250 words Summarize the following research paper and comment on the direction they could go next! (your own Idea)--> It means What direction should the research go/n nature COMMUNICATIONS ARTICLE https://doi.org/10.1038/s41467-021-21047-0 OPEN Check for updates Activatable polymer nanoagonist for second near- infrared photothermal immunotherapy of cancer Yuyan Jiang, Jiaguo Huang, Cheng Xu¹ & Kanyi Pu① 1,2区 Nanomedicine in combination with immunotherapy offers opportunities to treat cancer in a safe and effective manner; however, remote control of immune response with spatiotemporal precision remains challenging. We herein report a photothermally activatable polymeric pro- nanoagonist (APNA) that is specifically regulated by deep-tissue-penetrating second near- infrared (NIR-II) light for combinational photothermal immunotherapy. APNA is constructed from covalent conjugation of an immunostimulant onto a NIR-II semiconducting transducer through a labile thermo-responsive linker. Upon NIR-II photoirradiation, APNA mediates photothermal effect, which not only triggers tumor ablation and immunogenic cell death but also initiates the cleavage of thermolabile linker to liberate caged agonist for in-situ immune activation in deep solid tumor (8 mm). Such controlled immune regulation potentiates sys- temic antitumor immunity, leading to promoted cytotoxic T lymphocytes and helper T cell infiltration in distal tumor, lung and liver to inhibit cancer metastasis. Thereby, the present work illustrates a generic strategy to prepare pro-immunostimulants for spatiotemporal regulation of cancer nano-immunotherapy. 1 School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore. 2 Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore, Singapore. email: kypu@ntu.edu.sg NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 mmunotherapy that boosts host immune system to fight against tumor has revolutionized cancer treatment 1,2. How- ever, single model immunotherapy often suffers from limited response rate and occasional immune-related adverse effects (irAEs) such as cytokine storm, hematopoietic system dysfunc- tion, and organ failure³. Nanomedicine thus has been brought into cancer immunotherapy in the hope to address these critical concerns in view of its tunable physicochemical properties to reinforce drug-immune cell interactions, and optimal pharma- cological profiles (e.g., biodistribution and retention) of immu- notherapeutic molecules 4-6. More importantly, nanomedicine serves as an assembly platform to allow additional therapeutic modalities (e.g., chemotherapy, phototherapy, radiotherapy, etc.) to be seamlessly integrated with immunotherapy to enhance therapeutic efficacy7-11. With the promise to improve immune response at no expense of breaking the systemic immune toler- ance, cancer nano-immunotherapy has been recently under extensive development. Further enhancement in the specificity of cancer immu- notherapy to facilitate its clinical translation mainly lies in the development of smart immunotherapeutic nanoagents with controlled activation 12-14. Till now, immunotherapeutic nanoa- gents have been programmed to unleash therapeutic action in response to biochemical indexes or cancer biomarkers such as pH, reactive oxidative species, and enzymes 15-18. For instance, human interleukin-15 super-agonist complex was engineered with disulfide-containing cross-linker for activatable release in response to T-cell receptor activation so as to improve therapeutic window of adjuvant cytokine therapy19. As another example, programmed cell death protein 1 antibody was specifically con- jugated to magnetic nanoclusters via pH-responsive benzoic- imine bond, leading to selective activation in the acidic intratu- moral microenvironment for potentiation of adoptive T-cell therapy 20. However, owing to their reliance on the difference of biomarkers at basal and pathological levels, the bioavailability and regional selectivity of immune activation for such immunother- apeutic nanoagents remain to be improved. In contrast to endogenous biomarkers, external stimuli are independent of the physiological environment and thus hold