In vivo imaging dye & probes

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1 In vivo imaging dye & probes Near-Infrared Fluorescent Dyes Description The Flamma NIR Flours series from BioActs is a Near-Infrared fluorescent dye product for animal imaging. BioActs products have high fluorescence intensity, high water solubility and low toxicity. The NIR Fluorescent Dyes products from BioActs offers fluorescence wavelength ranges from 600 to 900 nm, and small amount of interference by biomolecular autofluorescence occurs. With the effect of these long wavelengths, it can be used as an effective imaging product for In-vivo experiments. Flamma Fluors Absorbance Spectra Flamma Fluors Emission Spectra [Figure 1] Various absorption and fluorescence wavelengths of BioActs NIR products

BioActs' NIR Dye contains bioreactor such as NHS ester, Maleimide, Isothiocyanate, etc., so you can select the proper reactor you want and conduct effective experiments.

Product name Emission color Ex Max (nm) Em Max (nm) Common filter set Excitation laser line Size PWC1201 Flamma 648 Carboxylic acid Red 648 663 Cy 5 594, 633 nm 5mg, 25mg PWC1501 Flamma 675

2 BioActs' NIR Dye contains bioreactor such as NHS ester, Maleimide, Isothiocyanate, etc., so you can select the proper reactor you want and conduct effective experiments. [Figure 2] Selection of reactors for binding of biomaterials with amines [Figure 3] Selection of reactors for binding of biomolecules to Thiol Product List Cat. No. Product name Emission color Ex Max (nm) Em Max (nm) Common filter set Excitation laser line Size PWC1201 Flamma 648 Carboxylic acid Red Cy 5 594, 633 nm 5mg, 25mg PWC1501 Flamma 675 Carboxylic acid Far red Cy , 680 nm 5mg, 25mg PWC1308 Flamma 749 Carboxylic acid NIR Cy nm 5mg, 25mg PWC1603 Flamma 774 Carboxylic acid NIR Cy nm 5mg, 25mg POC1616 ICG Carboxylic acid NIR Cy nm 5mg, 25mg PWS1215 Flamma 648 NHS ester Red Cy 5 594, 633 nm 1mg, 5mg, 25mg

3 PWS1515 Flamma 675 NHS ester Far red Cy , 680 nm 1mg, 5mg, 25mg PWS1301 Flamma 749 NHS ester NIR Cy nm 1mg, 5mg, 25mg PWS1603 Flamma 774 NHS ester NIR Cy nm 1mg, 5mg, 25mg POS1604 ICG NHS ester NIR Cy nm 1mg, 5mg, 25mg PWSN1215 Flamma 648 Sulfo-NHS ester Red Cy 5 594, 633 nm 1mg, 5mg, 25mg PWSN1515 Flamma 675 Sulfo-NHS ester Far red Cy , 680 nm 1mg, 5mg, 25mg PWSN1301 Flamma 749 Sulfo-NHS ester NIR Cy nm 1mg, 5mg, 25mg PWSN1603 Flamma 774 Sulfo-NHS ester NIR Cy nm 1mg, 5mg, 25mg POSN1604 ICG Sulfo-NHS ester NIR Cy nm 1mg, 5mg, 25mg PWA1215 Flamma 648 Vinylsulfone Red Cy 5 594, 633 nm 1mg, 5mg, 25mg PWA1515 Flamma 675 Vinylsulfone Far red Cy , 680 nm 1mg, 5mg, 25mg PWA1603 Flamma 774 Vinylsulfone NIR Cy nm 1mg, 5mg, 25mg POA1616 ICG Vinylsulfone NIR Cy nm 1mg, 5mg, 25mg KWI1215 Flamma 648 Isothiocyanate Red Cy 5 594, 633 nm 1mg, 5mg, 25mg KWI1515 Flamma 675 Isothiocyanate Far red Cy , 680 nm 1mg, 5mg, 25mg PWI1603 Flamma 774 Isothiocyanate NIR Cy nm 1mg, 5mg, 25mg POI1616 ICG Isothiocyanate NIR Cy nm 1mg, 5mg, 25mg In vivo behavior experiment of NIR Dye near-infrared products - The experimental procedure for in-vivo imaging of the product is as follows. - Preparation: Balb/c nude male 5weeks old (We carry out the experiment at week 6 after 1 week stabilization period. - Animal experiments: Dilute each fluorescent substance to a concentration of 0.03 mg / ml. is Inject 100 μl into the prepared animal by intravenous injection. Observed Fluorescent images at 1 minute, 30 minutes, 1 hour, 2 hours, 3 hours, and 1 day intervals. (Observation equipment: IVIS)

