PEPTIDIC SCAFFOLDS FOR TARGETED DELIVERY OF PROTEASE-SENSITIVE
3. Results and Discussion
3.3. Intracellular fate of PS in cells - Confocal Microscopy
Figure 2. Intracellular localization of probes AC203 (A), AC560 (B) , AC594 (C) and AC226 (D) (in red) after 2 hours of incubation. Lysotracker green® (in green) is used to mark lysosomes and DAPI (in blue) was used as a marker for nuclei.
Figure 2 presents the intracellular localization of all probes assessed after 2 hours of incubation. All probes, except AC595 (negative control) showed strong fluorescence.
Intracellular accumulation was observed as a form of vacuolar structures. This confirms that the cellular uptake is dependent on the peptidic sequence used and its recognition by target proteases in this case, hence giving specificity of these probes towards protease expressing cells. Similar vacuolar localization was observed using polymeric prodrugs29. Furthermore probe AC594 which targets cathepsin B showed a good co-localization with stained lysosomes. These observations are in accordance with cathepsin B intracellular localization since it is known to participate in the early and late stages of endolysosomal breakdown of proteins and have higher concentration of cathepsin B is found in early endosomes and less in
168 lysosomes37. Interestingly, AC226 showed vacuolar but also cytoplasmic localization that was not present for the other probes tested. This observation suggests a fast cellular uptake of this probe as compared to the other probes tested. However, all probes studied in this paper exhibited good cellular activation and uptake with high specificity toward PC-3 cells giving hope for the in vivo diagnosis of prostate cancer.
4. Conclusions
The high quenching efficiency of the probes as well as their cellular activation and accumulation with high specificity towards PC-3 cells is of great opportunity for the in vivo diagnosis of prostate cancer and many other protease-related tumors. Tuning these probes for other target proteases can be easily achieved by varying the protease cleavable peptidic sequence and may open new horizons in in vivo imaging of cancer and other overexpressing protease diseases such as rheumatoid arthritis, Alzheimer’s disease, stroke and atherosclerosis38.
Acknowledgements
This work has been supported in part by the SNF grants 205320_13830, CR32I3_129987, CR32I3_147018, 31003A_149962, CR32I3_150271, and 205321_12683
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References:
1. Celli JP, Spring BQ, Rizvi I, et al: Imaging and photodynamic therapy: mechanisms, monitoring, and optimization. Chem Rev 110:2795-838, 2010
2. Gabriel D, Zuluaga MF, Lange N: On the cutting edge: protease-sensitive prodrugs for the delivery of photoactive compounds. Photochem Photobiol Sci 10:689-703, 2011 3. Law B, Tung CH: Proteolysis: a biological process adapted in drug delivery, therapy,
and imaging. Bioconjug Chem 20:1683-95, 2009
4. Lovell JF, Liu TW, Chen J, et al: Activatable photosensitizers for imaging and therapy. Chem Rev 110:2839-57, 2010
5. Hanahan D, Weinberg RA: The hallmarks of cancer. Cell 100:57-70, 2000
6. Hanahan D, Weinberg RA: Hallmarks of cancer: the next generation. Cell 144:646-74, 2011
7. Lee M, Fridman R, Mobashery S: Extracellular proteases as targets for treatment of cancer metastases. Chem Soc Rev 33:401-9, 2004
8. Lopez-Otin C, Bond JS: Proteases: multifunctional enzymes in life and disease. J Biol Chem 283:30433-7, 2008
9. Morris R, Winyard PG, Blake DR, et al: Thrombin in inflammation and healing:
relevance to rheumatoid arthritis. Ann Rheum Dis 53:72-9, 1994
10. Nakano S, Ikata T, Kinoshita I, et al: Characteristics of the protease activity in synovial fluid from patients with rheumatoid arthritis and osteoarthritis. Clin Exp Rheumatol 17:161-70, 1999
11. So AK, Varisco PA, Kemkes-Matthes B, et al: Arthritis is linked to local and systemic activation of coagulation and fibrinolysis pathways. J Thromb Haemost 1:2510-5, 2003
12. Lee S, Park K, Kim K, et al: Activatable imaging probes with amplified fluorescent signals. Chem Commun (Camb):4250-60, 2008
13. Sekar RB, Periasamy A: Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 160:629-33, 2003
170 14. Lo PC, Chen J, Stefflova K, et al: Photodynamic molecular beacon triggered by fibroblast activation protein on cancer-associated fibroblasts for diagnosis and treatment of epithelial cancers. J Med Chem 52:358-68, 2009
15. Lovell JF, Chen J, Huynh E, et al: Facile synthesis of advanced photodynamic molecular beacon architectures. Bioconjug Chem 21:1023-5, 2010
16. Zheng G, Chen J, Stefflova K, et al: Photodynamic molecular beacon as an activatable photosensitizer based on protease-controlled singlet oxygen quenching and activation.
