2).

In Vitro Cytotoxicity Study. 4T1 murine breast cancer cells (1× 104 cells per well) were severally seeded into 96-well plates for 24h. Next, different concentrations of P0 NPs and P0RGD NPs (1, 2.5, 5, 10 and 20 µg/mL) were respectively added to culture media. After 24h incubation, the cells were washed three times with PBS and then treated with 100 μL of 0.5 mg/mL MTT working solution for 4 h. For PTT treatment, the cells were irradiated with 808 nm laser (2.0 W cm−2) for 10 min. After overnight culture, the cells were further incubation with MTT solution. After removal of MTT solution, DMSO (100 μL) was added to completely dissolve the formazan crystals. The microplate reader was used to measure the absorbance at 490 nm (Themo Multiscan MK3, USA). Cell viability was characterized by the ratio of the absorbance of the cells with CP NP treatment to that of the cells treated with culture medium alone.


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Quantitative Real-Time PCR Analysis. To evaluate the performance of PTT treatment on immune response, the mRNA expression level of IL-1β, IL-6, IL-12, TNF-α and

CXCL-1 in RAW264.7 cells were tested by semi-quantitative real-time PCR analysis (RT-qPCR). The cells were treated with 0.1 µg/mL P0 NPs and P0RGD NPs for 24 h. For PTT treatment, the RAW264.7 cells were irradiated with 808 nm laser (2.0 W cm−2) for 10 min. After overnight culture, both the cells and the total RNA were collected using TRIzol reagent (Takara Biochemicals, China). ReverTra Ace® qPCR RT Kit (Toyobo, Tokyo, Japan) was used to synthesize first standard cDNA. The mRNA expressions ofIL-1β, IL-6, IL-12, TNF-α and CXCL-1 were investigated via the SYBR green system (Toyobo, Tokyo, Japan) with a standard protocol: 1 cycle (95 °C, 1 min), 40 cycles (95 °C, 15 s) and 1 cycle (60 °C, 1 min).38 The relative gene expressions were characterized using the 2−ΔΔCt method and subsequently normalized to the expression of Gapdh, which is used as the housekeeping gene. Table S2 illustrates the sequences of quantitative polymerase chain reaction (q-PCR) primers.



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Animal Model Construct and Immune Response Assay. Female Balb/c mice with 6 to 8week age were received from China National Laboratory Animal Resource Center. All animals for the in vivo studies were handled, anesthetized, and killed according to the regulation of China Animal Protection Law and the operation protocols were approved by the Zhejiang University of Technology Animal Care and Use Committee.

The 4T1 xenograft tumor models were subcutaneously injected 1 × 106 4T1 cells on the right flank. The mice were randomly divided into five group (n = 8) when the tumor sizes reached about 100 mm3 for different treatment: only PBS (і), P0 NPs (ii), P0RGD NPs (iii), P0 NPs + laser (iv), P0RGD NPs + laser (v). The mice were intravenously injected with 100 μL of PTT nanoparticle solution (2 mg/kg BW), while the control group was treated with PBS (100 μL). For PTT treatment, the mice were locally treated with 808 nm laser irradiation (2 Wcm-2) for 20 min after intravenous injection of P0 NPs and P0RGD NPs for 8 h.

After the first PTT treatment, the 4T1 tumor tissues were collected at 48, 96 and 168 h. For cytokine assay, the tumor tissues were lysed and centrifuged to remove connective tissues and cell debris. The protein levels of IL-10 and IL-12p70 were measured with ELISA following 14 ACS Paragon Plus Environment

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manufacturer’s protocol. For tumor infiltrating lymphocytes assay, the tumors were collected at 48 h after the first treatment and cut into small pieces and lysed with collagenase Ι (1mg/mL) for 2 h. After red blood cells (RBC) using the RBC lysis buffer was removed, the prepared singlecell suspension was analyzed by staining with fluorescence-labeled antibodies against CD4+, CD8+, and CD3+ for flow cytometry analysis following the standard protocol. For systemic immune response assay, the sera were collected at 48, 96 and 168 h after the first PTT treatment. The protein levels of mouse interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α) were also analyzed with ELISA kits according to the manufacturer's protocol. To quantify the activated CD4+ and CD8+ T cells in spleen after different treatments, the spleen tissues were collected at 48 h after the first PTT treatment. Single cell suspension was prepared and then stained with fluorescence labeled antibodies against CD4+, CD8+ and CD69+ at 4 C for 30 min before FCM analysis.