injection, elimination from the body was

4 In-vivo Experiment result [Figure 4] After 1 day of Flamma 749 Carboxylic acid injection, elimination from the body was confirmed [Figure 5] After 1 day of Flamma 774 Carboxylic acid injection, elimination from the body was confirmed [Figure 6] After 1 day of ICG Carboxylic acid injection, elimination from the body was confirmed)

5 References - Paclitaxel loaded hyaluronic acid nanoparticles for targeted cancer therapy: In vitro and in vivo analysis (Reju G. Thomas,, MyeongJu Moon, SeJy Lee, Yong Yeon Jeong, International Journal of Biological Macromolecules, 2015, Volume 72, Pages ) - Photo-crosslinked hyaluronic acid nanoparticles with improved stability for in vivo tumor-targeted drug delivery (Hong Yeol Yoona, Heebeom Koo, Ki Young Choi, Ick Chan Kwon, Kuiwon Choi, Jae Hyung Park, Biomaterials, 2013, Volume 34, Issue 21, Pages ) - Co-delivery of VEGF and Bcl-2 dual-targeted sirna polymer using a single nanoparticle for synergistic anti-cancer effects in vivo (So Jin Lee, Simmyung Yook, Ji Young Yhee, Hong Yeol Yoon, Myung-Goo Kim, Sook Hee Ku, Sun Hwa Kim, Jae Hyung Park, Ji Hoon Jeong, Ick Chan Kwon, Seulki Lee, Hyukjin Lee, Kwangmeyung Kim, Journal of Controlled Release, 2015, Volume 220, Part B, Pages ) - Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model (Reju George Thomas, Myeong Ju Moon, Jo Heon Kim, Jae Hyuk Lee, Yong Yeon Jeong, PLoS ONE, 2015, 10(12): e ) - Notch1 targeting sirna delivery nanoparticles for rheumatoid arthritis therapy (Min Ju Kim, Jong-Sung Park, So Jin Lee, Jiyeon Jang, Jin Su Park, Seung Hyun Back, Gahee Bahn, Jae Hyung Park, Young Mo Kang, Sun Hwa Kim, Ick Chan Kwon, Dong-Gyu Jo, Kwangmeyung Kim, Journal of Controlled Release, 2015, Volume 216, Pages ) - Cancer-targeted MDR-1 sirna delivery using self-cross-linked glycol chitosan nanoparticles to overcome drug resistance (Ji Young Yhee, Seungyong Song, So Jin Lee, Sung-Gurl Park, Ki-Suk Kim, Myung Goo Kim, Sejin Son, Heebeom Koo, Ick Chan Kwon, Ji Hoon Jeong, Seo Young Jeong, Sun Hwa Kim, Kwangmeyung Kim, Journal of Controlled Release, 2015, Volume 198, Pages 1 9) - A new fluorescence/pet probe for targeting intracellular human telomerase reverse transcriptase (htert) using Tat peptide-conjugated IgM (Kyung oh Jung, Hyewon Youn, Seung Hoo Kim, Young-Hwa Kim, Keon Wook Kang, June-Key Chung, Biochemical and Biophysical Research Communications, 2016, Volume 477, Issue 3, Pages ) - Controlled Release of Hepatocyte Growth Factor from MPEG-b-(PCL-ran-PLLA) Diblock Copolymer for Improved Vocal Fold Regeneration (Jae Won Choi, Yeon Soo Kim, Ju Kyeong Park, Eun Hye Song, Ji Hoon Park, Moon Suk Kim, Yoo Seob Shin, Chul-Ho Kim, Macromolecular Bioscience, 2016, DOI: /mabi )