potential for more precise spatiotemporal and dosage control over immune regulation 12,21. Near-infrared light (NIR-I, 650-950 nm) is a popular external stimulus for the construction of smart nanomedicine owing to its minimal invasiveness and easy operation²². In particular, NIR-I light-responsive nanoagents have been developed to serve as the signal transducers to convert incident photons into regulatory signals, such as singlet oxygen to trigger the activation of indoleamine 2,3-dioxygenase 1 inhibitor, or high-energy emission to liberate CpG21,23. Superior to NIR-I light, second NIR light (NIR-II, 1000-1300 nm) has been recently revealed to possess even better biological transparency and fur- ther ameliorated phototoxicity with a lower maximum permis- sible exposure limit 24. However, few NIR-II nanotransducers are available, which include gold nanostructures25, transition metal- based nanoparticles26, and naphthalocyanine derivatives27. Recently, semiconducting polymer nanoparticles (SPNs) com- posed of highly л-conjugated backbones have formed a promising class of NIR-II nanoagents 28,29. With their excellent photo- thermal performance and intrinsically benign compositions, SPNs have been exploited for deep-tissue molecular photoacoustic imaging³ photothermal therapy (PTT)³², and photothermal ferrotherapy33. However, the integration of SPNs with NIR-II light for spatiotemporal photoregulation of immunotherapy has yet to be explored. 30,31 We herein report the synthesis of an activatable polymer nanoagonist (APNA) for NIR-II light-regulated photothermal immunotherapy of cancer (Fig. 1). APNA is composed of a NIR- 2 II light-absorbing semiconducting polymer backbone as photo- thermal transducer, conjugated with a potent toll-like receptor type 7 and 8 (TLR7/8) agonist (Resiquimod: R848) as the immunostimulant through a thermolabile cleavable linker (2,2'- azobis [2-(2-imidazolin-2-yl)propane]: VA-044) (Fig. 1a). Mainly acting on antigen-presenting cells (APCs) such as dendritic cells (DCs), R848 helps upregulate secretion of crucial proin- flammatory cytokines and enhance maturation or polarization of APCs, priming T lymphocytes³4. Upon focal NIR-II photo- irradiation, APNA mediates photothermal effect to directly ablate tumor, and elicit immunogenic cell deaths (ICDs) of cancer cells to promote antitumor immunity; it also in situ triggers the cleavage of thermolabile linker at the tumor site to activate the TLR7/8 agonist so as to further potentiate antitumor immune response (Fig. 1b). Such an APC-mediated spatiotemporal potentiation of cancer immunotherapy enables complete eradi- cation of the primary tumor in deep tissue (~8 mm) and efficient inhibition of both distal tumor and lung metastasis without eli- citing obvious systemic adverse effects. Furthermore, the under- lying mechanisms of photothermal activation of immune response along the gradient of photothermal depth and in situ photothermal temperature in the deep beds of tumor are unveiled, providing guidelines for the development of photo- thermal immunotherapy. Results Synthesis and in vitro characterization. To obtain the NIR-II- absorbing semiconducting polymer precursor pBODO-Br, Stille polycondensation was used to copolymerize three monomers: 4,8-bis[5-bromo-4-(2-octyldodecyl)-2-thienyl]-benzo[1,2c:4,5c']bis [1,2,5]thiadiazole (BBT), 2,5-bis(6-bromohexyl)-3,6-bis(5-bro- mothiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (DPP- Br), and (4,8-bis((2-ethylhexyl)oxy)benzo[1,2b:4,5-b'] dithiophene- 2,6-diyl)bis(trimethylstannane) (OT) (Fig. 2a). Because of the strong-electron withdrawing of BBT and the electron-rich OT, PBODO-Br has a charge transfer backbone and narrowed band gap, showing strong absorption in the NIR-II window (Supple- mentary Fig. 1). Gel permeation chromatography analysis indi- cated that pBODO-Br had a number average molecular weight (Mn) of 9826 (Supplementary Fig. 2). pBODO-Br was further transformed to pBODO-N3 through substitution of bromide with azide for post-functionalization (Supplementary Fig. 3). To con- jugate the thermo-responsive linker VA-044 with the immuno- agonist R848, VA-044 was first modified with 