In Vivo Antitumor Activity. The 4T1 xenograft tumor models were constructed and the mice were randomly divided into five groups for different treatments: only PBS (і), P0 NPs (ii), P0RGD NPs (iii), P0 NPs + laser (iv), P0RGD NPs + laser (v). The mice were injected with 100 μL of PBS or CP NPs every three day for 5 times with and without



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irradiation. After different treatment, the tumor volume was continuously measured and calculated using the equation: tumor volume = 1/2 × length × width2.39 The mouse body weights were also measured. Five days after last laser treatment, the mice were euthanized. At the following, the tumors were dissected, weighed, and then imaged.

For histological assay, tumor tissue treatment was done as follows. The tissue was fixed using paraformaldehyde (4%) for 4 h, processed into paraffin, and sectioned at a thickness of 5 μm. The one-step TUNEL apoptosis assay kit was used to assess the apoptosis and necrosis levels of tumor tissues in all treated groups. Fluorescence microscopy (Leica DM IL LED FLUO) was used to capture images of Apoptotic cells.

Statistical Analysis. All the collected data were characterized as means ± standard error of the mean (SEM). The statistical analysis results were obtained with GraphPad Prism 5 (GraphPad Software, La Jolla, CA). The one-way analysis of variance (ANOVA) using StatView 5.0.1 was to express the statistical differences between replicates. The significant difference was strictly accepted at *p < 0.05 and **p < 0.01 or ***p < 0.001.

Results and Discussion 16 ACS Paragon Plus Environment

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Uniform CP NPs Fabrication and Their Photothermal Performance

In this study, we synthesized a conjugated polymer (P0) by Stille coupling polymerization (supporting information), which has intrinsically rigid backbones, high planarity and strong donor-acceptor strength, which is favorable for non-radiative decay and high photothermal conversion efficiency.14,40 The polymer P0 exhibits a number average molecular weight of 3.4 ×104, and a polymer dispersity index of 2.2. The purified products were characterized by NMR to confirm the right structure with good purity (Figure S1). For synthesis of monodisperse NPs, we have fabricated a microfluidic glass capillary mixer with coaxial flow. Figure 1a represents the schematic diagram of microfluidic glass capillary mixer with two inlets for the synthesis of NPs through conventional nanoprecipitation (100% water in anti-solvent) and modified nanoprecipitation. Each inlet is connected with a syringe controlled by a programmable syringe pump. Syringe pump A is used to control the flow of outer fluid (anti-solvent) which contains mixture of water and MeOH, and syringe pump B controls the flow rate of inner fluid, organic



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solvent (10% CHCl3 + 90% THF). The solvent stream comprises of CP, DSPEPEG2000 and DSPE-PEG2000-MAL. In the mixing zone, inner fluid (solvent) is rapidly mixed with outer fluid (anti-solvent), resulting in NPs through self-assembly process due to super-saturation, and then the mixture is kept for solvent evaporation. Using microfluidic mixer, we have synthesized CP NPs of different sizes by varying the total flow rate from 1 to 15 mL min-1 (Reynolds no 21.2 to 318.2) and the amount of MeOH from 0% to 75% through modified nanoprecipitation (Table S1).33 By varying the amount of MeOH in an anti-solvent at Re 318 (flow rate 15 mL min-1), different sizes of CP NPs were obtained. With an increase of X from 0 to 25%, continuous reduction in the size and PDI of NPs is noticed with a significant drop in the size to 52 nm through modified nanoprecipitation with 25% MeOH in an anti-solvent. Whereas, with an increase of X from 40% to 75%, the size of NPs increases from 55 nm to 177 nm (Figure 1b). As shown in Figure 1c, by varying Reynolds no (Re) from 21.2 to 318.2, reduction in the size of P0 NPs from 187 nm to 52 nm is noticed with PDI in the range of 0.15 to 0.20. The continuous reduction in the size of NPs with Re can be attributed to the fast and uniform mixing in the system through increasing flow rate.25 While in 18 ACS Paragon Plus Environment