6 AngioFlamma series Description Integrin is a heterodimeric cell-surface receptor that binds to an arginine-glycine-aspartate (RGD) sequence and mediates adhesion and fixation between cells and substrates outside the cell. Integrin is also associated with cell signaling and gene expression associated with cell growth, migration and survival. Integrin is used as a marker in disease studies because it is involved in blood clotting, inflammatory reactions and cancer manifestations. BioActs' AngioFlamma products are based on RGD peptides and are fluorescent probe materials with Flamma Fluors fluorescence products that are used in vivo inflammatory reactions, detection of tumor and angiogenesis Product List Cat. No. Product name Emission color Ex Max (nm) Em Max (nm) Common filter set Excitation laser line ARW1025 AngioFlamma FAM Green FITC 488 nm 0.5mg, 1mg, 5mg ARW1011 AngioFlamma 552 Yellow TRITC 488, 532 nm 0.5mg, 1mg, 5mg ARR1001 AngioFlamma TAMRA Orange TRITC 488, 532 nm 0.5mg, 1mg, 5mg ARW1028 AngioFlamma 560 Orange TRITC 488, 532 nm 0.5mg, 1mg, 5mg ARW1215 AngioFlamma 648 Far red Cy 5 594, 633 nm 0.5mg, 1mg, 5mg ARW1501 AngioFlamma 675 NIR Cy , 680 nm 0.5mg, 1mg, 5mg ARW1301 AngioFlamma 749 NIR Cy nm 0.5mg, 1mg, 5mg ARW1601 AngioFlamma 774 NIR Cy nm 0.5mg, 1mg, 5mg ARO1601 AngioFlamma ICG NIR Cy nm 0.5mg, 1mg, 5mg Size In-vivo Imaging Protocol - The experiment procedure for in-vivo imaging of the product proceeds as follows. - PREPARING XENOGRAFT TUMOR MODEL FOR IMAGING 1. MDA-MB-231 human breast cancer cells (1.0 x 10 7 ) should be injected subcutaneously on the female nude mouse shoulder. 2. After the tumor cells have grown to about mm 3, administer the drug. - INJECTION and FLUORESCENCE IMAGING 1. Fluorescence imaging was performed up to 24 hours after injection using the explore

Optix system (ART Advanced Research Technologies

excitation (λex = 675 nm) and emission (λem =

Prepared mice were injected intravenously with

[Figure 7] Breast cancer cell imaging using

Chitosan Polyamidoamine Dendrimer Complex

7 Optix system (ART Advanced Research Technologies Inc., Montreal, Canada) and Flamma 675 products, excitation (λex = 675 nm) and emission (λem = 698 nm) filter sets. 2. Prepared mice were injected intravenously with 100 μg / 200 μl of AngioFlamma 675 product and images were taken at set times. In-vivo Imaging Result 10min 3h 6h 9h 12h 24h [Figure 7] Breast cancer cell imaging using AngioFlamma 675 References - Zwitterionic Chitosan Polyamidoamine Dendrimer Complex Nanoparticles as a ph-sensitive Drug Carrier (Karen C. Liu, Yoon Yeo, Mol. Pharmaceutics, 2013, 10 (5), pp )

ApoFlamma series Description The ApoFlamma series is a family of fluorescent probe products for detecting apoptotic cells, allowing the observation of cells through fluorescence imaging by binding to

