Smart Nanocarrier Based on PEGylated Hyaluronic Acid for Cancer Therapy

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1 (Supporting information for ACS Nano) Smart Nanocarrier Based on PEGylated Hyaluronic Acid for Cancer Therapy Ki Young Choi,, Hong Yeol Yoon,, Jong-Ho Kim, Sang Mun Bae, Rang-Woon Park, Young Mo Kang, In-San Kim, Ick Chan Kwon, Kuiwon Choi, Seo Young Jeong, Kwangmeyung Kim,**, Jae Hyung Park, * Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul , Republic of Korea, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul , Republic of Korea, Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon , Republic of Korea, School of Medicine, Kyungpook National University, Daegu , Republic of Korea *Corresponding author: Jae Hyung Park, Ph.D. Theranostic Macromolecules Research Center Department of Polymer Science and Engineering Sungkyunkwan University, Suwon , Republic of Korea Tel: ; fax: ; jhpark1@skku.edu **Co-corresponding author: Kwangmeyung Kim, Ph.D. Biomedical Research Institute Korea Institute of Science and Technology, Seoul , Korea Tel: ; fax: ; kim@kist.re.kr S1

2 In Vitro Cellular Uptake of HA-NPs. SCC7 cells were cultured in RPMI1640 medium (Gibco, Grand Island, NY, USA) and MDA-MB-231 and NIH-3T3 cells were cultured in DMEM medium. All culture media contained 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and culturing conditions consisted of 37 o C in a humidified 5% CO 2 atmosphere. After cell attachment, the medium was replaced with 2 ml of serum-free culture medium containing Cy5.5-labeled nanoparticles (20 µg/ml), followed by incubation for 10 and 60 min. The cells were then washed twice with PBS (ph 7.4) and fixed with a 4% paraformaldehyde solution. The intracellular localization of nanoparticles was observed using an IX81-ZDC focus drift compensating microscope (Olympus, Tokyo, Japan) with excitation and emission wavelengths of 673 nm and 692 nm, respectively. In order to monitor cellular uptake of P-HA-NPs in different cell lines, Cy5.5-labeled NPs were incubated with cancer cells (MDA-MB-231 and SCC7) and normal fibroblast cells (NIH-3T3) for 10 and 90 min. The cancer cells used in this study have been demonstrated to over-express CD44 on their surfaces compared to normal cells (including NIH-3T3), which are reported not to over-express CD As shown in Supporting Information Figure S1, fluorescent signals were detected in the cytoplasm of cancer cells at 10 min post incubation and became stronger after 90 min. In contrast, when Cy5.5-labeled HA-NPs were incubated with fibroblasts (NIH-3T3), much weaker signals were observed after both 10 and 90 min. These results suggest that the P-HA-NPs prepared in this study selectively bound to CD44 and were rapidly internalized into the cancer cells via receptor-mediated endocytosis, while rarely being internalized by normal cells. The cellular imaging results are also in good agreement with our previous report 6. S2

3 Figure S1. Cellular uptake behavior of Cy5.5-labeled P-HA-NPs in vitro. Confocal images of normal fibroblast cells (NIH-3T3) and cancer cells (MDA-MB-231 and SCC7) incubated with Cy5.5-labeled P-HA-NPs at a concentration of 20µg/ml. Cellular uptake profile was examined under a fluorescent confocal microscope using the Z-stack method at pre-determined time points. S3

4 Figure S2. Size distribution of P-HA-NP, DOX-P-HA-NP and CPT-P-HA-NP. S4

5 Figure S3. TEM images of CPT-P-HA-NP incubated with Hyal. S5

6 References 1. Lee, H.; Mok, H.; Lee, S.; Oh, Y. K.; Park, T. G., Target-specific intracellular delivery of sirna using degradable hyaluronic acid nanogels. J Control Release 2007, 119 (2), Luo, Y.; Prestwich, G. D., Synthesis and selective cytotoxicity of a hyaluronic acidantitumor bioconjugate. Bioconjug Chem 1999, 10 (5), Sheridan, C.; Kishimoto, H.; Fuchs, R. K.; Mehrotra, S.; Bhat-Nakshatri, P.; Turner, C. H.; Goulet, R., Jr.; Badve, S.; Nakshatri, H., CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis. Breast Cancer Res 2006, 8 (5), R Choi, K. Y.; Min, K. H.; Na, J. H.; Choi, K.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y., Self-assembled hyaluronic acid nanoparticles as a potential drug carrier for cancer therapy: synthesis, characterization, and in vivo biodistribution. J Mater Chem 2009, 19 (24), Choi, K. Y.; Chung, H.; Min, K. H.; Yoon, H. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y., Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010, 31 (1), Choi, K. Y.; Min, K. H.; Yoon, H. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Choi, K.; Jeong, S. Y., PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials 2011, 32 (7), S6