Organic Semiconducting Photoacoustic. Nanodroplets for Laser-Activatable Ultrasound. Imaging and Combinational Cancer Therapy

Transcription

1 Organic Semiconducting Photoacoustic Nanodroplets for Laser-Activatable Ultrasound Imaging and Combinational Cancer Therapy Wei Tang, Zhen Yang *, Sheng Wang, Zhantong Wang, Jibin Song, Guocan Yu, Wenpei Fan, Yunlu Dai, Jingjing Wang, Lingling Shan, Gang Niu, Quli Fan *, Xiaoyuan Chen * Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, MD 20892, USA Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, Nanjing , China * zhen.yang3@nih.gov, iamqlfan@njupt.edu.cn, shawn.chen@nih.gov 1

2 Figure S1. Chemical structures of PDI amphiphilic molecules: PEG-based PDI (1) and amine functional PEG-based PDI (2). Figure S2. Absorbance spectra of the added and un-encapsulated ZnF 16 Pc photosensitizer in the preparation of PS-PDI-PAnDs. 2

3 Figure S3. DLS data of the PS-PDI-PAnDs in (a) aqueous, (b) PBS, (c) 10% FBS, and (d) DMEM solution after 3 days incubation at 4, 29, and 37. No significant size changes were observed in all conditions, suggesting excellent colloidal stability of the PS-PDI-PAnDs. (e) Photographs of the above-mentioned solutions after 3 days incubation. (f) photograph of a mixture of PFC and PS-PDI-PAnP solution. Two obvious layers were found in the mixture. Figure S4. (a) TEM and (b) DLS analysis of PS-PDI-PAnPs and PDI-PAnPs. 3

4 Figure S5. Normalized absorption spectra of PS-PDI-PAnDs, PS-PDI-PAnPs, PDI-PAnPs, and PSs. 4

5 Figure S6. (a) Echo intensity in B-mode of PS-PDI-PAnDs at different concentrations before and after laser irradiation based on Figure 1h. (b) B-mode US images and (c) Quantitative echo intensity in B-mode of PS-PDI-PAnDs, PS-PDI-PAnPs, PDI-PAnPs (all at the same concentration of 1 mg PDI/mL), and PBS control before and after the laser irradiation. N.S., not statically significant. ***, P <

6 Figure S7. MTT assay results at 24-h after the treatments. (a) U87MG cells treated with PS-PDI- PAnDs, PS-PDI-PAnPs, and PDI-PAnPs at different concentrations without irradiation. (b) U87MG cells treated with PS-PDI-PAnDs, PS-PDI-PAnPs, and PDI-PAnPs at different concentrations, followed by a 200-second of 671-nm laser irradiation at 0.1 W/cm 2. Figure S8. MTT assay results after 24 h of co-incubation with the PS-PDI-PAnDs. (a) HUVEC human endothelial cells showed a cell viability of 88% even at a high concentration of 400 µg PDI/mL. (b) NIH/3T3 mouse embryo fibroblast cells remained over 75 % cell viability at 400 µg PDI/mL. All these observations suggested excellent biocompatibility of the PS-PDI-PAnDs to normal cells. 6

7 Figure S9. Comparison of 1 O 2 generation between PS-PDI-PAnDs and PS-PDI-PAnPs (a) in a cap-open cuvette or (b) in an argon-filled cuvette. The cuvette was irradiated with a 671-nm laser at 0.1 W/cm 2 for 10 min. SOSG was used as a 1 O 2 indicator. Figure S10. In vitro oxygen release profiles of the PS-PDI-PAnDs. The oxygen concentrations in de-oxygenated aqueous solution (pre-treated with N 2 ) were measured by a portable pinpoint dissolved oxygen meter (PENTAIR, # DO62, USA) in a sealed 50 ml centrifugation tube. Right after adding the oxygen-saturated PS-PDI-PAnDs, the oxygen concentration rapidly increased and gradually saturated within 5 min, indicating effective oxygen loading and gradual but sustained oxygen release of the PFC. A subsequent incubation of the solution at 42 led to a burst increase of dissolved oxygen concentration, which may attribute to the vaporization of the PFC, leading to a burst-like oxygen release. In contrast, N 2 pre-treated water showed little difference in oxygen concentration as the temperature changes. 7