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traditional nanoprecipitation method, it was found that large NP aggregates appeared with broad size distribution and multiple peaks (Figure S3), which is due to the slow and inefficient mixing of solvent and anti-solvent.42 a

Anti-Solvent (X% MeOH + (100-X)% H 2O)

A

PumpC2A H5

C4H9

C4H9

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P0 solution with organic Solvent (10% CHCl 3 + 90% THF)

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PFTTQ

C8H17

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P O O NH4

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(OCH2CH2)45-OCH3

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P0

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+ DSPE-PEG2000

DSPE-PEG-MAL DSPE-PEG2000

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120

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Scale: 200 nm

Figure 1. (a) Schematic diagram of microfluidic glass capillary mixer for the synthesis of monodisperse P0RGD NPs through modified nanoprecipitation. (b) Variation in the size



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and PDI of NPs by varying the composition of MeOH in anti-solvent during the synthesis at Re 318. (c) Variation in the size and PDI of NPs synthesized by using modified nanoprecipitation with 25% MeOH in an anti-solvent at different Re. (d) TEM images of monodispersed (i) P0 NPs and (ii) P0RGD NPs synthesized by using modified nanoprecipitation with 25% MeOH in an anti-solvent, respectively.

For biological applications, P0 NPs synthesized through modified nanoprecipitation (X = 25% MeOH) and at Re 318 with the size of 52 nm encapsulated into DSPE-PEG2000 and DSPE-PEG2000¬MAL were used. To realize P0 NPs with tumor-specific targeting efficacy, c-RGD, a peptide for αVβ3 integrin receptors, was chemically grafted onto the NP surface to form P0RGD NPs via Michael addition reaction with a reaction yield of about 41.2% (Figure S4). The spherical P0RGD NPs exhibited sizes of about 55 and 60 nm using dynamic laser light scattering (DLS) and transmission electron microscope (TEM), respectively (Figure 1d (ii)). As shown in Figure. S2, P0 has an intrinsic large extinction coefficient of 73.8 L g-1 cm-1 at 741 nm in the NIR window, and no obvious fluorescent signals, which indicates


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that the excitation energy of P0 would favorably dissipate through non-radiative decay to generate thermal energy.17 The photothermal effect of P0 NPs upon laser irradiation was subsequently investigated in vitro. The temperature changes of P0 NPs at different concentrations were real-time monitored under continuous laser illumination at the power density of 1.0 and 2.0 W cm-2, respectively. As represented in Figure S3a, the temperature of the sample P0 NPs (50 µg/mL) quickly increased from 25 °C to 50 °C (∆T ~ 25 °C) after laser irradiation (1.0 W cm-2) for 10 minutes, while under the same condition, the water temperature only slightly increased from 25 °C to 33 °C. Moreover, it is obvious to note that the temperature of 50 µg/mL CP NP suspension increased from 25 °C to 65 °C (∆T ~ 40 °C) after continuous 808 nm irradiation (2.0 W cm-2) for 10 min (Figure S5b and S5c). The temperature elevation depends on the NP concentration and laser source power. According to previous research, irreversible cellular damage was observed after 1 h of continuous heating at 46 °C, while to achieve the same therapeutic effect, only 4-6 min of heating is required at 50-52 °C.43 The photothermal results suggest that P0 NPs could efficiently generate hyperthermia effect to cause irreversible cancer cell damages. Furthermore, the photothermal conversion is 21 ACS Paragon Plus Environment