7-iodoheptanoic acid, followed by Steglich esterification reaction with R848 to afford caged agonist VR (Supplementary Figs. 4, 5). A bifunctional poly(ethylene glycol) (PEG, Mw=2000) with one propargyl terminal and the other amine terminal (alkyne-PEG-NH2) was synthesized and linked to VR via EDC/NHS coupling to obtain alkyne-PEG-VR conjugate (Supplementary Figs. 6, 7). Thereafter, PBODO-N3 was grafted with alkyne-PEG-VR to afford semi- conducting pro-agonist pBODO-PEG-VR through click reaction (Supplementary Fig. 8). The amphiphilicity of pBODO-PEG-VR allowed its spontaneous assembly in aqueous solution to form nanoparticles termed as APNA (Fig. 1a). To prepare the control nanoparticle without pro-agonist termed as APNC, pBODO-N3 was conjugated with alkyne terminated PEG (pBODO-PEG), fol- lowed by self-assembly in aqueous solution. Optical and colloidal properties of APNA were studied and compared with APNC. Both APNA and APNC had similar absorption spectra with maxima at 690 nm in NIR-I window and 1060 nm in NIR-II window (Fig. 2b), showing that conjugation of pro-agonist had negligible influence on NIR-II light-harvesting property. Whereas dynamic light scattering (DLS) indicated a larger hydrodynamic size of APNA (71 nm) than APNC (48 nm) NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 a N C2H5 C4H9 C2H5 C4H9 R = N=N -N S 0.5 n C8H17 C10H21 0.5 NN C2H5 C4H9 C₂H C4H9 NIR-II Photothermal Unit b 0 Photothermal depth (mm) 9 4 7 8 PBODO-PEG-VR i. NIR-II photoirradiation O Primary Tumor Tumor temperature (°C) 50 45 43 42 41 Self- assembly ARTICLE PEG Thermolabile linker Caged agonist ΑΡΝΑ NH₂ ii. Photothermia Immunogenic cell death Photothermal activation TAAS DAMPS (HMGB1, etc.) Inhibition of lung metastasis v. T cell infiltration iv. T cell activation CD8+ T Cell Distant Tumor Abscopal Effect CD4+ T Cell Lymph node iDC Activated agonist iii. DC Maturation Migration MDC Fig. 1 Scheme of APNA-mediated NIR-II photothermal immunotherapy. a Chemical structure of pBODO-PEG-VR and preparation of APNA. b Mechanism of antitumor immune response by APNA-mediated NIR-II photothermal immunotherapy. TAAS tumor-associated antigens, DAMPs damage-associated molecular patterns, iDC immature DC, mDC mature DC, HMGB1 high-mobility group box 1 protein. (Fig. 2c), probably owing to the presence of hydrophobic agonist molecules. Transmission electron microscopy (TEM) further confirmed spherical morphology for both APNA and APNC (Fig. 2c). In addition, zeta potential measurement indicated a relatively neutralized surface charge of APNA (-13 mV) in contrast with APNC (-29 mV) (Supplementary Fig. 9a). Both nanoparticles showed negligible size change during storage in aqueous solutions for 2 months (Supplementary Fig. 9b), suggesting their excellent colloidal stability. According to high performance liquid chromatography (HPLC) calibration curve, the drug loading capacity of APNA was ca. 5.3%, suggesting that each pBODO-PEG-VR molecule contained ~3.3 equivalents of R848 pro-drug. Photothermal property of APNA was evaluated and compared with APNC. Upon continuous NIR-II (1064 nm) photoirradia- tion, both APNA and APNC induced significant temperature rise of aqueous solution (Fig. 2d). After irradiation for 6 min, the maximum solution temperatures of APNA and APNC nanopar- ticles were 78 and 76 °C, respectively. Such phenomenon implied the negligible impact of agonist conjugation on the photothermal transduction capability of semiconducting backbone. Indeed, APNA and APNC had similar photothermal conversion efficiency as high as 84.4% at 1064 nm (Supplementary Fig. 10). In addition, negligible changes in maximal solution temperatures were observed for both APNA and APNC nanoparticles throughout five heating and natural cooling cycles, suggesting their excellent photostability as photothermal agents. Photothermal activation of pro-agonist from APNA was evaluated and analyzed by HPLC (Fig. 2e, f). Without photo- irradiation, no elution peak related to free R848 (TR = 17.9 min) from APNA nanoparticles could be measured (Fig. 2f). After continuous photoirradiation of APNA solution for 10 min, an elution peak at 24.7 min assigned to the released