8 Figure S11. Impact of the oxygen release rates on PDT efficiency. (a) Intracellular oxygen level in hypoxic U87MG cells was detected with an oxygen indicator [Ru(dpp) 3 ]Cl 2 after incubated overnight at 37 with PS-PDI-PAnPs, PS-PDI-PAnDs, or PS-PDI-PAnDs followed by a 200 s 42 exposure after the incubation. Red fluorescence of the [Ru(dpp) 3 ]Cl 2 was significantly diminished in the PS-PDI-PAnD-treated cells, indicating alleviated oxygen deficiency in the U87MG cells, which was attributed to the gradual oxygen release of the PS-PDI-PAnDs. Moreover, subsequent co-incubation of the hypoxic U87MG cells with the PS-PDI-PAnDs at 42 further quenched the fluorescence signals of the indicator due to the burst oxygen release. In comparison, co-incubation with PS-PDI-PAnPs made no difference in the intracellular oxygen level because of no oxygen release during the incubation. These observations confirmed controllable oxygen release of the PS-PDI-PAnDs. Scale bar, 20 µm. (b) Oxygen release ratedependent PDT efficiency was investigated on hypoxic U87MG cells by MTT assays. Cells were received the same treatments as in (a). After the 24 hr of co-incubation, the cells were irradiated with a 671-nm laser at 0.1 W/cm 2 for 200 s at room temperature or 42 in separate groups. The phototoxicity was strongly dependent on the oxygen release rate. Without oxygen release (PS- PDI-PAnPs group), little phototoxicity was induced. With gradual oxygen release (PS-PDI- PAnDs group), the cell viability after photoirradiation was 22.5 % at 100 µg PDI/mL. With burst-like oxygen release (PS-PDI-PAnDs, 42 group), significant reduction in cell viability was observed, showing a cell viability after photoirradiation of 45.6 % and 14.0 % at concentration of 50 and 100 µg PDI/mL, respectively. These observations suggested that O 2 played an important role for enhancing PDT and the oxygen release rate displayed a positive correlation with the PDT efficiency. 8

9 Figure S12. (a) Blood circulation and (b) tissue distribution (at 48-h p.i.) of the PS-PDI-PAnDs in health balb/c mice. Figure S13. (a) Ex vivo NIRF images of dissected tumors and major organs at 48-h p.i. of PS- PDI-PAnDs. (b) Column histograms of fluorescence activities in different organs. Quantitative data was calculated based on the ex vivo imaging results shown in (a). 9

10 Figure S14. Ex vivo PA images of the dissected tumors and major organs at 48-h p.i. of the PS- PDI-PAnDs. Figure S15. (a) Thermographic images of mice at the end of a 671-nm laser irradiation. The irradiation duration was fixed at 10 minutes, but the irradiation fluences was elevated from 0.1 to 0.3 W/cm 2. PS-PDI-PAnDs or PS-PDI-PAnPs were i.v. injected into the mice 24 hours before the irradiation in separate groups. (b) Temperature curves at tumor areas during the photoirradiations in (a). 10

11 Figure S16. Tumor oxygenation changes during the Oxy-PDT. (a) PA images of oxygenated hemoglobin (λ = 850 nm) before injection, at 24 h p.i., and after 30 min of irradiation (671 nm, 0.2 W/cm 2, 10 min) at 24 h p.i. Significant signal increase was observed after injection of the PS- PDI-PAnDs and the PA signal remained at the same level after the irradiation. In contrast, injection of the PS-PDI-PAnPs didn t improve the tumor oxygenation. (b) Quantitative analysis based on PA signals in (a). A ~ 1.9 times enhancement was measured in the irradiated and PS- PDI-PAnD-treated tumors, while a ~20 % oxygen level drop was found in the irradiated and PS- PDI-PAnP-treated ones. (c) Hypoxia-inducible factor 1- α (HIF1-α) staining on tumors collected 30 min after the irradiation. The results were in good accordance with the oxygenated hemoglobin PA imaging. A high level of positive HIF1-α fluorescence signal was observed in the PS-PDI-PAnP-treated tumors, but negligible fluorescence was detected in the PS-PDI-PAnDtreated ones. Scale bar, 20 µm. All these results demonstrated enhanced tumor oxygen level in Oxy-PDT comparing to conventional PDT. 11

12 Figure S17. Mice body weight growth curves. No significant weight drop was observed in all treatment groups. 12

13 Figure S18. H&E staining on major organs after the therapy. No abnormalities were observed. Scale bar, 100 µm. 13

14 Figure S19. (a) Thermographic images of mice treated with a photoirradiation (671-nm, 0.5 W/cm 2 or 0.2 W/cm 2, 10 min) at 24 h p.i. of the PS-PDI-PAnPs. (b) Temperature curves at tumor regions based on (a). 14

15 Figure S20. Tumor growth curves of PS-PDI-PAnPs and PS-PDI-PAnDs plus (a) a 0.5 W/cm 2 irradiation or (b) 0.2 W/cm 2 irradiation. The combinational PDT/PTT and conventional PDT showed a TGI of 92.1 ± 4.8 % and 62.5 ± 11.4 % on day 18, which indicated a significant lower rate compared to that of the corresponding Oxy-PDT related treatment in Figure 5c (combinational Oxy-PDT/PTT, TGI = 100 %. Oxy-PDT, TGI = 67.5 ± 9.1 %). (c) H&E staining on tumors acquired at 24-h after the therapy. Scale bar, 100 µm. *, P < ***, P <

16 Figure S21. (a) Mice body weight growth curves during the treatment with PS-PDI-PAnPs plus a 0.2 or 0.5 W/cm 2 laser irradiation. No significant weight drop was observed in the treatment groups compare to the PBS control. (b) H&E staining on major organs after the therapy with PS- PDI-PAnPs plus a 0.2 or 0.5 W/cm 2 laser irradiation. No pathological abnormalities were found. Scale bar, 100 µm. 16