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Correlation of Subcellular Compartmentalization of HPMA Copolymer-Mce6 Conjugates with Chemotherapeutic Activity in Human Ovarian Carcinoma Cells

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Abstract

Purpose. Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e6 monoethylenediamine (Mce6) conjugates was explored to aid in the design of second-generation PDT delivery systems.

Methods. Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce6 was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce6, P-NLS(FITC)-GG-Mce6) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce6, P-NLS(FITC)-GFLG-Mce6). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC50), cell associated drug concentration (CAD) and confocal microscopy.

Results. P-GFLG-Mce6 possessed enhanced chemotherapeutic activity compared to P-GG-Mce6 indicating enzymatically released Mce6 was more active than copolymer-bound Mce6. Lysosomes appeared less sensitive to photodamage as observed by a higher IC50. Nuclear-directed HPMA copolymer-Mce6 conjugates (P-NLS(FITC)-GG-Mce6 P-NLS(FITC)-GFLG-Mce6) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce6 conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce6) or amino groups (P-NH2-GG-Mce6) also displayed increased chemotherapeutic activity.

Conclusions. Nuclear delivery was observed for P-NLS(FITC)-GG-Mce6 and P-NLS(FITC)-GFLG-Mce6 indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.

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REFERENCES

  1. J. Moan and K. Berg. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem. Photobiol. 53:549-553 (1991).

    Google Scholar 

  2. Q. Peng, J. Moan, and J. M. Nesland. Correlation of subcellular and intratumoral photosensitizer localization with ultrastructural features after photodynamic therapy. Ultrastruct. Pathol. 20:109-129 (1996).

    Google Scholar 

  3. Q. Peng, J. Moan, L. W. Ma, and J. M. Nesland. Uptake, localization, and photodynamic effect of meso-tetra(hydroxyphenyl)porphine and its corresponding chlorin in normal and tumor tissues of mice bearing mammary carcinoma. Cancer Res. 55:2620-2626 (1995).

    Google Scholar 

  4. Y. Matsumura and H. Maeda. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS. Cancer Res. 46:6387-6392 (1986).

    Google Scholar 

  5. H. F. Dvořák, J. A. Nagy, J. T. Dvořák, and A. M. Dvořák. Identification and characterization of the blood vessels of solid tumors that are leaky to circulating macromolecules. Am. J. Pathol. 133:95-109 (1988).

    Google Scholar 

  6. J. W. Baish, P. A. Netti, and R. K. Jain. Transmural coupling of fluid flow in microcirculatory network and interstitium in tumors. Microvasc. Res. 53:128-141 (1997).

    Google Scholar 

  7. J. G. Shiah, Y. Sun, C. M. Peterson, and J. Kopeček. Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e(6) and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts. J. Control. Release 61:145-157 (1999).

    Google Scholar 

  8. J. Kopeček, P. Kopecková, T. Minko, and Z. Lu. HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action. Eur. J. Pharm. Biopharm. 50:61-81 (2000).

    Google Scholar 

  9. Z. R. Lu, P. Kopečková, and J. Kopeček. Polymerizable Fab' antibody fragments for targeting of anticancer drugs. Nat. Biotechnol. 17:1101-1104 (1999).

    Google Scholar 

  10. T. V. Akhlynina, D. A. Jans, A. A. Rosenkranz, N. V. Statsyuk, I. Y. Balashova, G. Toth, I. Pavo, A. B. Rubin, and A. S. Sobolev. Nuclear targeting of chlorin e6 enhances its photosensitizing activity. J. Biol. Chem. 272:20328-20331 (1997).

    Google Scholar 

  11. S. K. Bisland, D. Singh, and J. Gariépy. Potentiation of chlorin e6 photodynamic activity in vitro with peptide-based intracellular vehicles. Bioconjug. Chem. 10:982-992 (1999).

    Google Scholar 

  12. N. L. Krinick, Y. Sun, D. Joyner, J. D. Spikes, R. C. Straight, and J. Kopeček. A polymeric drug delivery system for the simultaneous delivery of drugs activatable by enzymes and/or light. J. Biomater. Sci. Polym. Ed. 5:303-324 (1994).

    Google Scholar 

  13. R. Duncan and J. B. Lloyd. Pinocytosis in the rat visceral yolk sac. Effects of temperature, metabolic inhibitors and some other modifiers. Biochim. Biophys. Acta 544:647-655 (1978).

    Google Scholar 

  14. J. Kopeček and H. Bažilová. Poly[N-(2-hydroxypropyl)methacrylamide]. 1. Radical polymerization and copolymerization. Europ. Polym. J. 9:7-14 (1973).

    Google Scholar 

  15. P. Rejmanová, J. Labský, and J. Kopeček. Aminolyses of monomeric and polymeric p-nitrophenyl esters of methacryloylated amino acids. Makromol. Chem. 178:2159-2168 (1977).

