ROS-Inducing Micelles Sensitize Tumor-Associated Macrophages to

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ROS-Inducing Micelles Sensitize Tumor-Associated Macrophages to TLR3 Stimulation for Potent Immunotherapy Lanlan Liu, Huamei He, Ruijing Liang, Huqiang Yi, Xiaoqing Meng, Zhikuan Chen, Hong Pan, Yifan Ma, and Lintao Cai Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00239 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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ROS-Inducing Micelles Sensitize Tumor-Associated Macrophages to TLR3 Stimulation for Potent Immunotherapy Lanlan Liu†,1,2, Huamei He†,1, Ruijing Liang†,1, Huqiang Yi1,3, Xiaoqing Meng1,2, Zhikuan Chen1,2, Hong Pan1,2, Yifan Ma1,4,* and Lintao Cai1,* 1

Guangdong Key Laboratory of Nanomedicine, CAS Key Lab for Health Informatics, Institute of

Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences, Shenzhen 518055, PR China 2

University of Chinese Academy of Sciences, Beijing 100049, PR China

Present Addresses 3

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

Shenzhen 518055, PR China 4

HRYZ BIOTECH CO, Shanghai 200122, PR China

*Corresponding authors e-mail: [email protected], [email protected] †

These authors contributed equally to this work.

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ABSTRACT: One approach to cancer immunotherapy is the repolarization of immunosuppressive tumor-associated macrophages (TAMs) to anti-tumor M1 macrophages. The present study developed galactose-functionalized

zinc

protoporphyrin

IX

(ZnPP)

grafted

poly(L-lysine)-b-poly(ethyleneglycol) polypeptide micelles (ZnPP PM ) for TAM-targeted immunopotentiator delivery, which aimed at in vivo repolarization of TAMs to anti-tumor M1 macrophages. The outcomes revealed that ROS-inducing ZnPP PM demonstrated specificity for the in vitro and in vivo targeting of macrophages, elevated the level of ROS, and lowered STAT3 expression in BM-TAMs. Poly I:C (PIC, a TLR3 agonist)-loaded ZnPP PM (ZnPP PM/PIC) efficiently repolarized TAMs to M1 macrophages, which were reliant on ROS generation. Further, ZnPP PM/PIC substantially elevated the activated NK cells and T lymphocytes in B16-F10 melanoma tumors, which caused vigorous tumor regression. Therefore, the TAM-targeted transport of an immunologic adjuvant with ZnPP-grafted nanovectors may be a potential strategy to repolarize TAMs to M1 macrophages in situ for effective cancer immunotherapy. Keywords: tumor-associated macrophages; zinc protoporphyrin IX; immunologic adjuvant; targeted delivery; cancer immunotherapy


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■ INTRODUCTION Tumor-associated macrophages (TAMs) are known as alternatively activated M2 macrophages whose functions are severely impaired in the presentation of tumor-associated antigens, stimulation of antitumor responses of natural killer (NK) cells and T cells, and the lysis of tumor cells.1-4 In contrast to M1 macrophages, which are strong eliminators of pathogens and tumor cells, TAMs are immunosuppressive and control tumor advancement,5,

6

and as a result, they are deemed as a

potential target for tumor therapy. Current continuous experimental and pre-clinical TAM-targeted evaluations have made some encouraging advances; however, a primary issue for creating clinical TAM-targeted therapy is having the best mode of therapeutic administration at the precise area.7 Therefore, it is particularly desirable to plan a rational administration procedure and choose optimized therapeutics to reprogram TAMs (with pro-tumor function) into M1 macrophages (with anti-tumor function) to control the tumor microenvironment and increase the effectiveness of immunotherapy. Toll-like receptor (TLR) agonists, which are possible therapeutic reagents for cancer immunotherapy, are a group of molecules that can stimulate different immune cells and vigorously improve innate and adaptive immune responses. TLR agonists, in particular Poly I:C (PIC, a TLR3 agonist), can encourage the repolarization of TAMs to M1 macrophages.8, 9 Nevertheless, it is a challenge to target and transport PIC into TAMs. Thus, it is particularly desirable to create nanotechnology-based systems for TAMs-targeted delivery of PIC to repolarize TAMs and enhance anticancer immune responses. Reactive oxygen species (ROS), which are the byproducts of intracellular oxidative phosphorylation, have a pivotal part in controlling signal transduction pathways and gene expression,



and they are involved in innate and adaptive immune reactions.10-14 ROS is also an important modulator in macrophage polarization and activation.15 Prior evaluations have revealed that the decrease of ROS inhibited repolarization of TAMs to M1 macrophage.16-19 Zinc protoporphyrin IX (ZnPP) is well documented as an inhibitor of heme oxygenase-1 (HO-1), it has been shown to specifically increase the ROS in some cancer cells such as hepatoma and colon cancer cells.20, 21 In the present study, we generated ROS-inducing nanovectors in which ZnPP was grafted to galactose-functionalized poly(ethyleneglycol)-b-poly(L-lysine) (PLL) block copolymers, through an amide bond to obtain cationic ZnPP polypeptide micelles (ZnPP PM). ZnPP in the core of the polypeptide micelles could lead to the increased level of ROS. ZnPP PM encapsulated negatively charged PIC with positively charged PLL blocks through electrostatic adsorption to form ZnPP PM/PIC nanocomplexes. As TAMs express elevated levels of macrophage galactose-specific C-type lectin (MGL),22 the grafted galactose groups could modulate the identification and uptake of nanocomplexes by TAMs. The results showed that ZnPP PM/PIC successfully targeted into macrophages, thereby increasing the level of ROS and decreasing STAT3 expression in BM-TAMs. More importantly, TAM-targeted delivery of PIC with ZnPP PM synergistically repolarized TAMs into M1 macrophages, and evoked strong anti-tumor immune reactions, thus causing substantial tumor suppression.