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calculated to be around 40 % (Figure S6), which is comparable to the reported organic and inorganic photothermal nanoparticles (Table S3). The photothermal stability of NPs was studied by cyclic photothermal treatment (Figure S5d). It is noticed that after 3 cycles of 808 nm laser illumination (2.0 W cm-2) for 5 min and subsequent cooling for10 min, the photo-thermal heating performance of P0 NPs remained robust (∆T from 40 °C to 37 °C). It is suggested that P0 NPs as efficient photothermal agents can be used for repeated photothermal treatment. The Cytotoxicity and Inflammatory Response Induced by PTT Treatment in vitro

To investigate the photothermal effect on 4T1 cells, the toxicity of P0 NPs and P0RGD NPs at different concentrations was measured after incubation with 4T1 cells for 24 h, respectively. As shown in Figure 2, two NPs demonstrated no obvious cytotoxicity even at the concentration up to 20 µg/mL, suggesting their comparable biocompatibility, which is highly desirable in photo-trigged cancer therapy.44 However, under an 808 nm laser irradiation, the cell viability treated with P0 NPs or P0RGD NPs decreased sharply in a dose-dependent manner after photothermal treatment. The cell viability in 10 and


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20 µg/mL P0RGD NPs group decreased by 46% and 69%, respectively, while the cell viability in 10 and 20 µg/mL P0 NPs group was reduced by 37% and 46%, respectively. Moreover, the photothermal toxicity of P0RGD NPs to 4T1 cells was much more effective than that of P0 NPs, which highlights that RGD could enhance the cellular uptake and benefit for the PTT treatment (Figure S7 and S8).

Figure 2. In vitro cell viability of 4T1 cells after incubation with different concentrations of P0 NPs and P0RGD NPs for 24 h with and without irradiation.

Macrophages are versatile and plastic effector cells of the immune system, which are generally defined as two extremes: classically activated M1 and alternatively activated M2 cells. TAMs commonly refer to an alternative M2 phenotype, exhibiting anti-



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inflammatory and pro-tumor effects.45-46 Pro-inflammatory immune response is beneficial to regulate tumor microenvironment. We thus examined whether PTT treatment assisted by CP NPs would trigger pro-inflammatory response in macrophage. Herein, a mouse macrophage cell line RAW264.7 monocyte was used, which has been well described as a cell line to study immune response. As shown in Figure 3, higher mRNA expression levels of IL-1β, IL-6 and CXCL-1 were observed upon PTT treatment with 0.1 µg/mL P0RGD NPs, as compared to control groups and P0 NPs, respectively. The immune activation effect of P0RGD NPs was found to be stronger than that P0 NPs. We also noticed that the mRNA expression levels of IL-1β, IL-6 and CXCL-1 in P0RGD group without laser treatment were higher than control, which may be due to the endocytosis of the nanoparticles. For example, Man-HA-MnO2 NPs (5 M MnO2, equal to 0.4 μg/mL MnO2) was reported to induce macrophage polarization.37 We further investigated the effect of 1 and 10 μg/mL P0RGD NPs on macrophage activation. As shown in Figure S9, similar mRNA expression levels of IL-1β, IL-6 and CXCL-1 were observed upon PTT treatment with 0.1 and 1 µg/mL P0RGD NPs. It is worth to note that upon treatment with 10 µg/mL P0RGD, the mRNA levels of examined genes showed no 24 ACS Paragon Plus Environment

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statistical differences when compared with control, which may be due to the cell cytotoxicity caused by photothermal effect of P0RGD NPs (Figure S10). Inflammatory response is the function of macrophage to respond to the stimuli and higher concentrations of foreign nanoparticles were reported to be able to cause cell cytotoxicity and induce immune tolerance.47-48

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Figure 3. The mRNA expressions of (a) IL-1β, (b) IL-6, (c) IL-12, (d) TNF-α, (e) CXCL-1 in RAW264.7 cells quantitatively tested by real-time PCR analysis after 24 h incubation with 0.1 µg/mL P0 NPs and P0RGD NPs with and without photothermal laser irradiation



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(808 nm, 2 W/cm2 for 10 min). All data are shown as means ± SEM (n = 6, *p < 0.05 and **p < 0.01 with control groups as comparison).