8 ApoFlamma series Description The ApoFlamma series is a family of fluorescent probe products for detecting apoptotic cells, allowing the observation of cells through fluorescence imaging by binding to apoptotic cells. Among the ApoFlamma series, the ApoFlamma PS products allows fluorescence imaging to be observed by specifically binding to the phosphatidylserine exposed to the outer membrane of the apoptotic cell. The ApoFlamma H product allows fluorescent imaging to be observed by specifically binding to Histone 1 released to the outside by the apoptotic process. Both product lines can be observed by in vivo experiments. In particular, near-infrared fluorescence products can be used without being affected by the autofluorescence of animal. [Figure 8] Imaging using ApoFlamma PS 648 of the Etoposide-treated Hela cell

Orange 543 575 TRITC 488, 532 nm 10 doses, 50 doses, 200 doses APW1011 ApoFlamma PS 552 Yellow 550 565 TRITC 488, 532 nm 10 doses, 50 doses, 200 doses APW1215 ApoFlamma PS 648 Red 648 663 Cy 5 594,

9 [Figure 9] Imaging with ApoFlamma PS 675 of B16F10 cells treated with Etoposide Product List Cat. No. Product name Emission color Ex Max (nm) Em Max (nm) Common filter set Excitation laser line APW1025 ApoFlamma PS FAM Green FITC 488 nm 10 doses, 50 doses, 200 doses APP1001 ApoFlamma PS TAMRA Orange TRITC 488, 532 nm 10 doses, 50 doses, 200 doses APW1011 ApoFlamma PS 552 Yellow TRITC 488, 532 nm 10 doses, 50 doses, 200 doses APW1215 ApoFlamma PS 648 Red Cy 5 594, 633 nm 10 doses, 50 doses, 200 doses APW1501 ApoFlamma PS 675 Far red Cy , 680 nm 10 doses, 50 doses, 200 doses APW1301 ApoFlamma PS 749 NIR Cy nm 10 doses, 50 doses, 200 doses APW1601 ApoFlamma PS 774 NIR Cy nm 10 doses, 50 doses, 200 doses APO1601 ApoFlamma PS ICG NIR Cy nm 10 doses, 50 doses, 200 doses AHW1025 ApoFlamma H FAM Green FITC 488 nm 10 doses, 50 doses, 200 doses AHH1001 ApoFlamma H TAMRA Orange TRITC 488, 532 nm 10 doses, 50 doses, 200 doses AHW1011 ApoFlamma H 552 Yellow TRITC 488, 532 nm 10 doses, 50 doses, 200 doses AHW1215 ApoFlamma H 648 Red Cy 5 594, 633 nm 10 doses, 50 doses, 200 doses AHW1501 ApoFlamma H 675 Far red Cy , 680 nm 10 doses, 50 doses, 200 doses AHW1301 ApoFlamma H 749 NIR Cy nm 10 doses, 50 doses, 200 doses AHW1601 ApoFlamma H 774 NIR Cy nm 10 doses, 50 doses, 200 doses AHO1601 ApoFlamma H ICG NIR Cy nm 10 doses, 50 doses, 200 doses Size

- When the tumor size of the mice was grown to 1 cm, injected intravenously with 100 μl of ApoFlamma (50 μm each) via tail vein after isoflurane anesthesia.

10 In-vivo Imaging Protocol - The experiment procedure for in-vivo imaging of the product proceeds as follows. - Six-week-old Balb/c female nude mice were injected subcutaneously with 1 x 10 8 MDA231 tumor cells suspended in RPMI medium containing 10% FBS in the right femur. - When the tumor size of the mice was grown to 1 cm, injected intravenously with 100 μl of ApoFlamma (50 μm each) via tail vein after isoflurane anesthesia minutes after the injection of ApoFlamma, the tumor was observed via the explorer Optix (GE healthcare, USA) imaging equipment. In-vivo Imaging Result [Figure 10] Imaging Apoptosis process of drug-injected tumor cells using ApoFlamma H 774

11 [Figure 11] Imaging Apoptosis process of rheumatoid using ApoFlamma product [Figure 12] Imaging images of protective drugs using ApoFlamma H 774 with Parkinson's disease model induced by injection of MPTP drug.