agonist was observed. Such product from the photothermal cleavage was further confirmed by liquid chromatography-mass spectrometry (LCMS) and proton nuclear magnetic resonance spectroscopy ('H NMR) (Supplementary Figs. 11, 12). Through subsequent hydrolysis by esterase, the parent R848 was able to be liberated from the photothermally activated pro-agonist in an intact form, which was validated by both HPLC profiles and LCMS (Fig. 2f, Supplementary Fig. 13). Thereafter, the relationship between photothermal temperature and photothermal activation rate was investigated (Fig. 2g). When the photothermal temperature was kept at 37°C for 20 min under photoirradiation, a maximal agonist activation ratio of 8.5% was achieved. Whereas such ratio was dramatically promoted to 74% when photothermal tempera- ture was elevated to 45 °C or 55 °C and maintained for 20 min. Notably, such high activation ratio was achieved in 5 min with NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications 3 ARTICLE Absorption a HOOC N NN NEN N ii N=N NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 VA-044 VCOOH VR COOH COOH Br C2H5 C4H9 C2H5 C4H9 OT C8H17 C10H21 + N...N S Br S. C8H17 C10H21 DPP-Br Br C2H5 CH9 N NH2 !!! Болон Alkyne-PEG-VR S. N N C2H5C4H9 iv NN -Br N..N C8H17 SC8H17 C10H21 C10H21 N.. N S C10H21 BBT C2H5 C4H9 pBODO-Br Br H₂N C2H5C4H9 HK 0. S0.5 n C2H5 C4H9 №3 vi vii C2H5 C4H9 PBODO-PEG-VR ΑΡΝΑ C8H11C10H21 -$0.5 C2H5 C4H9 PBODO-N3 N3 S 0.5 n C2H5 C4H9 b 0.8 ΑΡΝΑ APNC 0.6 0.4 0.2 0.0 00 e 600 800 Wavelength (nm) 1064 nm C 25 viii ix PBODO-PEG APNC d 100- APNC ΑΡΝΑ 80 Number (%) ΑΡΝΑ 20 APNC 15 10 5 APNA APNC 50 nm Temperature (°C) 0 N% 60 40 20 1000 1 10 100 1000 10000 Diameter (nm) Caged agonist R848 APNA - laser ΑΡΝΑ Activated agonist H₂N APNA + laser H₂N R848 Esterase H₂N APNA + laser + esterase 0 10 20 Time (min) g 0.8 Drug Release Ratio 0.6 0.4 0.2- 0 720 1440 2160 2880 3600 Time (s) 37 °C 45 °C - 55 °C 0.0 0.0 0 5 10 15 Laser irradiation time (min) 20 Fig. 2 In vitro characterization of APNA. a Synthetic routes of APNA and APNC. (i) 7-iodoheptanoic acid, sodium hydride (NaH), dry tetrahydrofuran (THF), 25 °C, 2d. (ii) R848, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine (DMAP), dry acetonitrile (ACN), 25 °C, 3 d. (iii) alkyne-PEG-NH2, EDC, N-hydroxysuccinimide (NHS), dry THF, 25 °C, 2d. (iv) Pd2(dba)3, tri(o-tolyl)phosphine, chlorobenzene, 100 °C, 2 h. (v) sodium azide, dimethylformamide (DMF)/THF, 25 °C, 2d. (vi) alkyne-PEG-VR, CuBr, N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA), dry THF, 25 °C, 2d. (vii) aqueous self-assembly of pBODO-PEG-VR. (viii) methoxy-PEG-alkyne, CuBr, PMDETA, dry THF, 25 °C, 2d. (ix) aqueous self-assembly of pBODO- PEG. b Absorption spectra of APNA and APNC in 1× PBS solution. c DLS profiles of APNA and APNC nanoparticles. Inset: TEM images of APNA and APNC nanoparticles. d Photostability studies of APNA and APNC ([pBODO] = 20 μg mL −¹) by photothermal heating (1064 nm photoirradiation, 1 W cm-2, 6 min) and cooling (natural cooling, 6 min) cycles. e Schematic illustration of photothermally triggered agonist release from APNA and subsequent hydrolysis by esterase. f HPLC analysis of photothermally triggered agonist release from APNA ([pBODO] = 50 μg mL−1). 1064 nm photoirradiation: 1 W cm-2, 10 min. g Relationship of photothermal activation ratio and different photothermal temperatures ([pBODO] = 50 µg mL−1). The photothermal temperature was controlled by switching power density of photoirradiation. Data were expressed as mean ± SD. Error bars indicated standard deviations of three independent measurements. 