Immune Checkpoint Blockade for Anti-Metastatic Cancer Immunotherapy Rui Ge, Cangwei Liu, Xue Zhang, Wenjing Wang, Binxi Li, Jie Liu, Yi Liu, Hongchen

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1 Supporting Information Photothermal-Activatable Fe 3 O 4 Superparticles Nano-Drug Carriers with PD-L1 Immune Checkpoint Blockade for Anti-Metastatic Cancer Immunotherapy Rui Ge, Cangwei Liu, Xue Zhang, Wenjing Wang, Binxi Li, Jie Liu, Yi Liu, Hongchen Sun, Daqi Zhang,, * Yuchuan Hou, ǁ, * Hao Zhang,, * and Bai Yang State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun , P. R. China, Department of Oral Pathology, School and Hospital of Stomatology, Jilin University, Changchun , P. R. China, Department of Thyroid Surgery, China-Japan Union Hospital, Jilin University, Changchun , P. R. China, and ǁ Department of Urinary Surgery, The First Hospital of Jilin University, Changchun , P. R. China. *Address correspondence to hao_zhang@jlu.edu.cn; daqizhang@yeah.net; hou63@163.com S-1

2 Figure S1. (a) TEM image of OA-stabilized Fe 3 O 4 NPs. (b) The size distribution of Fe 3 O 4 -R837 SPs calculated from TEM observation. (c) DLS size distribution of Fe 3 O 4 -R837 SPs. (d) Time-dependent size and PDI changes of Fe 3 O 4 -R837 SPs in PBS. (e) M-H curves of Fe 3 O 4 NPs and Fe 3 O 4 -R837 SPs. S-2

3 Calculation S1. The average number of Fe 3 O 4 NPs in a superparticle (Q) is calculated according to the following equation: Q = VSPη V NP SP 4 3 π RSPη SP = 3 4 eq 1 3 π RNP 3 R SP and R NP are the radius of superparticles and nanoparticles, which can be determined by TEM images. R SP is nm and R NP is 5.8 nm. η SP is the unit cell space utilization of SPs. According to the previous literature, 1 η SP is %. The calculated average number of Fe 3 O 4 NPs in a superparticle (Q) is REFERENCE (1) Zhuang, J.; Wu, H.; Yang, Y.; Cao, Y. C. Controlling Colloidal Superparticle Growth Through Solvophobic Interactions. Angew. Chem. Int. Ed. 2008, 47, S-3

4 Figure S2. (a) Photothermal effect of the irradiation of 150 µg/ml aqueous suspension of Fe 3 O 4 -R837 SPs with 808 nm laser. The suspension is irradiated for 1155 s, and cooled to room temperature under ambient environment. (b) Time constant for heat transfer from the system is determined to be τ s = s by applying the linear time data from the cooling period (after 1155 s) versus negative natural logarithm of driving force temperature. S-4

5 Calculation S2. The photothermal transduction efficiency (η) of Fe 3 O 4 -R837 SPs is calculated by eq 2: hs( T T ) Q η = A808 P(1 10 ) max surr 0 eq 2 Where h is heat transfer coefficient, S is the surface area between container and environment, T max and T surr are the maximal steady-state temperature of the SPs and the ambient temperature, Q 0 is the heat generated by water and quartz cell under laser irradiation, P is the incident laser power, and A 808 is the absorbance of SPs at 808 nm. To possess h, the lasting temperature decrease should be recorded at fixed time intervals (Figure S2a). A linear fit of time (t) and ln(θ) is made and θ represents (T-T surr )/(T max -T surr ) (Figure S2b). h can be calculated from the slope of fitted eq 3: t Σm C i p, i = ( ln ) + b eq 3 hs θ Where m i and C p,i are the mass and heat capacity of the irradiated system. S-5

6 Figure S3. (a) UV-vis absorption spectra of R837 in water with the concentration of 2.5~10 µg/ml. (b) The regression equation is determined by the absorbance at 322 nm versus the feeding concentration of R837. The encapsulation efficiency (c) and loading efficiency (d) of R837 by Fe 3 O 4 SPs at different feeding concentration. The concentration of R837 is fixed at 200 µg/ml. S-6

7 Figure S4. (a) UV-vis absorption spectra of R837 released from Fe 3 O 4 -R837 SPs with 808 nm NIR irradiation. (b) The released percentage of R837 from Fe 3 O 4 -R837 SPs with or without 808 nm NIR irradiation. The laser power density is 1.0 W/cm 2. (c) The released percentage of R837 from Fe 3 O 4 -R837 SPs with 808 nm NIR irradiation at different power densities for 10 min. S-7