[Figure 13] Apoptotic cells by camptothecin imaging with ApoFlamma PS FAM References - Monitoring the correlation between I-uptake and apoptosis using Apoptosis-targeting peptide-1 (ApoPep- 1) (Kyung

supplement 1, 1761) - Relationship between Apoptosis Imaging and Radioiodine Therapy in Tumor Cells with Different Sodium Iodide Symporter Gene Expression (Kyung Oh Jung, Hyewon Youn, Young-Hwa Kim,

12 [Figure 13] Apoptotic cells by camptothecin imaging with ApoFlamma PS FAM References - Monitoring the correlation between I-uptake and apoptosis using Apoptosis-targeting peptide-1 (ApoPep- 1) (Kyung Oh Jung, Seung Hoo Kim, Hyewon Youn, Keon Kang, Dong Soo Lee, June-Key Chung, J Nucl Med, 2011, vol. 52, no. supplement 1, 1761) - Relationship between Apoptosis Imaging and Radioiodine Therapy in Tumor Cells with Different Sodium Iodide Symporter Gene Expression (Kyung Oh Jung, Hyewon Youn, Young-Hwa Kim, Seunghoo Kim, Juri Na, Yong-il Kim, Jin Woo Park, Keon Wook Kang, Dong Soo Lee, June-Key Chung, Molecular Imaging, 2014: pp 1 9) - Sodium [18F]Fluoride PET/CT in Myocardial Infarction (Jeong Hee Han, Sue Yeon Lim, Min Su Lee, Won Woo Lee, Molecular Imaging and Biology, 2015, Volume 17, Issue 2, pp ) - A novel method to detect articular chondrocyte death during early stages of osteoarthritis using a noninvasive ApoPep-1 probe (Xiangguo Che, Lianhua Chi, Clara Yongjoo Park, Gyoung-Ho Cho, Narae Park, Seong-Gon Kim, Byung-Heon Lee, Je-Yong Choi, Arthritis Research & Therapy, 2015, 17:309) - In Vivo Near-Infrared Fluorescence Imaging of Apoptosis Using Histone H1-Targeting Peptide Probe after Anti-Cancer Treatment with Cisplatin and Cetuximab for Early Decision on Tumor Response (Hyun- Kyung Jung, Kai Wang, Min Kyu Jung, In-San Kim, Byung-Heon Lee, PLoS ONE, 2014, 9(6): e100341) - In vivo imaging of myocardial cell death using a peptide probe and assessment of long-term heart function (Bodhraj Acharya, Kai Wang, In-San Kim, WoongChol Kang, Chanil Moon, Byung-Heon Lee, Journal of Controlled Release, 2013, Volume 172, Issue 1, Pages )

NpFlamma HGC series Description As people s interest in cancer increase, the importance of imaging cancer in order to develop an anticancer drug along with the diagnosis of cancer is increasing day

13 NpFlamma HGC series Description As people s interest in cancer increase, the importance of imaging cancer in order to develop an anticancer drug along with the diagnosis of cancer is increasing day by day. NpFlamma HGC series is the nano-particle complex of amphiphilic polymer and near-infrared phosphor that enable to selectively accumulate in the cancer tissues due to high permeability of angiogenesis of cancer tissues. Therefore, it is easy for passive targeting tumor imaging using near-infrared penetration. In addition, it is possible to contrast blood vessel through injecting probe into the blood vessel. NpFlamma HGC Series inside super hydrophobic material enables to embed hydrophobic drug and accordingly it can be used for efficient transfer of tumor drug through interlinking with passive targeting action. NpFlamma HGC Series is the nano-particle produced based on chitosan, which has almost no toxicity and side effect, and also has a good merit having a long half-life, high stability and high water solubility. It seems that the use of this product enables more effective optical image. [Figure 14] An effective tumor imaging is available using NpFlamma HGC products group.