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■EXPERIMENTAL SECTION Materials and general instruments. NH2-PEG-COOH, dry N,N′-Dimethylformamide (DMF), hydrogen bromide 33 wt % solution in glacial acetic acid, protoporphyrin IX zinc (II) (ZnPP) and D-(+)-galactose were purchased from Sigma-Aldrich (USA). ε-benzyloxycarbonyl-L-lysine (Lys(Z)) was obtained from GL Biochem (China). Lactobionic acid was purchased from J&K Scientific Ltd (China). All of the other solvents were of analytical grade and utilized without additional purification. The Dulbecco's modified Eagle's medium was provided by Corning Company. Hoechst 33358 was obtained from Invitrogen (USA). Anti-mouse monoclonal antibodies labeled with fluorochromes (CD11b, CD86, CD8a, CD69, CD4, and NK 1.1) came from eBioscience (USA). A One-Step TUNEL Apoptosis Assay Kit was bought from Beyotime (China). C57BL/6J mice (6 weeks of age) were acquired from Guangdong Province Laboratory Animal Center (China) and kept in the institutional facility for animal care under specific pathogen-free (SPF) conditions. The evaluation procedure was analyzed and authorized by the Institutional Animal Care and Use Committee of Shenzhen Institutes of Advanced Technology. The structures of the copolymers were analyzed by 1H NMR spectra and FT-IR spectra. 1H NMR spectra were obtained with a Bruker 400-MHz nuclear magnetic resonance device with CF3COOD as a solvent. FT-IR spectra were documented by Bruker Vertex 70 spectrophotometer with the KBr pellet technique. To quantify the hydrodynamic size and zeta potential of PIC complexes in water, a Nano-ZS ZEN3600 (Malvern Instruments) was used at 25°C. The morphologies of micelles were noted by TEM with a FEI Tecnai G2 F20 S-Twin microscope. UV-vis absorption spectra were quantified via UV/vis spectrometry (Lambda25, Perkin−Elmer, USA). Synthesis and characterization of ZnPP PM and PIC delivery. Firstly, T-PLL was synthesized via



ring-opening polymerization of Lys(Z) with triethylamine as initiators accompanied by the deprotection of Z groups using HBr/HAc solution.23 To prepare galactose-functionalized PEG (Gal-PEG-COOH), lactobionic acid (0.14 g) was mixed with excess EDC and NHS in anhydrous DMSO at 25 °C for 12 h in dark, and then reacted with NH2-PEG-COOH (0.8 g) for 24 h. The PLL-PEG-Gal block copolymers were synthesized by further condensation reaction of Gal-PEG-COOH (0.3 g) and T-PLL (0.14 g) with EDC and NHS solution. Finally, ZnPP-PLL-PEG-Gal block copolymers (ZnPP PM) were obtained by the condensation of PLL-PEG-Gal (0.53 g) with ZnPP (9 mg). The final products were purified by dialysis and collected by freeze-dry. ZnPP PM were dissolved in H2O and then subtly combined with PIC solution for 5 min to gather PIC-loaded polypeptide micelle (ZnPP PM/PIC) nanocomplexes at different w/w ratios. After that, nanocomplexes were placed on 1% agarose gel in Tris-acetate-EDTA (TAE) buffer with GelRed (Biotium, USA.) for electrophoresis at 100 V for 20 min. PIC was viewed with a Dolphin-Doc Molecular Imaging System (Wealtec, USA). In vitro uptake of ZnPP PM by macrophages and tumor cells. The macrophages (RAW264.7 cells) and melanoma cells (B16-F10) were separately cultured in Dulbecco's modified Eagle's medium (DMEM) and RMPI 1640 (Corning) augmented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Hyclone) at 37°C under 5% CO2. The cells used in the cellular uptake were seeded in 24-well plates or 8-well chamber cover slip at a density of 1 × 105 and 1 × 104 cells/well respectively. After incubating for 12 h, the cells were treated with ZnPP or ZnPP PM and incubated for another 1 h. Then Hoechst 33358 was used to stain the nuclei for 5 min and fluorescent images were gathered via a confocal laser scanning microscope (TCS SP5II, Leica, Ernst-Leitz-Strasse,