    Google Scholar 

  16. H. R. Yen, J. Kopeček, and J. D. Andrade. Optically controlled ligand delivery. 1. Synthesis of water-soluble copolymers containing photocleavable bonds. Makromol. Chem. 190:69-82 (1989).

    Google Scholar 

  17. K. Ulbrich, C. Koňák, Z. Tuzar, and J. Kopeček. Solution properties of drug carriers based on poly[N-(2-hydroxypropyl)methacrylamide] containing biodegradable bonds. Makromol. Chem. 188:1261-1272 (1987).

    Google Scholar 

  18. J. D. Spikes, N. L. Krinick. and J. Kopeček. Photoproperties of a mesochlorin e6-N-(2-hydroxypropyl)methacrylamide copolymer conjugate. J. Photochem. Photobiol. A: Chem. 70:163-170 (1993).

    Google Scholar 

  19. D. Kessel, Y. Luo, P. Mathieu, and J. J. Reiners Jr. Determinants of the apoptotic response to lysosomal photodamage. Photochem. Photobiol. 71:196-200 (2000).

    Google Scholar 

  20. D. Kessel and R. D. Poretz. Sites of photodamage induced by photodynamic therapy with a chlorin e6 triacetoxymethyl ester (CAME). Photochem. Photobiol. 71:94-96 (2000).

    Google Scholar 

  21. K. W. Woodburn, Q. Fan, D. R. Miles, D. Kessel, Y. Luo, and S. W. Young. Localization and efficacy analysis of the phototherapeutic lutetium texaphyrin (PCI-0123) in the murine EMT6 sarcoma model. Photochem. Photobiol. 65:410-415 (1997).

    Google Scholar 

  22. W. N. Leung, X. Sun, N. K. Mak, and C. M. Yow. Photodynamic effects of mTHPC on human colon adenocarcinoma cells: photocytotoxicity, subcellular localization and apoptosis. Photochem. Photobiol. 75:406-411 (2002).

    Google Scholar 

  23. K. Berg. and J. Moan. Lysosomes as photochemical targets. Int. J. Cancer 59:814-822 (1994).

    Google Scholar 

  24. J. Moan, K. Berg, H. Anholt, and K. Madslien. Sulfonated aluminium phthalocyanines as sensitizers for photochemotherapy. Effects of small light doses on localization, dye fluorescence and photosensitivity in V79 cells. Int. J. Cancer 58:865-870 (1994).

    Google Scholar 

  25. P. D. Wilson, R. A. Firestone, and J. Lenard. The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole. J. Cell Biol. 104:1223-1229 (1987).

    Google Scholar 

  26. T. T. Lah, M. Kokalj-Kunovar, B. Strukelj, J. Pungercar, D. Barlic-Maganja, M. Drobnic-Kosorok, L. Kastelic, J. Babnik, R. Golouh, and V. Turk. Stefins and lysosomal cathepsins B, L and D in human breast carcinoma. Int. J. Cancer 50:36-44 (1992).

    Google Scholar 

  27. A. A. Rosenkranz, D. A. Jans, and A. S. Sobolev. Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency. Immunol. Cell Biol. 78:452-464 (2000).

    Google Scholar 

  28. D. A. Jans and S. Hubner. Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol. Rev. 76:651-685 (1996).

    Google Scholar 

  29. N. S. Soukos, M. R. Hamblin, and T. Hasan. The effect of charge on cellular uptake and phototoxicity of polylysine chlorin(e6) conjugates. Photochem. Photobiol. 65:723-729 (1997).

    Google Scholar 

  30. D. Kessel, Y. Luo, Y. Deng, and C. K. Chang. The role of subcellular localization in initiation of apoptosis by photodynamic therapy. Photochem. Photobiol. 65:422-426 (1997).

    Google Scholar 

  31. W. P. Thorpe, M. Toner, R. M. Ezzell, R. G. Tompkins, and M. L. Yarmush. Dynamics of photoinduced cell plasma membrane injury. Biophys. J. 68:2198-2206 (1995).

    Google Scholar 

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Authors and Affiliations

  1. Departments of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, 30S 2000E Room 301, Salt Lake City, Utah, 84112

    Monica Tijerina, Pavla Kopečková & Jindŕich Kopeček

  2. Department of Bioengineering, University of Utah, Salt Lake City, Utah, 84112

    Pavla Kopečková

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  1. Monica Tijerina
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  2. Pavla Kopečková
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  3. Jindŕich Kopeček
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Correspondence to Jindŕich Kopeček.

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Tijerina, M., Kopečková, P. & Kopeček, J. Correlation of Subcellular Compartmentalization of HPMA Copolymer-Mce6 Conjugates with Chemotherapeutic Activity in Human Ovarian Carcinoma Cells. Pharm Res 20, 728–737 (2003). https://doi.org/10.1023/A:1023425300829

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