To further investigate the effect of CP NPs on macrophage polarization, the peritoneal macrophages were isolated. The results revealed that the mRNA levels of IL-1β, IL-6, IL-12, TNF-α were up-regulated in PTT treatment with P0RGD NPs, which were also confirmed by ELISA assay (Figure S11a andS11b).In addition, the mRNA levels of M1related biomarker iNOS was significantly increased and M2-related CD206 and Arg-1 were significantly decreased (Figure S7b).These results suggest that P0RGD NPs under laser irradiation can induce tumor cell death and pro-inflammatory immune response. PTT with CP NP Exerts Anti-Tumor Immunity and Therapeutic Efficacy

Recent studies have revealed that suppressive tumor microenvironment is correlated with compromised therapeutic efficiency and tumor metastasis 49-50 Based on the in vitro study, we subsequently studied whether the PTT treatment would have any effect in TAM polarization level in tumor tissue. P0 NPs or P0RGD NPs was intravenously


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injected into BALB/c mice-bearing subcutaneous 4T1 tumors. After that, the 808nm laser irradiation (2 W cm-2) was locally applied to the tumors for 20 min. The 4T1 tumors were collected at 48, 96 and 168 h of first PTT treatment to elucidate the changes of cytokine secretion by using ELISA. It is known that M1 macrophages secrete high amount of pro-inflammatory cytokines IL12, while M2 macrophages produce high amount of IL-10. As shown in Figure 4a, the secretion of IL-12 in the supernatant of tumor lysates significantly increased almost 2-fold for P0RGD NPs-injected mice plus laser irradiation at 48 h, meanwhile, there is no significant change in sub-group. We also noticed that there was no statistical difference in the secretion of IL-10 in 4T1 tumors at 48, 96 and 168 h (Figure 4b). The elevated IL-12 secretion suggests that PTT treatment with P0RGD NPs is capable to induce M1 polarization within tumors, which would inhibit cancer metastasis.51



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P0 NPs

PBS 107

0.47 %

0.86 %

0.12 %

0.45 %

2.94 %

CD3-APC

106 105 104 0

0.02 %

0.58 %

104

105

106

107

0

104

105

106

107

104

0

105

106

107

0

104

105

106

107

1.40 %

0

104

105

107

106

CD8-PE-Cy7

d 107

2.59 %

2.14 %

2.40 %

2.86 %

4.86 %

106

CD3-APC

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

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105 104 0

0.34 %

0.05 %

0

104

105

106

107

0

104

105

106

107

0.88 %

2.85 %

0

104

105

106

107

0

104

105

106

107

2.11 %

0

104

105

106

107

CD4-FITC

Figure 4. The protein levels of IL-12p70 (a) and IL-10 (b) in the supernatant of tumors at 48, 72 and 168 h after the first PTT treatment analyzed by ELISA; (c) Flow cytometry results of cytotoxic T lymphocytes (CTL) infiltration in tumor tissues at 48 h after first PTT treatment. CD3+CD8+ cells were defined as CTLs; (d) Flow cytometry results of helper T cells in tumor tissues. CD3+CD4+ cells represent the helper T cells.


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The number of cytotoxic T lymphocytes (CTL) within the tumor micro-environmental is an essential prognostic factor for tumor cell killing. Thus, the populations of different groups of T cells in tumors at 48 h of first PTT treatment were investigated by flow cytometry. As shown in Figure 4c, neither P0 NPs or P0RGD NPs injection alone could promote CD8+ CTL infiltration into the tumors. In contrast, there are more CTL infiltrated in tumor after P0RGD NPs assisted photothermal treatment. T Meanwhile, it was also found that CD4+ ratios were greatly enhanced in tumors of mice with post-injection of P0RGD and PTT treatment (Figure 4d and Figure S12). Altogether, these results indicate that single round of PTT with P0RGD NPs is able to up-regulate the percentage of infiltrating T cells (CD4+ or CD8+) at tumor site.