8 Figure S5. (a) Time-dependent cellular uptake of Fe 3 O 4 SPs and Fe 3 O 4 -R837 SPs in 4T1 cells. (b) Efflux of Fe 3 O 4 SPs and Fe 3 O 4 -R837 SPs by 4T1 cells. (c) Confocal images showing the time-dependent cellular uptake of Fe 3 O 4 -R837 SPs in 4T1 cells. The scale bar is 200 µm. S-8

9 Figure S6. PTT of 4T1 cells in vitro. (a) 4T1 and 293 cells are incubated with different concentrations of Fe3O4 SPs or Fe3O4-R837 SPs for 24 h, and cell viabilities are estimated through standard MTT assay. (b) 4T1 cells are incubated with 150 µg/ml Fe3O4-R837 SPs for 2 h, and then they are irradiated by an 808 nm laser with the power density of 0.5, 1, 1.5 and 2 W/cm2 for 0, 5, 10, 15 and 20 min. (c) Confocal fluorescence and bright images of FD (green, live cells) and PI (red, dead cells) co-stained cells after PTT for different time. The scale bar is 200 µm. The control group is the 4T1 cells without incubation with Fe3O4-R837 SPs and irradiation. S-9

10 Figure S7. (a-h) Apoptosis and/or necrosis of 4T1 cells treated with PBS, R837, Fe 3 O 4 SPs, and Fe 3 O 4 -R837 SPs group with or without irradiation by flow cytometry analysis. S-10

11 Figure S8. (a-b) Quantification the secretion of pro-inflammatory cytokine in DC medium suspension with different treatment. - and + in the figures mean the treatment without or with irradiation, respectively. Error bars are based on the standard deviation of three parallel samples. S-11

12 Figure S9. (a-c) Pro-inflammatory cytokine levels of IL-6 (a), TNF-α (b), and IFN-γ (c) in sera from mice isolated on day 3 post-treatment. Error bars are based on standard deviations of three parallel samples. S-12

13 Figure S10. (a) The tumor volume growing trend for control, anti-pd-l1, Fe 3 O 4 -R837 SPs with irradiation and Fe 3 O 4 -R837 SPs with irradiation plus anti-pd-l1 groups. (b) Tumor weights at the end point of different groups. Arrows represent the time of materials injection (black) and NIR irradiation (red). + in the figure refers to the treatment with irradiation. Error bars are based on the standard deviation of five parallel samples. S-13

14 Figure S11. The average body weight trend for each group. Arrows represent the time of materials injection (black) and NIR irradiation (red). - and + in the figure means the treatment without or with irradiation, respectively. Error bars are based on the standard deviation of five parallel samples. S-14

15 Table S1. The serum biochemistry data between healthy mice and synergistic therapy group. Complete blood counts: total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), total protein (TP), albumin (ALB), globulin (GLO), albumin/ globulin (A/G), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bile acid (TBA), cholinesterase (CHE), prealbumin (PA), blood urea nitrogen (BUN), supercarbonate (HCO 3 ), uric acid (UA), creatinine (CREA), serum phosphorus (P), serum magnesium (Mg), total calcium (tca). Three mice are used in each group for the experiment. TBIL (µmol/l) DBIL (µmol/l) IBIL (µmol/l) TP (g/l) ALB (g/l) GLO (g/l) A/G Reference range Healthy control 1.65± ± ± ± ± ± ±0.12 Therapy 2.63± ± ± ± ± ± ±0.27 ALT (U/L) AST (U/L) ALP (U/L) GGT (U/L) TBA (µmol/l) CHE (U/L) PA (mg/l) Reference range Healthy control 61.00± ± ± ± ± ± ±20.58 ` Therapy 71.00± ± ± ± ± ± ±15.64 BUN (mmol/l) HCO 3 (mmol/l) UA (µmol/l) CREA (µmol/l) P (mmol/l) Mg (mmol/l) tca (mmol/l) Reference range Healthy control 6.75± ± ± ± ± ± ±0.34 Therapy 7.54± ± ± ± ± ± ±0.19 S-15

16 Figure S12. (a-c) Pro-inflammatory cytokine levels in the sera of mice treated with different treatment groups from day 0 to day 7. Arrows represent the time of materials injection (black) and NIR irradiation (red). - and + in the figure means the treatment without or with irradiation, respectively. Error bars are based on the standard deviation of five parallel samples. S-16