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Germany). The uptake of ZnPP or ZnPP PM was determined by flow cytometry to quantify the proportion of ZnPP positive cells after incubating for 1 h. Culture and stimulation of mouse bone marrow-derived TAMs. Following existing methods24, mouse bone marrow cells were cultured in DMEM supplemented with 10% FBS and 20% L929 cell suspension in 10 mL medium with or without 20% B16-F10 cell suspension at 37 °C under 5% CO2. Cells replaced new fresh medium after culturing for 5 days and gained mouse bone marrow-derived macrophages (BMMs) and mouse bone marrow-derived TAMs (BM-TAMs) after culturing for 7 days. On the sixth day, BMMs were cultured with LPS (100 ng/mL) for 24 h to obtain M1 type macrophages. On the seventh day, cells were seeded in 24-well plates or 12-well plates at a density of 1.5 × 105 and 3 × 105 cells/well respectively. Following incubating for 12 h, the cells were treated with ZnPP PM, PIC, ZnPP PM/PIC, N-acetyl-L-cysteine (NAC), NAC+ ZnPP PM/PIC, N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) or PM for another 24 h or 48 h. For ROS measurement, cells were rinsed with PBS and then incubated with DCFH-DA in serum-free DMEM medium for 30 min, and intracellular fluorescence was established with flow cytometry. The concentration of cytokines (IL-12p70 and TNF-α) in medium suspension was established by ELISA kits (BioPMend, San Diego, CA, UAS) based on typical procedures. Quantitative real time PCR (QRT-PCR). For analyzing the mRNA expression of iNOS, Msr2 and Arg1 in BM-TAMs, total RNA was removed with Trizol reagent (Life Technologies, USA), and cDNA was synthesized from RNA with a TOYOBO inverse transcription kit based on the company’s instructions. Real-time PCR was established with SYBR green qPCR mix (TOYOBO) on a light cycler 480II (Roche, USA). Primer sequences are listed in Supporting Information Table S1. Western Blotting Analysis. Cells were cultured under various circumstance and collected, washed



three times with PBS. Then cells were lysed with RIPA lysate buffer comprised of a cocktail of protease inhibitors. The concentrations of total protein were quantified by a BCA protein assay kit (Beyotime, China). Identical amounts of lysate were divided by SDS-PAGE and then moved onto PVDF membranes. Immunoblotting was performed with anti-mouse P-STAT3 and anti-mouse STAT3 primary antibodies accompanied by horseradish peroxidase-labeled goat anti-rabbit or anti-mouse secondary antibody. Finally, the PVDF membranes were hatched in ECL working solution before imaging with enhanced chemiluminescent system (Pierce). Animal Experiments. C57BL/6J mice (6 weeks of age) were subcutaneously injected with 1 × 106 B16-F10 cells suspension on the right buttock. Mouse were divided into four groups randomly and every group was intratumorally inoculated with PBS, ZnPP PM (100 µg/mouse), PIC (10 µg/mouse), ZnPP PM/PIC (ZnPP PM: PIC=100 µg: 10 µg in one mouse). A total of four dosages for tumor model from day 8 to day 14. In order to monitor the treatment effects, the tumor volume was measured with a digital caliper every other day. Tumor volume was determined as follows: tumor volume = (length × width2)/2. For humane reasons, animals were killed when the tumor volume was above 2000 mm3. At two days following the final treatment, tumors and tumor-draining lymph nodes (TDLN) were collected and digested to acquire a single-cell suspension.25 To evaluate active T cells and NK cells, cell suspensions were stained with a combination of anti-mouse-CD4-FITC, anti-mouse-CD69-PE, anti-mouse-CD8a-APC, and anti-mouse-NK1.1-PE-Cy5, and flow cytometry was used to quantify the populations of immune cells in the cell suspensions. Tumors were excised two days following the final treatment and snap frozen for cryosection. Cell apoptosis in tumor sections was established by a TUNEL assay and stained with TUNEL Apoptosis Assay Kit according to manufacturer's instruction. To investigate TAMs or


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tumor-infiltrating CD8+ T cells, tumor sections were labeled with anti-mouse CD11b-FITC or anti-mouse CD8a (53-6.7), eFluor 660 antibodies at room temperature for 2 h. The fluorescent images of tumor sections were documented via confocal laser scanning microscopy. Biosafety evaluation. Blood samples were gathered at 72 h following the final treatment, and liver function was assessed by quantifying the serum level of AST/ALT with AST and ALT activity assay kit. Renal function was assessed by quantifying the BUN/CRE with colorimetry based on the company’s protocol. The heart, liver, spleen, lung, and kidney were gathered for hematoxylin and eosin (H&E) staining, and an Olympus microscope was used to evaluate the histology of the various organs. Statistical analysis. Data were evaluated with Graphpad Prism software and presented as the means ± standard deviations (SD). One-way ANOVA or Student's t test was utilized to evaluate the differences between the groups. P values of < 0.05 were considered to be statistically significant.



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■RESULTS AND DISCUSSION Preparation

and

Characterization

of

ZnPP

PM

block

copolymers.

To

prepare

macrophage-targeted PLL-PEG-Gal block copolymers, EDC/NHS-activated lactobionic acid was conjugated with NH2-PEG-COOH to obtain Gal-PEG-COOH; this compound contained galactose groups for macrophage-specific targeting through the recognition of TAMs’ galactose receptors. The PLL-PEG-Gal block copolymers were synthesized by condensation reaction of Gal-PEG-COOH and T-PLL. In addition, the ROS-inducing ZnPP-PLL-PEG-Gal nanovectors were synthesized by further condensation reaction of PLL-PEG-Gal and ZnPP (Scheme 1). To characterize the structures of copolymers, 1H NMR and FT-IR analysis were employed. The 1