b

a

Con P0 NPs P0RGD NPs P0 NPs + laser P0RGD NPs + laser

PBS

CD69-APC

c

106

2.41 %

1.88 %

6.66 %

5.15 %

P0RGD NPs + laser

P0 NPs + laser

P0RGD NPs

P0 NPs

0.32 %

1.33 %

1.00 %

104

102

100

102

104

106

100

102

104

106

6.30 %

9.47 % 100

102

104

106

100

102

104

106

11.1 % 100

102

104

106

CD8-PE-Cy7

d

0.72%

1.43 %

106

CD69-APC

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

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1.25 %

1.07%

1.02%

104

102 13.3 % 100

102

104

106

10.3 11.6% % 100

102

104

106

29.6 7.15% % 100

102

104

106

26.8 %

32.7 13.8% % 100

102

104

106

100

102

104

106

CD4-FITC

Figure 5.The cytokine levels of tumor necrosis factor α (TNF-α) (a) and interferon-γ (IFN-γ) (b) in serum from mice at 48, 72 and 168 h after first PTT treatment were determined by ELISA; Representative scatter plots of (c) activated CD8+ T cells (CD8+CD69+) and (d) activated CD4+ T cells (CD4+CD69+)measured by flow cytometry at 48 h after the first PTT treatment. Data are shown as means ± SEM (n = 3,


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*p < 0.05, **p < 0.01 and ***p < 0.001 with control groups as comparison). Con presents control groups.

TNF-α is a powerful pro-inflammatory cytokine and IFN-γ is an essential effector molecule to mediate T cell antitumor effect.45 We further evaluated the secretion of TNF-α and IFN-γ in serum at 48, 96 and 168 h after first PTT treatment. As shown in Figure 5a and Figure 5b, significantly increased expressions of TNF-α and IFN-γ are observed upon treatment with P0RGD NPs with laser irradiation. From these results, the highest level of IFN-γ secretion was found to be at 48 h post-treatment of PTT. While at 168 h, the secretion of IFN-γ upon PTT treatment with P0RGD NPs restored to normal level. These results suggest that CP assisted PTT treatment could induce secretion of TNF-α and IFN-γ in serum, which are helpful to effectively enhance antitumor immunity. It is well known that spleen is an important immune organ outside of the tumor microenvironment. The populations of T cells in spleen were investigated to study the level of systemic immune response. To understand the impact of PTT with P0RGD on



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anti-tumor T-cell immunity in spleen, the activated CD8+CD69+ T cells and CD4+CD69+ T cells effector cells in spleen were checked by flow cytometry after different treatments. The populations of CD8+CD69+ and CD4+CD69+ T cells in spleen were not greatly up-regulated after PTT treatment with P0RGD NPs, as compared to the control, which indicates that CP assisted PTT treatments play a less important role in anti-tumor immunity in spleen (Figure 5c, 5d and Figure S13).


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Figure 6. In vivo photothermal therapy of the following groups: PBS, P0 NPs, P0RGD NPs, P0 NPs+ laser and P0RGD NPs + laser. The tumor growth curves(a) and body weight curves (b) after different treatments. Tumor weight(c) and weight of spleen and liver (d) of sacrificed mice from the above mentioned five groups on day 19. (e)



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Representative photos of 4T1 tumors from mice collected on day 19 in different group. (f) Fluorescence characterization of cell apoptosis in tumors after 19-day post-treatment using deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) to stain tumor sections. Scale bar: 50 µm. Data were shown as means ± SEM (n = 5, *p < 0.05, **p < 0.01 and ***p < 0.001 with control group as comparison).

Motivated by the excellent in vitro photothermal therapeutic efficacy and CP NPs caused CTL-mediated cellular immunity, we further evaluated their tumor therapeutic efficiency. As presented in Figure 6a, the treatment of P0 NPs and P0RGD totally failed to inhibit the tumor growth, indicating that CP NPs alone did not possess effective antitumor effect. As compared to the other groups, the “P0 NPs + laser” and “P0RGD + laser” groups exhibited significant antitumor efficacy. The final tumor weight and the photograph of tumor in different groups after different treatments also proved that P0RGD under irradiation could greatly inhibit 4T1 tumor growth (Figure 6c and 6e). At the same time, the mice weight was measured every two days during the 19-day treatment. As presented in Figure 6b, the mice in all groups with different treatments