4 NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications d NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 C a DAPI Lysosome SPN Merge DAPI Lysosome SPN Merge 120 APNAF PBS APNCF 20 μm - Cell Viability (%) 100- 80 60 60 40 60 APNC - Laser APNA Laser 40 APNC + Laser APNA + Laser 20 20 μm 0 5 10 ARTICLE 20 50 Concentration (μg mL-1) e PBS R848 APNC - Laser R848 Incubation 105 4.12% 9.57% 105 12.3% 64.1% 105 2.89% 13.7% CD80 CD86 ↑t 104- 104- 104- iDC MDC 103- 103- APNA-Laser 103- 0 0 0 CD80 D CD861 80.0% -103 6.27% 21.3% 2.32% 81.4% -103 1.97% -103 iDC mDC -103 0 103 104 105 -103 0 103 104 105 -1030 103 104 105 APNC+Laser APNC + Laser APNA - Laser APNA + Laser CD80 105- 3.13% CD86 20.2% 105- 10.6% 30.1% 105 4.35% 57.5% iDC mDC APNA+Laser CD80 CD86 iDC mDC CD80-FITC 104 104- 104- 103- 103- 103- 0 72.7% 3.94% -103 0 -103 0 56.7% 2.52% 34% 4.18% -103 -103 0 103 104 105 -103 0 103 -103 0 103 104 105 → CD86-PE 104 105 Fig. 3 In vitro APNA-mediated photothermal immunotherapy. a, b Confocal fluorescence images of cellular internalization (24 h) of APNAF and APNCF ([pBODO] = 10 μg mL-1) in 4T1 cancer cells a and BMDCs b. APNA and APNC were labeled with NCBS (2.5 w/w%). Blue fluorescence indicated nuclei staining by 4', 6-diamidine-2'-phenylindole dihydrochloride (DAPI). Green fluorescence indicated lysosomes staining by LysoTracker Green DND-26. Red fluorescence denoted as SPN channel indicated NIR fluorescence from APNAF or APNCF. Experiments were performed in triplicate with similar results. c Cell viability assay of 4T1 cells at 12 h after treatment with APNC or APNA at different concentrations with or without 1064 nm photoirradiation (1 W cm-2, 6 min) (n = 3). d Schematic illustration of pre-photothermal activation of APNA and subsequent in vitro stimulation of iDC. e Flow cytometry analysis of BMDC maturation (gated on CD11c+ DCs) at 48 h after various treatments. [R848] = 4.4 µg mL−1; [pBODO] = 10 μg mL −1; 1064 nm photoirradiation, 1W cm−², 6 min. Data were expressed as mean ± SD. photothermal temperature at 55 °C, whereas extended to 10 min at 45 °C. These results indicated the agonist could be selectively activated by photothermal heating mediated by the backbone of APNA. In vitro photothermal ablation and DC maturation. Cellular uptake of APNA and APNC was evaluated on 4T1 murine breast cancer cells and bone marrow-derived DCs (BMDCs). A fluor- escent dye (silicon 2,3-naphthalocyanine bis(trihexylsilyloxide)) (NCBS) was doped into nanoparticles (2.5 w/w%) to obtain fluorescent derivatives (APNA and APNCF) (Supplementary Figs. 14, 15). After treating 4T1 cells with APNAF or APNCF for 24 h, strong NIR fluorescence was observed from cellular plasma (Fig. 3a), indicating effective endocytosis of both nanoparticles in 4T1 cells. Similar uptake was detected for BMDCs (Fig. 3b, Supplementary Fig. 16). Furthermore, the nanoparticle signal overlapped well with the green fluorescence from lysosome staining, suggesting that endocytosed nanoparticles mainly resi- ded in lysosomes, wherein TLR7/8 was highly expressed 35,36 Without photoirradiation, both APNA and APNC induced neg- ligible cytotoxicity to 4T1 cells (Fig. 3c). However, with NIR-II photoirradiation, both nanoparticles triggered significant cell death relative to the control group. For instance, at 50 μg mL the cell viability of APNA and APNC treated-group decreased to 24% and 25%, respectively. ' The ability of APNA to photothermally trigger in vitro immune activation was investigated on DCs, a crucial species of APCs whose maturation impacts the activation of both innate and adaptive immunity37. After photoirradiation of APNA or APNC solution for 10 min, the solutions were transferred to the culture media of immature BMDCs, followed by detection of their maturation by flow cytometry analysis on the expression of the costimulatory molecules CD80 and CD86 (Fig. 3d, Supplementary Fig. 17). The post-photoirradiated APNA triggered upregulation of CD80 and CD86 (57.5%), which was 1.9-fold and 2.8-fold higher than unirradiated APNA (30.1%) or the post- photoirradiated control (APNC) (20.2%) (Fig. 3e). Furthermore, the DC maturation induced by APNA after photothermal activation was similar to free R848 at 4.4 µg mL-¹. Consistently, mixed lymphocyte reaction indicated the superior T-cell stimula- tion capacity of pre-activated APNA-treated DCs, as revealed by the higher percentage of CD3+CD8+ T cells and elevated IFN-y production relative to other groups (Supplementary Fig. 18). These results show that the immune-stimulating ability of APNA was effectively activated by NIR-II photoirradiation. NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications 5

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