H NMR spectra of PLL-PEG-Gal (Figure 1A) and ZnPP-PLL-PEG-Gal (Figure 1B) revealed a

characteristic shifts of PLL (δ1.3−1.6, δ4.6−4.8), PEG (δ3.6−3.8) and Gal (δ3.3, 4.6) in the block copolymers. The inset in the lower left of Figure 1B showed the peaks around δ6.1-6.3, which were attributable to the tethered ZnPP on ZnPP-PLL-PEG-Gal. By calculating the peak area ratios of each molecule’s characteristic peaks using 1H NMR spectra,23, 24, 26, 27 the polymerization degrees of PLL and Gal were 28 and 3 respectively. Furthermore, four ZnPP were grafted to each copolymer according to their characteristic peaks of PEG (δ3. 6−3.8) and ZnPP (δ6.1−6.3) (Figure 1B). The copolymer structures were further characterized by FT-IR (Figure 1C,D). After tethered with ZnPP, the peak at 3432 cm-1 nearby enhanced significantly, which was attributed to the O-H stretching vibration of -COOH in ZnPP. The tethering also increased the peak at 1650 cm-1 which corresponds to the in-plane bending vibration of N-H. The peak at 1105 cm-1 was attributed to the C-N stretching vibration of primary amine in PLL-PEG-Gal, which became weaker after tethered with ZnPP in Figure 1D. All these data suggest the successful synthesis of the target products.


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Preparation and Characterization of ZnPP PM/PIC Nanocomplexes. ZnPP PM block copolymers were dissolved in H2O and concurrently produced self-assembled ZnPP PM nanoparticles (Figure 2A). The TEM images revealed a homogeneous dispersion of spherical nanoparticles with a mean diameter of 15 ± 5 nm (Figure 2B). To encase PIC, cationic ZnPP PM nanoparticles were combined with PIC at various w/w ratios to produce the ZnPP PM/PIC nanocomplexes (Figure 2A). Next, the PIC condensation capability of ZnPP PM nanoparticles were assessed, as this is pivotal for nanoparticle-based immunologic adjuvant transport. The outcomes revealed that ZnPP PM fully averted PIC migration at the w/w ratio of four or greater (Figure 2C), suggesting it was effective to encapsulate PIC in ZnPP PM nanoparticles. Upon association with PIC, the ZnPP PM/PIC nanocomplexes (w/w = 10) was observed to have a hydrodynamic size of 30 ± 2.47 nm and a zeta potential of +28 mV (Figure 2D). The hydrodynamic size of ZnPP PM nanoparticles was greater than that of the TEM assessment because of their hydrophilicity and altered secondary structure in H2O. It is noteworthy that the size of ZnPP PM/PIC nanocomplexes did not significantly change compared with ZnPP PM nanoparticles (Figure 2D,E). However, the zeta potential of ZnPP PM nanocomplexes decreased from +28 mV to +16.6 mV after encapsulating with PIC. This zeta potential decrease could be due to the shielding effect of the PIC (Figure 2D). Meanwhile, as shown in UV-vis spectra, ZnPP PM/PIC exhibited the characteristic absorption peaks of ZnPP (426 nm) and PIC (256 nm) (Figure 2F). The above results suggest the successful preparation of ZnPP PM/PIC nanocomplexes. A flawless transport system should produce a minimal toxic impact. Here, the cytotoxicity of ZnPP PM nanoparticles was assessed on RAW 264.7 cells with a CCK8 assay. The outcome revealed that 5-40 µg/mL of the nanovectors did not lower the cell viability. While 60–80 µg/mL of



ZnPP PM moderately lowered the cell viability, the total cell viability was still above 70% at after 24 h of treatment (Figure 2G). It can therefore be inferred that cationic ZnPP PM nanoparticles demonstrated great biocompatibility for macrophages. We also investigated the stability of ZnPP PM nanoparticles in different media (water, PBS, and RMPI 1640 + 10% FBS). The outcome showed that the size of ZnPP PM did not significantly change during a 6-day storage at 4 °C in either of the medium (Supporting Information, Figure S1), suggesting a great stability of ZnPP PM nanoparticles. Cellular uptake of ZnPP PM nanovectors. We first assessed the cellular uptake of the nanovectors by RAW264.7 cells. These cells were incubated with free ZnPP or ZnPP PM. The internalization of free ZnPP by the RAW264.7 cells was barely detectable, indicating poor cellular uptake of free ZnPP. In contrast, ZnPP PM dramatically increased the fluorescence signals of ZnPP in the RAW264.7 cells (Figure 3A). PM-facilitated macrophage ZnPP uptake could be due to their positively charged surface and amphiphilic structure.28, 29In addition, the galactose groups on ZnPP PM were designed to target the C-type lectin receptors (e.g., MGL) found on macrophages.22 We further analyzed the cellular uptake of ZnPP PM in melanoma cells. A weak intracellular ZnPP signal was observed in the melanoma cells, and this signal was about 3-fold lower than that in RAW 264.7 cells (Figure 3B,C). In addition, the pretreatment of BMMs with D-galactose partially diminished the uptake of PM-encapsulated ZnPP (Supporting Information, Figure S2), indicating that PM-facilitated ZnPP uptake involves the recognition of surface galactose moieties by macrophages. These data suggest that the ZnPP PM exhibited specific targeting ability to macrophages. ZnPP PM/PIC repolarized TAMs into antitumor M1 macrophages in vitro. ROS is a by-product of mitochondria respiratory chain, and play a role as an important secondary messenger in both