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were shown no obvious body weight loss, indicating the low side toxic effect of all the treatments. To further investigate the cytokine secretion in tumor tissues after the last PTT treatment, the secretions of IL12, TNF-α and IL10 in tumors were also examined. None of these groups showed statistical difference (Figure S14), which suggests that repeated PTT treatments would cause immune resistance. The immune resistance may be a protection mechanism to avoid overly immune activation or imbalance of immune system for a long time, which is harmful to the organism.52 Spleen correlates to immunization and generally becomes abnormal under various pathological states like cancer. It is noticed that the spleen weight recovered to normal state in the “P0RGD + laser” group, while there was obvious spleen weight increasement in the control groups. This suggests that the P0RGD assisted photothermal immunotherapy is capable to achieve satisfactory therapeutic effect. To further evaluate the antitumor efficacy of our NPs, the mice after treatment in all groups were sacrificed to collect tumors, which were subsequently sliced for immunehistochemical analyses with TdT-mediated dUTP-biotin nick end labeling (TUNEL). The fluorescence TUNEL staining results as shown in Fig. 6f revealed that the treatment of 35 ACS Paragon Plus Environment


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“P0RGD + laser” group led to significantly larger necrotic areas in the tumors than in other groups and induced the most effective cell apoptosis. Collectively, these results indicate that P0RGD NPs are efficient photothermal agents for cancer therapy. Considering the safety of therapeutic powder (808 nm irradiated at 2.0 W cm-2 for 20 min), the photothermal temperature induced by 808 nm laser at tumor region and its adjacent normal tissue was monitored. The tumor temperature rapidly increased to 50.3°Cwithin 20 min, while the adjacent normal tissues exhibited a slight temperature change to 33.6°C after the same time of laser irradiation (Figure S15), which indicated the strong photothermal effect of P0RGD NPs on tumor tissues and less harm to adjacent normal tissue. To further study the biocompatibility of CP NPs in vivo, the major mice organs like livers and spleens in different treatment groups were collected, sectioned and studied by H&E staining. No significant tissue damage and inflammatory lesion is found in the major organs (Figure S16). To further investigate the potential toxicology of P0RGD NPs under laser irradiation, blood of untreated healthy mice and P0RGD treated mice under laser irradiation was collected. The assay of complete blood panel results reveals


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no statistical differences in these indicators between P0RGD NPs treated group and the control group (Figure S17). These results suggest good biocompatibility of P0RGD NPs. Conclusions

In summary, we demonstrate that uniform conjugated polymer nanoparticles have been successfully fabricated using a homemade microfluidic mixer. The obtained P0RGD NPs exhibited excellent photo-thermal conversion performance and triggered remarkable 4T1 tumor cell death in vitro under NIR light irradiation. More importantly, after first PTT treatment, P0RGD NPs could elicit anti-tumor immune response in tumor and finally exert effective therapeutic effect in vivo. To the best of our knowledge, it is the first time that the relationship between CP NPs assisted photothermal therapy and tumor microenvironment modulation has been studied in detail ACKNOWLEDGMENT

Z.W. and B.G. contributed equally to this work. The authors are grateful to National Natural Science Foundation of China (No. 51603186), the Scientific Innovation Program for University Students in Zhejiang Province (No. 2018R403073), the Singapore NRF



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Competitive Research Program (R279-000-483-281), National University of Singapore (R279-000-482-133) and NRF Investigatorship (R279-000-444-281) for financial support.

Supporting Information. Supplementary methods and results are available free of charge via the Internet at http://pubs.acs.org. The primers used for RT-qPCR, the NMR and UV/vis spectra of P0, BCA assay, photothermal property of CP NPs, cellular uptake, RAW264.7 cell viability assay, RT-qPCR analysis, the cytokines secretion, H&E staining, hematology and blood biochemical assay.

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SYNOPSIS TOC


Microfluidics-Prepared Uniform Conjugated Polymer Nanoparticles for

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