A Nanographene‐Porphyrin Hybrid for Near‐Infrared‐Ii Phototheranostics

Abstract Photoacoustic imaging (PAI)‐guided photothermal therapy (PTT) in the second near‐infrared (NIR‐II, 1000–1700 nm) window has been attracting attention as a promising cancer theranostic platform. Here, it is reported that the π‐extended porphyrins fused with one or two nanographene units (NGP‐1 and NGP‐2) can serve as a new class of NIR‐responsive organic agents, displaying absorption extending to ≈1000 and ≈1400 nm in the NIR‐I and NIR‐II windows, respectively. NGP‐1 and NGP‐2 are dispersed in water through encapsulation into self‐assembled nanoparticles (NPs), achieving high photothermal conversion efficiency of 60% and 69%, respectively, under 808 and 1064 nm laser irradiation. Moreover, the NIR‐II‐active NGP‐2‐NPs demonstrated promising photoacoustic responses, along with high photostability and biocompatibility, enabling PAI and efficient NIR‐II PTT of cancer in vivo.

Technology, USA).Photoacoustic (PA) images were taken by an ultra-high resolution multimode ultrasonic photoacoustic imaging system (Vevo LAZR, Visual Sonics, Canada).Confocal laser scanning microscopy (CLSM) images were taken on a confocal laser scanning microscope (Nikon A1R HD25, Japan).The MTT assay was performed on a microplate reader (Tecan Infinite M1000 PRO, Switzerland).
Preparation of NGP-1-NPs and NGP-2-NPs.Water dispersible nanoparticles of NGP-1 and NGP-2 (NGP-1-NPs and NGP-2-NPs, respectively) were prepared through a standard nanoprecipitation method. [2]0.5 mg of NGP-1 and 5 mg of DSPE-PEG2000 were dissolved in 1 mL of tetrahydrofuran (THF) to obtain a homogeneous solution, which was quickly added into 9 mL of DI water under vigorously stirring.The resulting mixture was stirred at room temperature with continuous nitrogen bubbling for 48 h to completely remove THF.The obtained dispersion of NGP-1-NPs was concentrated to 1 mL by centrifugal filter units (100 kDa, Millipore) at 2000 rpm (revolutions per minute) for 5 min, and then diluted with DI water to 9 mL, followed by concentration to 1 mL again by the centrifugation.This procedure was repeated for three times in total.This concentrated dispersion of NGP-1-NPs was stored in the dark at 4 °C.The concentrated NGP-2-NPs solution was prepared by the same procedure.

Dynamic Light Scattering (DLS) Analysis of
Temperature variations during these processes were recorded with the Ti400 thermal imaging camera.DI water was applied as control under the same experiment conditions.The photothermal stability of NGP-1-NPs and NGP-2-NPs was determined through laser irradiation for 5 cycles (laser on/off), and that of NGP-2-NPs (30 μg mL −1 ) and IR-1048-NPs (100 μg mL −1 ) was compared under 1064 nm laser (1 W cm −2 ).
Photothermal Conversion Efficiency Calculations.The photothermal conversion efficiencies (η) were determined according to the procedure described by Roper [3] and Li. [4]0.5 mL of NGP-2-NPs dispersion (30 μg mL −1 ) in DI water was irradiated with 1064 nm laser (1 W cm −2 ) for 10 min to reach a maximum equilibrium temperature (Tmax).Then, the laser was turned off and the sample was allowed to naturally cool to a temperature in equilibrium with its surroundings (Tsurr).The temperature variations of NGP-2-NPs dispersion during the natural cooling period were recorded with the Ti400 thermal imaging camera.Based on the obtained photothermal curves, η was calculated using the following equation: where ΔTmax is the maximum temperature change that is calculated according to Tmax -Tsurr, QDis is the heat related to the laser absorbance of DI water and container, I is laser power density (1 W cm −2 in this work), A1064 is the absorbance value of the sample at 1064 nm, h is the heat transfer coefficient, S is the surface area of the container, and the value of hS (W ℃ −1 ) was calculated with the following equation: where mD is the mass (0.5 g), CD is the heat capacity (4.2 J g −1 ℃ −1 ) of DI water used as the solvent, and τs is the sample system time constant, which was calculated with the following equation: where t is time starting from turning off the laser (0 s) during the cooling period, ΔT is the temperature change that is calculated according to Tt -Tsurr (Tt is the temperature of the system at the time of t), θ is the ratio of ΔT to ΔTmax during the cooling period.From the slope of the red line displayed in Figure 2f of the main text, τs was calculated to be 174.3s.The η of NGP-1-NPs was determined in the same way using 808 nm laser (1 W cm −2 ).