Proc Natl Acad Sci U S A 104:8989-94, 2007
17. Chen J, Stefflova K, Niedre MJ, et al: Protease-triggered photosensitizing beacon based on singlet oxygen quenching and activation. Journal of the American Chemical Society 126:11450-11451, 2004
18. Stefflova K, Chen J, Marotta D, et al: Photodynamic therapy agent with a built-in apoptosis sensor for evaluating its own therapeutic outcome in situ. Journal of Medicinal Chemistry 49:3850-3856, 2006
19. Liu TW, Akens MK, Chen J, et al: Imaging of Specific Activation of Photodynamic Molecular Beacons in Breast Cancer Vertebral Metastases. Bioconjugate Chemistry 22:1021-1030, 2011
20. Andreasen PA, Kjoller L, Christensen L, et al: The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 72:1-22, 1997
21. Cozzi PJ, Wang J, Delprado W, et al: Evaluation of urokinase plasminogen activator and its receptor in different grades of human prostate cancer. Hum Pathol 37:1442-51, 2006
22. Kim TD, Song KS, Li G, et al: Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer. BMC Cancer 6:211, 2006
23. Konecny G, Untch M, Pihan A, et al: Association of urokinase-type plasminogen activator and its inhibitor with disease progression and prognosis in ovarian cancer.
Clin Cancer Res 7:1743-9, 2001
24. Lah TT, Kokalj-Kunovar M, Drobnic-Kosorok M, et al: Cystatins and cathepsins in breast carcinoma. Biol Chem Hoppe Seyler 373:595-604, 1992
25. Sidenius N, Blasi F: The urokinase plasminogen activator system in cancer: recent advances and implication for prognosis and therapy. Cancer Metastasis Rev 22:205-22, 2003
171 26. Staack A, Tolic D, Kristiansen G, et al: Expression of cathepsins B, H, and L and their inhibitors as markers of transitional cell carcinoma of the bladder. Urology 63:1089-94, 2004
27. van der Burg ME, Henzen-Logmans SC, Berns EM, et al: Expression of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in benign, borderline, malignant primary and metastatic ovarian tumors. Int J Cancer 69:475-9, 1996
28. Chevalier A, Massif C, Renard PY, et al: Bioconjugatable azo-based dark-quencher dyes: synthesis and application to protease-activatable far-red fluorescent probes.
Chemistry 19:1686-99, 2013
29. Gabriel D, Zuluaga MF, Martinez MN, et al: Urokinase-plasminogen-activator sensitive polymeric photosensitizer prodrugs: design, synthesis and in vitro evaluation.
Journal of Drug Delivery Science and Technology 19:15-24, 2009
30. Zuluaga MF, Gabriel D, Lange N: Enhanced Prostate Cancer Targeting by Modified Protease Sensitive Photosensitizer Prodrugs. Molecular Pharmaceutics 9:1570-1579, 2012
31. Bogdanov AA, Lin CP, Simonova M, et al: Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment. Neoplasia 4:228-236, 2002 32. Tung CH, Mahmood U, Bredow S, et al: In vivo imaging of proteolytic enzyme
activity using a novel molecular reporter. Cancer Research 60:4953-4958, 2000
33. Jaffer FA, Kim DE, Quinti L, et al: Optical visualization of cathepsin K activity in atherosclerosis with a novel, protease-activatable fluorescence sensor. Circulation 115:2292-2298, 2007
34. Bouteiller C, Clave G, Bernardin A, et al: Novel water-soluble near-infrared cyanine dyes: Synthesis, spectral properties, and use in the preparation of internally quenched fluorescent probes. Bioconjugate Chemistry 18:1303-1317, 2007
35. Meyer Y, Richard JA, Delest B, et al: A comparative study of the self-immolation of para-aminobenzylalcohol and hemithioaminal-based linkers in the context of protease-sensitive fluorogenic probes. Organic & Biomolecular Chemistry 8:1777-1780, 2010 36. Richard JA, Meyer Y, Jolivel V, et al: Latent fluorophores based on a self-immolative
linker strategy and suitable for protease sensing. Bioconjugate Chemistry 19:1707-1718, 2008
37. Guha S, Padh H: Cathepsins: fundamental effectors of endolysosomal proteolysis.
Indian J Biochem Biophys 45:75-90, 2008
172 38. Choi KY, Swierczewska M, Lee S, et al: Protease-activated drug development.
Theranostics 2:156-78, 2012
173