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innate and adaptive immunity.11, 30 It may also play a complicated role in regulating macrophage polarization.15 In this study, BM-TAMs were produced for use as an in vitro model of TAMs.24 Herein, we observed that the level of ROS in LPS-induced M1 macrophages dramatically increased in comparison to unpolarized macrophages (BMMs) and BM-TAMs (Supporting Information, Figure S3). These results indicate the up-regulation of ROS in M1 macrophages could be a mechanism crucial to the promotion of macrophage polarization to immune-activated M1 phenotype. ZnPP PM administration increased the expression of ROS in BM-TAMs by about 2-fold, but not PM (Figure 4A). To further investigate whether ZnPP was involved in ROS generation, BM-TAMs were pretreated with the intracellular zinc chelator TPEN. As shown in Figure 4A, TPEN efficiently inhibited ZnPP PM-induced ROS production. Hence, ZnPP is an important component responsible for the ROS induction by ZnPP PM in BM-TAMs. BM-TAMs were treated with ZnPP PM with/without PIC for 48 h, the mRNA levels of M1 marker (iNOS) and M2 markers (Msr2 and Arg1) were examined with QRT-PCR. The outcomes revealed that PIC independently failed to impact M1 or M2 gene expression (Figure 4B-D), suggesting low responsiveness of BM-TAMs to TLR3 activation. However, the treatment of ZnPP PM/PIC significantly raised M1 macrophage marker (iNOS) 3-fold and suppressed M2 macrophage markers (Msr2 and Arg1) by 40%–70%, while ZnPP PM by itself did not impact M1 or M2 gene expression (Figure 4B-D). The impact of ZnPP PM/PIC on BM-TAM polarization was additionally assessed by cytokine production. It was noted that ZnPP PM had no significant impact on IL-12 and TNF-α production in BM-TAMs. Nevertheless, incubation with ZnPP PM/PIC significantly elevated the production of IL-12 and TNF-α by 4-fold and 2-fold, respectively (Figure 4E,F). These results indicate the synergistic effect of ZnPP PM and PIC on macrophage repolarization, which could be



attributed to TAM-targeted PIC delivery by ZnPP PM. It is well known that NAC blocks ROS generation, we investigated whether NAC affected ZnPP PM/PIC-induced macrophage polarization through elimination ROS. The pretreatment of BM-TAMs with NAC had no effect on either the expression of M1 or M2 genes or cytokine production in BM-TAMs. However, NAC weakened the repolarization to M1 macrophages by ZnPP PM/PIC (Figure 4B-F), suggesting the essential role of ROS induction by ZnPP PM on TLR3-induced macrophage repolarization. Hence, it could be proposed that ZnPP PM/PIC repolarized TAMs to M1 macrophages based on its ROS production. The signal transducer and activator of transcription 3 (STAT3) is a well-acknowledged possible negative regulator of inflammatory reactions31,

32

and it functions as a negative regulator for

macrophage activation.7 In our research, we noted that the protein levels of phosphorylated STAT3 (P-STAT3) in BM-TAMs were significantly elevated compared to that in BMMs and M1 macrophages (Figure 5A). These data suggest hyperactivated STAT3 signaling in BM-TAMs, which may be a primary mechanism encouraging TAM polarized to the immunosuppressive M2 phenotype. ZnPP PM administration significantly diminished P-STAT3 and STAT3 protein levels in BM-TAMs (Figure 5B). Meanwhile, free PIC also reduced P-STAT3 and STAT3 expression. But PIC encapsulation by ZnPP PM robustly reduced P-STAT3 and STAT3 expression as compared with PIC treatment (Figure 5C), which could be attributable to the ability of ZnPP PM in regulating gene expression by itself and facilitating the delivery of PIC into BM-TAMs. More interestingly, the inhibitory impact of ZnPP PM/PIC on the expression of STAT3 was significantly abolished by NAC in BM-TAMs (Figure 5D). These data suggest that the elevated ROS decrease STAT3 expression, which could be a key mechanism synergizing with PIC to promote polarization of TAM to the immunosuppressive M2 phenotype.


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Effect of ZnPP PM/PIC on repolarization of TAMs in vivo. TAM-targeting of ZnPP PM/PIC nanocomplexes in vivo was assessed in the B16-F10 melanoma tumor-bearing mice. Mice with tumors were i.t.-injected with ZnPP PM/PIC and then the immunofluorescent staining was performed on frozen tumor sections. The results showed that all most of ZnPP localized in the CD11b+ TAMs (Figure 6A), confirming TAMs was a primary target of ZnPP PM/PIC nanocomplexes but not tumor cells in vivo. The effect of ZnPP PM/PIC on TAM polarization was investigated after 4 dosages of treatment. As expected, flow cytometer analysis demonstrated that PIC moderately elevated M1 macrophage marker (CD86) in CD11b+ TAMs, whereas ZnPP PM/PIC increased CD86 expression more effectively than PIC did (Figure 6B). On the other hand, QRT-PCR assay observed that ZnPP PM/PIC significantly increased the M1 markers (IL-12 and iNOS) and decreased M2 marker (Msr2) (Figure 6C-E), showing that it was capable of TAM to M1 macrophage repolarization in vivo. Overall, ZnPP PM/PIC nanocomplexes not only exhibited specific targeting ability to TAMs but also repolarized TAMs into M1 macrophages in the tumor microenvironment. Anti-tumor activity of ZnPP PM/PIC in vivo. Since TAMs have primary parts in the tumor evolution process, the anti-tumor impact of ZnPP PM/PIC was assessed in B16-F10 tumor-bearing mice. The tumor-bearing mice were injected intratumorally with ZnPP PM/PIC nanocomplexes on day 8 following tumor implantation every two days, for a total of four dosages. While PIC injection slightly halted tumor advancement and prompted tumor cell apoptosis, ZnPP PM/PIC more efficiently halted tumor advancement (Figure 7A,B) and evoked substantial apoptosis in tumor tissues following the administration of four dosages (Figure 7C,D), suggesting its strong anti-tumor impact. The strong anti-tumor impact of ZnPP PM/PIC could be attributable to its ability to