Reactive Oxygen Species (ROSs) Production by NGP-1-NPs and NGP-2-NPs.
The production of ROSs by NGP-1-NPs and NGP-2-NPs under laser irradiation was detected with 2′,7′-dichlorofluorescin (DCFH) as the probe. [2]A dispersion of NGP-1-NPs (30 μg mL 4T1 or MCF-7 cancer cells were seeded in 96-well plates at a density of 4×10 3 cells/well and cultured in fresh cultured medium at 37 ℃ overnight to achieve the 60-70% confluence.For the dark groups, the above cells were further incubated with various concentrations of NGP-1-NPs or NGP-2-NPs (40, 30, 20, 10, and 0 μg mL −1 ) in fresh cell culture medium for another 24 h incubation at 37 ℃.Then, MTT (0.5 mg mL −1 in DMEM without FBS, 100 μL/well) was added with another 4 h incubation at 37 °C.After that, 100 μL of dimethyl sulfoxide (DMSO) was employed to thoroughly dissolved the produced formazan.After shaking the plates for 1 min, the absorbance of all of the wells at 490 nm was read with a microplate reader.The cell viability rate (VR) was calculated according to the following equation: where A is the absorbance of the experimental group and A0 is the absorbance of the control group (0 μg mL −1 ).For the laser irradiation groups, when replacing with fresh medium mixed with various concentrations of NPs for another 4 h incubation at 37 ℃, the cells were illustrated with 808 nm laser (1 W cm −2 ) or 1064 nm laser (1 W cm −2 ) for 10 min.After another 24 h incubation, MTT and DMSO were subsequently added as described above.Moreover, the dark cytotoxicity of NGP-2-NPs to normal L929 cells was also analyzed using MTT assay.405 nm), 500-600 nm (λex: 488 nm), and 660-700 nm (λex: 647 nm), respectively.

Cell Co-localization
Live/dead Cell Imaging.Live/dead cell staining assay was performed by confocal laser scanning microscopy.4T1 cancer cells were seeded in confocal dishes at a density of 8×10 4 cells/well and cultured in the medium at 37 ℃ for 12 h.The cells were incubated with NGP-1-NPs (40 μg mL −1 ) in fresh cell culture medium for 4 h, which were illuminated with 808 nm laser (1 W cm −2 ) for 10 min followed by another 4 h incubation.After removing the medium, the cells were incubated with fresh medium containing Calcein-AM (2 μM) and PI (4.5 μM) for 30 min at 37 ℃.CLSM images were acquired after washing with PBS for three times.The CLSM images of Calcein-AM and PI were collected at 500-540 nm (λex: 488 nm) and 570-650 nm (λex: 561 nm), respectively.

Figure S6 .
Figure S6.a) Photothermal stability of NGP-1-NPs under 808 nm laser irradiation (1 W cm −2 ) for five on/off cycles.b) Photothermal performance of NGP-1-NPs by cooling to room temperature with linear analysis.The concentration of NGP-1-NPs was 30 μg mL −1 , based on the amount of NGP-1.

Figure S9 .
Figure S9.Cell viability of L929 cells treated with various concentrations of NGP-2-NPs in dark for 24 h.Data shown are presented as mean ± standard deviation (n = 3).

Figure S10 .
Figure S10.a) Co-localization of NGP-1-NPs with DAPI, and LysoTracker after incubating with 4T1 cancer cells for 4 h.Scale bar: 40 μm.Cell viability of b) 4T1 cells c) and MCF-7 cells treated with various concentrations of NGP-1-NPs with or without 808 nm laser irradiation (1 W cm −2 ) for 10 min.d) Live/dead images of 4T1 cells costained with AM (green fluorescence for live cells) and PI (red fluorescence for dead cells) after incubation with PBS or NGP-1-NPs with or without 808 nm laser irradiation (1 W cm −2 ) for 10 min.Scale bar: 100 μm.The corresponding enlarged images for the white box region were also shown, Scale bar: 50 μm.Data shown in panels b and c are presented as mean ± standard deviation (n = 3).P-values are calculated by using one-way ANOVA with Tukey test, ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure S11 .
Figure S11.Body weight of mice in different treatment groups during the therapy period.Data are presented as mean ± standard deviation (n = 5).

Figure S12 .
Figure S12.Blood biochemistry tests of different treatment groups.Data are presented as mean ± standard deviation (n = 3).

Figure S13 .
Figure S13.Complete blood panel analysis of different treatment groups.Data are presented as mean ± standard deviation (n = 3).

Table S1 .
Summary of NIR-II absorbing materials

Table S2 .
Summary of NIR-I absorbing materials