advances.sciencemag.org/cgi/content/full/5/10/eaaw6870/DC1

Supplementary Materials for

Red blood cell–derived nanoerythrosome for antigen delivery

with enhanced cancer immunotherapy

Xiao Han, Shufang Shen, Qin Fan, Guojun Chen, Edikan Archibong, Gianpietro Dotti, Zhuang Liu*, Zhen Gu*, Chao Wang*

*Corresponding author. Email: cwang@suda.edu.cn (C.W.); guzhen@ucla.edu (Z.G.); zliu@suda.edu.cn (Z.L.)

Published 23 October 2019, Sci. Adv. 5, eaaw6870 (2019)

DOI: 10.1126/sciadv.aaw6870

This PDF file includes:

Fig. S1. Particle size and ζ potential of nano-Ag@erythrosomes at various ratios of RBC to B16F10 cell membrane. Fig. S2. TEM images of RBC vesicles and B16 vesicles. Fig. S3. Raw Western blot data according to Fig. 1D. Fig. S4. Signal of B16 and RBC membranes in major organs. Fig. S5. Ex vivo imaging of major organs after intravenous injection of nano-Ag@erythrosomes. Fig. S6. Flow cytometric analysis in macrophages in spleen. Fig. S7. Cytokine production in serum after intravenous injection of nano-Ag@erythrosomes. Fig. S8. B16F10-Luc tumor growth curve after mice were treated with nano-Ag@erythrosomes or B16 membrane vesicle with aPDL1. Fig. S9. In vivo therapeutic efficacy of nano-Ag@erythrosomes with aPDL1 in a B16F10-Luc lung metastasis model. Fig. S10. In vivo therapeutic efficacy of nano-Ag@erythrosomes with aPDL1 in a 4T1-Luc lung metastasis model.

Fig. S1. Particle size and ζ potential of nano-Ag@erythrosomes at various ratios of RBC to

B16F10 cell membrane. Particle size and zeta potential (n = 3) of nano-Ag@erythrosomes at various

ratios of RBC to B16F10 cell membrane. Data are means ± SEM.

Fig. S2. TEM images of RBC vesicles and B16 vesicles. TEM images of RBC vesicles and B16

vesicles. (Scare bar = 500 nm)

Fig. S3. Raw Western blot data according to Fig. 1D. (Photo Credit: Shufang Shen, Soochow

University)

Fig. S4. Signal of B16 and RBC membranes in major organs. Signal of B16 membrane and RBC

membrane in major organs (liver, lung, spleen, heart, and kidney). RBC membrane and B16 membrane

were labeled with DID and DIR respectively before membrane fusion. Nano-Ag@erythrosomes with a

R:T ratio of 20:1 was injected intravenously. In contrast, the simple mixture of RBC membrane vesicles

and B16 membrane vesicles at the same amount was used as a control group. A) The signal of RBC

membrane and B16 membrane in major organs of mice treated differently. B) and C) The average

radiant efficiency of RBC membrane and B16 membrane respectively. (n = 3). Data are means ± SD.

Statistical significance was calculated by Student’s t-test. **P < 0.01.

Fig. S5. Ex vivo imaging of major organs after intravenous injection of nano-Ag@erythrosomes.

(A) Ex vivo imaging of major organs at 1 h after i.v. injection of nano-Ag@erythrosomes at various

ratios and corresponding quantification results (B) (n = 3). Data are means ± SD.

Fig. S6. Flow cytometric analysis in macrophages in spleen. (A) Flow cytometric analysis of various

activation markers and PD-L1 in macrophages (gated on CD11b+ F4/80+) in spleen of untreated mice

and mice treated with DiD-labeled nano-Ag@erythrosome and (B) corresponding quantification results.

(n ≥ 3). Data are means ± SD. Statistical significance was calculated by Student’s t-test. *P < 0.05; **P

< 0.01; ***P < 0.005.

A

B

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*

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n.s.

***

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n.s. **

**

*

***

CD80 CD86 CD40 MHC II PDL1

Untreated (1)

MΦ without vesicle uptake (2)

MΦ with vesicle uptake (3)

Co

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1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

 

Fig. S7. Cytokine production in serum after intravenous injection of nano-Ag@erythrosomes.

Cytokines production in serum after 24 h and 48 h after i.v. injection of nano-Ag@erythrosomes.

Data are means ± SEM. Statistical significance was calculated by one-way analysis of variance

(ANOVA) with Tukey’s post hoc test.

TNF‐α IL17a IL‐1α IL23 IL10

0 vs. 24 (h) p = 0.2127 p = 0.1999 p = 0.0458 p = 0.2943 p = 0.1960

0 vs. 48 (h) p = 0.1623 p = 0.4916 p = 0.4687 p = 0.4494 p = 0.5727

IL27 IFN‐β GM‐CSF CCL2

0 vs. 24 (h) p = 0.2490 p = 0.2591 p = 0.3686 p = 0.233

0 vs. 48 (h) p = 0.4490 p = 0.6008 p = 0.3925 p > 0.999

 

* 

Fig. S8. B16F10-Luc tumor growth curve after mice were treated with

nano-Ag@erythrosomes or B16 membrane vesicle with aPDL1. (A) B16F10-luc tumor growth

curve after mice were treated with nano-Ag@erythrosomes or B16-membrane vesicle plus aPDL1.

(B-E) Individual growth curve. (n = 6). Data are means ± SD. Statistical significance was calculated

by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. *P < 0.05.

A

B C D E

Fig. S9. In vivo therapeutic efficacy of nano-Ag@erythrosomes with aPDL1 in a B16F10-Luc

lung metastasis model. (A) Schematic representation of treatment toward the B16F10-Luc metastasis

in lung tumor model. (B) In vivo bioluminescence imaging of the B16F10 tumor in control and treated

groups. Representative mice of 5 mice per treatment group was shown. (C) Representative lung

photographs and (D) quantification of lung metastasis nodules. (E) The survival curves of mice (n = 5)

in 40 days after various treatments indicated. Data are means ± SEM. Statistical significance was

calculated by Log-rank (Mantel-Cox) test. ***P < 0.005. (Photo Credit: Chao Wang, Soochow

University)

Fig. S10. In vivo therapeutic efficacy of nano-Ag@erythrosomes with aPDL1 in a 4T1-Luc lung

metastasis model. (A) Schematic representation of the treatment toward 4T1-Luc lung tumor model.

(B) In vivo bioluminescence imaging of the 4T1-Luc tumor in control and treated groups. Three

representative mice of 5 mice per treatment group were shown. (C) The survival curves of mice (n = 5)

in 40 days after various treatments indicated. Statistical significance was calculated by Log-rank

(Mantel-Cox) test. **P < 0.01.