[Figure 15] NpFlamma HGC products group can penetrate into tumor cell tissues to observe strong fluorescence specifically to cancer cell Product List Cat. No.

675 Far red 675 698 Cy 5.5 633, 680 nm 10 doses, 50 doses, 100 doses PNC1301 NpFlamma HGC 749 NIR 750 782 Cy 7 680 nm 10 doses, 50 doses, 100 doses PNC1601 NpFlamma HGC 774 NIR 777 802 Cy 7.

14 [Figure 15] NpFlamma HGC products group can penetrate into tumor cell tissues to observe strong fluorescence specifically to cancer cell Product List Cat. No. Product name Emission color Ex Max (nm) Em Max (nm) Common filter set Excitation laser line PNC1201 NpFlamma HGC 648 Red Cy 5 594, 633 nm 10 doses, 50 doses, 100 doses PNC1401 NpFlamma HGC 675 Far red Cy , 680 nm 10 doses, 50 doses, 100 doses PNC1301 NpFlamma HGC 749 NIR Cy nm 10 doses, 50 doses, 100 doses PNC1601 NpFlamma HGC 774 NIR Cy nm 10 doses, 50 doses, 100 doses PNC1801 NpFlamma HGC ICG NIR Cy nm 10 doses, 50 doses, 100 doses Size In-vivo Imaging Protocol - The experiment procedure for in-vivo imaging of the product is proceeded as follows. - Prepare the experiment through vortexing by putting DW or PBS into the NpFlamma HGC series powder (1does=120ug/100ul) - As fluorescent substance is weak against the light, it must be stored in the condition protected from light - Mouse fur may cause scattering, absorption etc. of excitation light when optical imaging is in process. Therefore, it is necessary to use nude mouse or remove the mouse fur for preparation of the test. - It is recommended to prepare 31G syringe as injection must be conducted through the vein of mouse. - Prepare 5 week-old mouse Balb/c-nude, male.

- Inject NpFlamma HGC series (120ug/100ul) intravenously into mouse.

15 - Inject SCC7 cell line (1x10 6 /0.1ml) subcutaneous of Balb/c-nude mouse. - When the volume of tumor becomes 60~80mm 3, take a time-zero image of each subject before injection of the agent. - Inject NpFlamma HGC series (120ug/100ul) intravenously into mouse. - The optimal imaging time point is 1hr, 3hr, 6hr, 9hr and 24 hr post injection of the NpFlamma HGC series. - When take a 24hr imaging post injection of NpFlamma HGC series, extract major organs (e.g liver, lung, spleen, kidney, heart, and tumor) and then take of ex-vivo imaging. In-vivo Imaging Result (IVIS)

16 [Figure 16] As result of conducting fluorescence observation for the mouse after injecting NpFlamma HGC products group up to max. 7 days, it was possible to confirm a strong fluorescence from cancer tissues until the 7 th day

NpFlamma HGC 675 (n=5) AngioSense 680EX (n=5) * 각 Organ 의 Liver 에대한형광 Ratio. Liver Lung Spleen Kidney Heart Tumor 1 1 0.940-0.509-1.812 2 1 0.

Liver Lung Spleen Kidney Heart Tumor 1 1 0.325-0.406 0.226 1.634 2 1 0.431-0.368 0.237 1.329 3 1 0.358-0.324 0.203 1.022 4 1 0.276 0.158 0.330 0.

Liver Lung Spleen Kidney Heart Tumor 1 1 1.632 - - - 2.271 2 1 0.879 - - - 2.292 3 1 0.834 - - - 2.171 4 1 0.682 - - - 2.762 5 1 1.476 - - - 1.

