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Additional file 1: Figure S1 (A) In vitro biocompatibility of O-CS-DOX NDs at varied concentrations for 24 h treatment. (B) In vitro biocompatibility of O-CS-DOX NDs at varied concentrations for 48 h treatment.

Additional file 1: Figure S1. A Tumor volume change per mouse at various treatments. B HE staining in main organs after the treatments. Scale bar: 100 µm. n=5. Figure S2. NO concentration after being triggered by ultrasound irradiation. n=3. Figure S3. A Fluorescent images of Hepa1-6 cells treated with DiO-labeled NDs under different pH values at 12, 24 h. NDs labeled with DiO appeared green dots, and cell nuclei counterstained by Hoechst were blue. Scale bar: 50 µm. B Quantitative histogram of FCM of Hepa1-6 cells treated with DiO-labeled NDs under different pH values at 12, 24 h. n=3.

Xiao Sun,1 Lu Guo,1 Mengmeng Shang,1 Dandan Shi,1 Ping Liang,2 Xuanxuan Jing,1 Dong Meng,1 Xinxin Liu,1 Xiaoying Zhou,1 Yading Zhao,1 Jie Li1 1Department of Ultrasound, Qilu Hospital of Shandong University, Jinan, People’s Republic of China; 2Department of Interventional Ultrasound, Chinese PLA General Hospital, Beijing, People’s Republic of ChinaCorrespondence: Jie LiDepartment of Ultrasound, Qilu Hospital of Shandong University, Wenhua Western Road, 107#, Jinan Tel/Fax +86-531-82166101Email jieli301@163.comPurpose: To synthesize and evaluate a novel folate-conjugated ultrasonic nanobubble (HA-FOL-NB) loading low-molecular-weight hyaluronic acid (LMW-HA) for specific tumor-associated macrophages (TAMs) targeting and reeducation.Methods: The characteristics, cytotoxicity, contrast-enhanced ultrasound imaging (CEUS), and targeting ability to TAMs of HA-FOL-NBs were investigated. The TAMs reprogramming function of HA-FOL-NBs combining ultrasound targeted nanobubble destruction was assessed as well.Results: HA-FOL-NBs (about 342nm) showed remarkable contrast enhancement images, and higher targeting ability due to the folate to folate receptor interactions. Combined with ultrasound targeted nanobubble destruction, HA-FOL-NBs could specifically deliver LMW-HA into TAMs, thus exhibited stronger reeducation effect compared with free LMW-HA.Conclusion: These folate-conjugated and LMW-HA-loaded nanobubbles, with targeted CEUS imaging and TAMs reeducation, are expected to be a potential approach for tumor therapy based on TAMs, especially folate receptor-positive ones.Keywords: ultrasound contrast agent, ultrasonic nanobubble, tumor-associated macrophage, tumor microenvironment, contrast-enhanced ultrasound imaging

Mengmeng Shang,1 Xiao Sun,1 Lu Guo,1 Dandan Shi,1 Ping Liang,2 Dong Meng,1 Xiaoying Zhou,1 Xinxin Liu,1 Yading Zhao,1 Jie Li1 1Department of Ultrasound, Qilu Hospital of Shandong University, Jinan 250012, People’s Republic of China; 2Department of Interventional Ultrasound, Chinese PLA General Hospital, Beijing 100853, People’s Republic of ChinaCorrespondence: Jie LiDepartment of Ultrasound, Qilu Hospital of Shandong University, 107 Wenhua Xi Road, Jinan 250012, People’s Republic of ChinaTel +86-531-82166101Fax +86-531-86927544Email jieli301@163.comBackground: Synergistic chemoradiotherapy (CRT) has become a primary effective curative approach for many solid cancers. However, CRT is still associated with several obstacles, including the increases in side effects and systemic toxicity. Incorporating nanocarriers into CRT is a new and exciting approach to solve these obstacles. The purpose of the present study was to design a unique pH- and ultrasound-responsive perfluoropentane-encapsulated, paclitaxel (PTX)-loaded carboxymethyl chitosan nanodroplets (NDs) for combined imaging and synergistic CRT.Materials and Methods: The NDs were prepared by a homogenization/emulsion method. Their physicochemical properties, echogenicity and biocompatibility were evaluated. PTX-loaded NDs with a high loading efficiency and encapsulation efficiency were prepared and their pH-responsive drug release profile was determined by dialysis sack method. Then, PC3 cells were exposed to (1) PTX (4 μg/mL), (2) NDs (30 μg/mL), (3) PTX-loaded NDs (34 μg/mL), (4) RT (6 Gy), (5) RT (10 Gy), (6) combination of PTX (4 μg/mL), ultrasound (0.5 W/cm2, 30 s) and RT (6 Gy), (7) combination of NDs (30 μg/mL), ultrasound (0.5 W/cm2, 30 s) and RT (6Gy), (8) combination of PTX-loaded NDs (30 μg/mL), ultrasound (0.5 W/cm2, 30 s) and RT (6 Gy). 24 hrs later, CCK-8 assay, flow cytometry and migration assay were carried out to evaluate their therapeutic effects in CRT.Results: The desired NDs were successfully prepared, which were with round, spherical shapes, relatively smooth surfaces, core-shell structures and uniform in sizes (< 300 nm with PDI< 0.3 when at pH≧6.0). The NDs exhibited good abilities in pH-dependent charge conversion, biocompatibility and ultrasound contrast echogenicity. The in vitro drug release from PTX-loaded NDs (the highest loading efficiency and encapsulation efficiency were 20.35% and 91.58%) was pH dependent and exhibited an initial burst followed by a sustained drug release. The results of the CCK-8 assay, flow cytometry and migration assay all showed PTX-loaded NDs combined ultrasound and RT significantly enhanced cell responses in CRT.Conclusion: The pH- and ultrasound-responsive PTX-loaded NDs, which exhibited a high echogenicity, drug delivery ability and radiosensitization ability, could be a feasible option for combined imaging and novel enhancing approach in synergistic CRT.Keywords: nanodroplets, ultrasound contrast agents, chemoradiotherapy, radiosensitizer, paclitaxel, chitosan