repolarize TAMs to tumoricidal M1 macrophages. Besides having direct tumor cytotoxicity, active macrophages can present tumor antigens to effector cells to trigger anti-tumor adaptive immune responses.33 In the present study, the treatment with ZnPP PM/PIC significantly increased the percentage of the activated effector cells (CD8+CD69+, CD4+CD69+, or NK1.1+CD69+ cells) in tumor and TDLNs by 2-3 folds (Figure 7E,F), which indicated its ability to contribute to tumor regression through increasing activated effector cells. Moreover, it has been shown that activated TAMs release a diverse array of soluble cytotoxic factors that could subsequently activate and recruit different types of effector cells for tumor inhibition.3,

24

In addition to activating CD8+ T cells, CD8+ T cell recruitment into the tumor

microenvironment is another key factor that is directly correlated with cancer curability and prognosis. The results showed that a small amount of red fluorescence originated from CD8+ T cells appeared in the tumor section with PIC or ZnPP PM treatment. However, large amount of red fluorescence emerged after ZnPP PM/PIC treatment. Overall, ZnPP PM/PIC not only strongly induced the activation of tumor-specific CD8+ T cells but also encouraged their migration into the tumor environment, and evoked strong anti-tumor immune reactions, thus causing substantial tumor suppression (Supporting Information, Figure S4). Biosafety of ZnPP PM/PIC in vivo. Nanoparticle-based therapy is always accompanied by a major concern about its biosafety. In the current study, it was shown that serum BUN/CRE (renal function) and serum ALT/AST (liver function) did not differ significantly between the PBS and ZnPP PM/PIC groups (Supporting Information, Figure S5A,B), indicating no significant ZnPP PM/PIC renal or liver toxicity. Further studies to detect the potential toxicity of ZnPP PM/PIC were carried out by hematoxylin and eosin (H&E) staining of the following major organs: heart, liver, spleen, lung and


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kidney. The staining did not indicate any lesions, degenerations or histopathological abnormalities (Supporting Information, Figure S5C). Altogether, these results above suggested the intratumoral administration of ZnPP PM/PIC nanocomplexes were well tolerated and biocompatible.

■CONCLUSION The repolarization of TAMs to M1 macrophages is a potential strategy for the therapy of cancer; however, it is still a challenge to achieve efficient TAM repolarization. In the present study, we developed the ROS-inducing polypeptide nanovectors for TAM-targeted immunologic adjuvant delivery. The administration of ZnPP PM/PIC efficiently encouraged the repolarization of TAMs (with anti-tumor function) to M1 macrophages (with anti-tumor function) through modulating the expression of ROS and STAT3 in TAMs, and increased the activated NK cells and T lymphocytes in tumors and TDLNs, which then caused vigorous tumor regression (Figure 8). Thus, TAM-targeted transport of the immunologic adjuvant with ZnPP-grafted nanovectors could be a potential approach to repolarize TAMs and prompt tumor regression. However, intratumoral injection has certain limitations in clinic studies, thereby there is urgent need to verify other modes of injection in our future studies.

■ ASSOCIATED CONTENT Supporting Information: The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Primer lists (Supplementary Table S1), The stability of ZnPP PM (Supplementary Figure S1),The cellular uptake of ZnPP in BMMs (Supplementary Figure S2),The production of ROS in BMMs, M1 and BM-TAMs (Supplementary Figure S3), Immunofluorescence microscopy of CD8+ T cells in tumor tissues (Supplementary Figure S4), Biosafety of ZnPP PM/PIC in vivo (Supplementary Figure S5; PDF).



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Acknowledgements: Lanlan Liu, Huamei He and Ruijing Liang contributed equally to this work. This work was supported by the National Natural Science Foundation of China (81371679, 81701816, 81671758, 31571013, 51502333 and 81501580), Key International S&T Cooperation Project (2015DFH50230), Natural Science Foundation of Guangdong Province (2017A030313079 and

2017A030313726),

Guangdong

Natural

Science

Foundation

of

Research

Team

(2016A030312006), Shanghai Science and technology Program (16DZ1910900), Dongguan project on Social science and Technology Development (2015108101019), Shenzhen Science and Technology

Program

(JSGG20160331185422390

and

JCYJ20160429191503002),

China

Postdoctoral Science Foundation (2017M612776) and SIAT Innovation Program for Excellent Young Researchers (Y7G005).

Competing financial interests: The authors declare no competing financial interests.