17 NpFlamma HGC 675 (n=5) AngioSense 680EX (n=5) * 각 Organ 의 Liver 에대한형광 Ratio. Liver Lung Spleen Kidney Heart Tumor * 각 Organ 의 Liver 에대한형광 Ratio. Liver Lung Spleen Kidney Heart Tumor NpFlamma HGC 749 (n=5) AngioSense 750EX (n=5) * 각 Organ 의 Liver 에대한형광 Ratio. Liver Lung Spleen Kidney Heart Tumor * 각 Organ 의 Liver 에대한형광 Ratio. Liver Lung Spleen Kidney Heart Tumor [Figure 17] As result of comparing ex-vivo image of organ after 24 hr for NpFlamma HGC product and the other company s product, it was confirmed that tumor accumulating efficiency against the other s organ was high against the other s tested organ, and that the accumulating trend of the other company s tested organ was low

18 References - New generation of multifunctional nanoparticles for cancer imaging and therapy (Kyeongsoon Park, Seulki Lee, Eunah Kang, Kwangmeyung Kim, Kuiwon Choi, Ick Chan Kwon, Advanced Functional Materials, 2009, Volume 19, Issue 10, Pages ) - Tumor-homing multifunctional nanoparticles for cancer theragnosis: Simultaneous diagnosis, drug delivery, and therapeutic monitoring (Kwangmeyung Kim, Jong Ho Kim, Hyungkyu Park, Yoo-Shin Kim, Kyeongsoon Park, Heayun Nam, Seulki Lee, Jae Hyung Park, Rang-Woon Park, In-San Kim, Kuiwon Choi, Sang Yoon Kim, Kinam Park, Ick Chan Kwon, Journal of Controlled Release, 2010, Volume 146, Issue 2, Pages ) - Glycol chitosan nanoparticles as specialized cancer therapeutic vehicles: Sequential delivery of doxorubicin and Bcl-2 sirna (Hong Yeol Yoon, Sejin Son, So Jin Lee, Dong Gil You, Ji Young Yhee, Jae Hyung Park, Maggie Swierczewska, Seulki Lee, Ick Chan Kwon, Sun Hwa Kim, Kwangmeyung Kim & Martin G. Pomper, Scientific Reports, 2014, 4, Article number: 6878) NpFlamma MMP series Description Cancer cell is influenced by ECM (extracellular matrix), ECM-related growth factors and cytokine and surrounding cells in the growth process of cancer cell. Especially four characteristics of cancer such as migration, invasion, metastasis and angiogenesis react according to the surrounding micro environment. In this case, MMP (matrix metalloprotease) that is the protease controls cell-cell and cell-ecm interactions playing an important role in the growth of cancer cell. Accordingly a number of researches have been proceeding a variety of researches using MMP. As this product generates fluorescence according to the absolute volume of MMP, it can be effectively accumulated in the cancer tissues and it is possible for optical imaging of the tissues where MMP is over-expressed. As this product enables to quickly check activity and suppression of protease through imaging, which can be applied to drug screening, and it is possible for real-time based cell imaging and non-invasive tissues imaging for cell and tissue. Especially in addition to the method of screening new drug such as inhibitor that inhibits over-expression of protease, it can be used usefully for early diagnosis of a variety of diseases and incurable diseases like autoimmune diseases that are MMP-related cancer, ostarthritis, rheumarthritis, dementia, etc. In addition, this products group is expected to be easy to use owing to have various fluorescent wavelengths.

19 [Figure 18] Operation principle of NpFlamma MMP Series and high reaction to Enzyme were confirmed [Figure 19] High restoring force of fluorescence of NpFlamma MMP Series according to conc. of Trypsin was confirmed

20 Product List Emission Cat. No. Product name Color Ex Max Em Max Common Excitation (nm) (nm) filter set laser line PNM0101 NpFlamma MMP-2,9 ICG NIR Cy nm 10 dose, 50 dose, 100 dose PNM0103 NpFlamma MMP-2,9 648 Red Cy 5 594, 633 nm 10 dose, 50 dose, 100 dose PNM0104 NpFlamma MMP-2,9 675 Far red Cy nm 10 dose, 50 dose, 100 dose PNM0105 NpFlamma MMP-2,9 749 NIR Cy nm 10 dose, 50 dose, 100 dose PNM0106 NpFlamma MMP-2,9 774 NIR Cy nm 10 dose, 50 dose, 100 dose Size In-vivo Imaging Protocol - The experiment procedure for in-vivo imaging of the product is proceeded as follows. - Prepare the experiment through vortexing by putting DW or PBS into the NpFlamma MMP series powder (1does=120ug/100ul) - As fluorescent substance is weak against the light, it must be stored in the condition protected from light - Mouse fur may cause scattering, absorption etc. of excitation light when optical imaging is in process. Therefore, it is necessary to use nude mouse or remove the mouse fur for preparation of the test. - It is recommended to prepare 31G syringe as injection must be conducted through the vein of mouse. - Prepare 5 week-old mouse Balb/c-nude, male. - Inject SCC7 cell line (1x10 6 /0.1ml) subcutaneous of Balb/c-nude mouse. - When the volume of tumor becomes 60~80mm 3, take a time-zero image of each subject before injection of the agent. - Inject NpFlamma MMP series (120ug/100ul) intravenously into mouse. - The optimal imaging time point is 1hr, 3hr, 6hr, 9hr and 24 hr post injection of the NpFlamma MMP series - When take a 24hours imaging post injection of NpFlamma MMP series, extract major organs (e.g liver, lung, spleen, Kidney, heart, and tumor) and then take of ex-vivo imaging.

product and the other s product, high tumor

21 In-vivo Imaging Result (IVIS) [Figure 20] As result of comparing the in-vivo images by hours for NpFlamma MMP product and the other s product, high tumor accumulating efficiency against the other s tested organ was confirmed

22 [Figure 21] As result of comparing the ex-vivo images of organ between NpFlamma MMP product and the other s product, high tumor accumulating efficiency against the other s organ was confirmed

23 References - Optimization of matrix metalloproteinase fluorogenic probes for osteoarthritis imaging (Ju Hee Ryu, Aeju Lee, Jin Hee Na, Seulki Lee, Hyung Jun Ahn, Jong Woong Park, Cheol-Hee Ahn, Byung-Soo Kim, Ick Chan Kwon, Kuiwon Choi, Inchan Youn, Kwangmeyung Kim, Amino Acids, 2011, Volume 41, Issue 5, pp ) - Dark Quenched Matrix Metalloproteinase Fluorogenic Probe for Imaging Osteoarthritis Development in Vivo (Seulki Lee, Kyeongsoon Park, Seung-Young Lee, Ju Hee Ryu, Jong Woong Park, Hyung Jun Ahn, Ick Chan Kwon, In-Chan Youn, Kwangmeyung Kim, Kuiwon Choi, Bioconjugate Chem., 2008, 19 (9), pp ) - Early Diagnosis of Arthritis in Mice With Collagen-Induced Arthritis, Using a Fluorogenic Matrix Metalloproteinase 3 Specific Polymeric Probe (Ju Hee Ryu, Aeju Lee, Jun-Uk Chu, Heebeom Koo, Chang-Yong Ko, Han Sung Kim, Soo-Young Yoon, Byung-Soo Kim, Kuiwon Choi, Ick Chan Kwon, Kwangmeyung Kim, Inchan Youn, ARTHRITIS & RHEUMATISM, 2011, Vol. 63, No. 12, pp ) - Polymeric Nanoparticle-Based Activatable Near-Infrared Nanosensor for Protease Determination In Vivo (Seulki Lee, Ju Hee Ryu, Kyeongsoon Park, Aeju Lee, Seung-Young Lee, In-Chan Youn, Cheol-Hee Ahn, Soon Man Yoon, Seung-Jae Myung, Dae Hyuk Moon, Xiaoyuan Chen, Kuiwon Choi, Ick Chan Kwon, Kwangmeyung Kim, Nano Lett., 2009, Vol. 9, No. 12, pp )

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Smart Nanocarrier Based on PEGylated Hyaluronic Acid for Cancer Therapy

(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,