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(18) Padgett, L.E.;Burg, A.R.;Lei, W.Q.;Tse, H.M. Loss of Nadph Oxidase-Derived Superoxide Skews Macrophage Phenotypes to Delay Type 1 Diabetes. Diabetes 2015,64,937-46. (19) Yi, L.;Liu, Q.;Orandle, M.S.;Sadiq-Ali, S.;Koontz, S.M.;Choi, U.;Torres-Velez, F.J.;Jackson, S.H. P47(Phox) Directs Murine Macrophage Cell Fate Decisions. Am J Pathol 2012,180,1049-58. (20) Fang, J.;Akaike, T.;Maeda, H. Antiapoptotic Role of Heme Oxygenase (HO) and the Potential of HO as a Target in Anticancer Treatment. Apoptosis 2004,9,27-35. (21) Tanaka, S.;Akaike, T.;Fang, J.;Beppu, T.;Ogawa, M.;Tamura, F.;Miyamoto, Y.;Maeda, H. Antiapoptotic Effect of Haem Oxygenase-I Induced by Nitric Oxide in Experimental Solid Tumour. Brit J Cancer 2003,88,902-9. (22) Huang, Z.;Zhang, Z.P.;Zha, Y.H.;Liu, J.L.;Jiang, Y.C.;Yang, Y.;Shao, J.;Sun, X.L.;Cai, X.;Yin, Y.;Chen, J.N.;Dong, L.;Zhang, J.F. The Effect of Targeted Delivery of Anti-TNF-Alpha Oligonucleotide into CD169(+) Macrophages on Disease Progression in Lupus-Prone MRL/Lpr Mice. Biomaterials 2012,33,7605-12. (23) Deng, J.Z.;Gao, N.N.;Wang, Y.A.;Yi, H.Q.;Fang, S.T.;Ma, Y.F.;Cai, L.T. Self-Assembled Cationic Micelles Based on PEG-PLL-PLLeu Hybrid Polypeptides as Highly Effective Gene Vectors. Biomacromolecules 2012,13,3795-804. (24) Liu, L.L.;Yi, H.Q.;He, H.M.;Pan, H.;Cai, L.T.;Ma, Y.F. Tumor Associated Macrophage-Targeted Microrna Delivery with Dual-Responsive Polypeptide Nanovectors for Anti-Cancer Therapy. Biomaterials 2017,134,166-79. (25) Liu, L.L.;Yi, H.Q.;Wang, C.;He, H.M.;Li, P.;Pan, H.;Sheng, N.;Ji, M.Y.;Cai, L.T.;Ma, Y.F. Integrated Nanovaccine with MicroRNA-148a Inhibition Reprograms Tumor-Associated Dendritic Cells by Modulating MiR-148a/DNMT1/SOCS1 Axis. J Immunol 2016,197,1231-41. (26) Luo, Z.C.;Wang, C.;Yi, H.Q.;Li, P.;Pan, H.;Liu, L.L.;Cai, L.T.;Ma, Y.F. Nanovaccine Loaded with Poly I:C and STAT3 SiRNA Robustly Elicits Anti-Tumor Immune Responses through Modulating Tumor-Associated Dendritic Cells in Vivo. Biomaterials 2015,38,50-60. (27) Yi, H.Q.;Liu, L.L.;Sheng, N.;Li, P.;Pan, H.;Cai, L.T.;Ma, Y.F. Synergistic Therapy of Doxorubicin and MiR-129-5p with Self-Cross-Linked Bioreducible Polypeptide Nanoparticles Reverses Multidrug Resistance in Cancer Cells. Biomacromolecules 2016,17,1737-47. (28) He, C.B.;Hu, Y.P.;Yin, L.C.;Tang, C.;Yin, C.H. Effects of Particle Size and Surface Charge on Cellular Uptake and Biodistribution of Polymeric Nanoparticles. Biomaterials 2010,31,3657-66. (29) Chevre, R.;Le Bihan, O.;Beilvert, F.;Chatin, B.;Barteau, B.;Mevel, M.;Lambert, O.;Pitard, B. Amphiphilic Block Copolymers Enhance the Cellular Uptake of DNA Molecules through a Facilitated Plasma Membrane Transport. Nucleic Acids Res 2011,39,1610-22. (30) Wang, C.;Li, P.;Liu, L.L.;Pan, H.;Li, H.C.;Cai, L.T.;Ma, Y.F. Self-Adjuvanted Nanovaccine for Cancer Immunotherapy: Role of Lysosomal Rupture-Induced Ros in MHC Class I Antigen Presentation. Biomaterials 2016,79,88-100. (31) Cheng, F.D.;Wang, H.W.;Cuenca, A.;Huang, M.;Ghansah, T.;Brayer, J.;Kerr, W.G.;Takeda, K.;Akira, S.;Schoenberger, S.P.;Yu, H.;Jove, R.;Sotomayor, E.M. A Critical Role for STAT3 Signaling in Immune Tolerance. Immunity 2003,19,425-36. (32) Yu, H.;Kortylewski, M.;Pardoll, D. Crosstalk between Cancer and Immune Cells: Role of STAT3 in the Tumour Microenvironment. Nat Rev Immunol 2007,7,41-51. (33) Lewis, C.E.;Pollard, J.W. Distinct Role of Macrophages in Different Tumor Microenvironments. Cancer Res 2006,66,605-12.


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Figures and figure legends:

Scheme 1. Synthetic route for (1) PLL-PEG-Gal and (2) ZnPP-PLL-PEG-Gal copolymers.



Figure 1. Characterization of copolymers. (A, B) 1H NMR spectra of PLL-PEG-Gal copolymers (A) and ZnPP-PLL-PEG-Gal copolymers (B). Insets in the upper are the chemical structural formulas of copolymer, and insets in the lower left are the magnified 1H NMR spectra of corresponding polymers. (C, D) FT-IR spectrum of PLL-PEG-Gal (C) and ZnPP-PLL-PEG-Gal copolymers (D).


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Figure 2. Preparation and characterization of PIC-loaded ZnPP PM nanoparticles. (A) Scheme of ZnPP PM and PIC-loaded ZnPP PM (ZnPP PM/PIC) nanoparticle preparation. (B, E) Transmission electron microscopy (TEM) images of ZnPP PM (B) and ZnPP PM/PIC (E) nanoparticles. The scale bars shown are 100 nm. (C) PIC binding ability of ZnPP PM/PIC by the agarose gel retardation assay. (D) Size and zeta potential of ZnPP-PM and ZnPP PM/PIC nanoparticles. (F) UV-vis spectra of PIC, ZnPP-PM and ZnPP PM/PIC. (G) Cytotoxicity of ZnPP-PM nanoparticles in RAW264.7 cells using CCK8 assay.



Figure 3. Cellular uptake of ZnPP PM nanovectors. (A, B) Fluorescence microscopy of macrophages (RAW 264.7) or melanoma cells (B16-F10) after incubation for 1 h with ZnPP or ZnPP PM. The scale bars shown are 25 µm. (C) Cellular uptake of ZnPP in macrophages (RAW 264.7) or melanoma cells (B16-F10) was determined by flow cytometry after cells encapsulated with ZnPP or ZnPP PM for 1 h.


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Figure 4. ZnPP-PM/PIC nanocomplexes repolarize TAMs to M1 macrophages depending on ROS production. (A) BM-TAMs were treated with ZnPP-PM, PM and TPEN either alone or in combination. Intracellular ROS production in BM-TAMs at 24 h using flow cytometry. (B-D) BM-TAMs were treated with ZnPP-PM, PIC, ZnPP PM/PIC, NAC with or without ZnPP PM/PIC for 48 h. The mRNA levels of iNOS, Msr2, Arg1 gene were determined by QRT-PCR. (E-F) The concentrations of IL-12 (E) and TNF-a (F) in culture supernatants were analyzed by ELISA. The asterisks indicate differences between PBS and other treatments are statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. #Differences between two groups are statistically significant, p < 0.05.



Figure 5. Effect of ZnPP PM/PIC on the expression of STAT3. (A) The protein levels of STAT3 and P-STAT3 in M1, BMMs and BM-TAMs were determined by Western blotting. (B-C) BM-TAMs were treated with PBS, ZnPP-PM, PIC or ZnPP PM/PIC for 48 h, the protein levels of STAT3 and P-STAT3 were determined by Western blotting (B). The mRNA level of STAT3 gene was measured by QRT-PCR (C). The asterisks indicate differences between PBS and other treatments are statistically significant. *p < 0.05, **p < 0.01. #Differences between two groups are statistically significant, p < 0.05.


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Figure 6. In vivo TAM repolarization by ZnPP PM/PIC. Tumor-bearing mice were i.t. injected with PBS, ZnPP PM, PIC or ZnPP PM/PIC as described in experimental section. Tumors were harvested two days after the last treatment. (A) Tumor tissues were labeled with Hoechst (blue) and anti-mouse-CD11b-FITC to identify the TAMs by confocal microscopy. The bar represents 50 µm. (B) The expression of CD86 in TAMs using flow cytometry. (C-E) The mRNA levels of IL-12, iNOS and Msr2 gene in TAMs were determined by QRT- PCR. The asterisks indicate differences between PBS and other treatments are statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001. #

Differences between two groups are statistically significant, p < 0.05.



Figure 7. Anti-tumor effect of ZnPP PM/PIC in vivo. B16 tumor-bearing mice were injected intratumorally with PBS, ZnPP-PM, PIC or ZnPP PM/PIC on day 8 following tumor implantation


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every two days. (A) Photographs of excised tumors on day 18. (B) Tumor growth inhibition curves in C57BL/6 mice, black arrows indicate the treatment time. (C) Representative photographs of TUNEL staining in different groups. The bar shown is 200 µm. (D) Quantitiative analysis of apoptotic cells was determined by image pro software. (E, F) Proportions of CD8+CD69+ cells, CD4+CD69+ cells and NK1.1+CD69+ cells in tumor (E) and TDLN (F) analyzed by flow cytometry. The asterisks indicate differences between PBS and other treatments are statistically significant. *p < 0.05, **p < 0.01. #Differences between two groups are statistically significant, p < 0.05.



Figure 8. Schematic illustration of ZnPP PM/PIC-induced anti-tumor immune response.


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ROS-Inducing Micelles Sensitize Tumor-Associated Macrophages to TLR3 Stimulation for Potent Immunotherapy Lanlan Liu†, Huamei He†, Ruijing Liang†, Huqiang Yi, Xiaoqing Meng, Zhikuan Chen, Hong Pan, Yifan Ma* and Lintao Cai*

“For Table of Contents Only”

ZnPP

PM/PIC

were

constructed

via

self-assembly

of

galactose-functionalized

poly(ethyleneglycol)-b-poly(L-lysine) (PLL-PEG-Gal) block copolymers grafted with ZnPP, and further entrapping PIC (a TLR3 agonist) through electrostatic adsorption. The results showed that TAM-targeted delivery of PIC with ZnPP PM synergistically repolarized TAMs (with pro-tumor function) to M1 macrophages (with anti-tumor function) by modulating the expression of ROS and STAT3, and evoked strong anti-tumor immune reactions, thereby causing substantial tumor suppression.


ROS-Inducing Micelles Sensitize Tumor-Associated Macrophages to

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