人類嗜酸性白血球陽離子蛋白(human eosinophil cationic protein，hECP)是由活化的嗜酸性白血球分泌且具有毒性之鹼性蛋白質。ECP能與細胞表面上硫酸乙醯肝素(heparan sulfate)結合，在細胞膜的脂肪筏(lipid raft)區域利用巨胞飲(macropinocytosis)機制進入細胞。先前本實驗室研究發現ECP中含有三段肝素結合區域(HBRs)，34RWRCK38 (HBR1)、75RSRFR79 (HBR2)、101RPGRR105 (HBR3)。本研究將ECP 結構和六糖肝素(haprin hexasaccharide)進行電腦模擬對接(docking)預測，發現胺基酸Arg34、Gln40、His64、Arg105與六糖肝素結合時貢獻的結合能最大。為了釐清這些胺基酸和肝素結合的重要性，將ECP的 Arg34、Gln40、His64、Arg105胺基酸個別點突變成丙氨酸(Ala)，並運用恆溫滴定熱量計(isothermal titration calorimetry，ITC)測量突變株ECP與肝素之結合力變化。運用細胞表面酵素連結免疫吸附分析(cell ELISA)，發現HBR1中Arg34、 Arg36、Lys38為最重要的肝素結合位。已知ECP序列中第32至41個胺基酸序段為具細胞穿透能力之胜肽(cell penetrating peptide，CPPecp)，具備多種生物活性。本研究發現CPPecp能促進rhodamine螢光標定之微脂體(rhodamine-labeled liposomes，RL)穿透入人類癌細胞株。此外，CPPecp協同微脂體包覆藥物(liposomal formulated drug，LFD)使用，能提升該藥物對於癌細胞之毒殺效果。本研究展現ECP中HBR和肝素結合之重要胺基酸，且證實CPPecp促進微脂質體進入細胞且增強微脂體修飾藥物之效用，可具體貢獻於微脂體修飾藥物之設計與應用。

Human eosinophil cationic protein (hECP) is a basic and cytotoxic granular protein released from activated eosinophils. ECP interacts with cellular surface heparan sulfate proteoglycans (HSPGs) and internalizes into cells through lipid raft-associated macropinocytosis. Three heparin binding regions (HBRs) on ECP, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), have been recently identified. In this study, binding energy of amino acids interacting with haprin hexasaccharide was estimated by docking simulation, and Arg34, Gln40, His64 and Arg105 in ECP were predicted to contribute the most. To determine the roles of these residues in heparin binding to ECP, mutant ECPs with single alanine replacement were generated, and heparin binding affinities of wild type and mutant ECPs were measured and compared by isothermal titration calorimetry. Further, cell ELISA showed that Arg34, Arg36 and Lys38 within HBR1 acted as key residues for heparin binding in ECP. In addtion a novel cell penetrating peptide (CPPecp) spanning residues 32 to 41 in ECP was recently demonstrated to possess multiple biological functions. CPPecp was able to increase rhodamine-labeled liposome (RL) penetration into human cells. Moreover, cytotoxicity of liposomal formulated drug (LFD) significantly enhanced in the presence of CPPecp in the cells. Taken together, we have demonstrated key residues in HBRs of ECP for heparin binding. Membrane HSPGs and phospholipid binding drive CPPecp to enhance cellular uptake of liposomes, which in turn may facilitate novel design and application for LFD delivery.

References
1 Nissen-Druey, C. and Speck, B. (1978) Differential counts of neutrophil, eosinophil, and macrophage colonies in cultures from human bone marrow and peripheral blood. Blut. 37, 241-248
2 Simon, H. U., Rothenberg, M. E., Bochner, B. S., Weller, P. F., Wardlaw, A. J., Wechsler, M. E., Rosenwasser, L. J., Roufosse, F., Gleich, G. J. and Klion, A. D. (2010) Refining the definition of hypereosinophilic syndrome. The Journal of allergy and clinical immunology. 126, 45-49
3 Valent, P., Klion, A. D., Horny, H. P., Roufosse, F., Gotlib, J., Weller, P. F., Hellmann, A., Metzgeroth, G., Leiferman, K. M., Arock, M., Butterfield, J. H., Sperr, W. R., Sotlar, K., Vandenberghe, P., Haferlach, T., Simon, H. U., Reiter, A. and Gleich, G. J. (2012) Contemporary consensus proposal on criteria and classification of eosinophilic disorders and related syndromes. The Journal of allergy and clinical immunology. 130, 607-612 e609
4 Lowe, D. G. (1988) Carcinoma of the cervix with massive eosinophilia. British journal of obstetrics and gynaecology. 95, 393-401
5 Cormier, S. A., Taranova, A. G., Bedient, C., Nguyen, T., Protheroe, C., Pero, R., Dimina, D., Ochkur, S. I., O'Neill, K., Colbert, D., Lombari, T. R., Constant, S., McGarry, M. P., Lee, J. J. and Lee, N. A. (2006) Pivotal advance: Eosinophil infiltration of solid tumors is an early and persistent inflammatory host response. J Leukocyte Biol. 79, 1131-1139
6 Bystrom, J., Amin, K. and Bishop-Bailey, D. (2011) Analysing the eosinophil cationic protein--a clue to the function of the eosinophil granulocyte. Respiratory research. 12, 10
7 Ackerman, S. J., Loegering, D. A., Venge, P., Olsson, I., Harley, J. B., Fauci, A. S. and Gleich, G. J. (1983) Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin. Journal of immunology. 131, 2977-2982
8 Rosenberg, H. F. (2008) RNase A ribonucleases and host defense: an evolving story. J Leukoc Biol. 83, 1079-1087
9 Olsson, I. and Venge, P. (1974) Cationic proteins of human granulocytes. II. Separation of the cationic proteins of the granules of leukemic myeloid cells. Blood. 44, 235-246
10 Beintema, J. J. and Kleineidam, R. G. (1998) The ribonuclease A superfamily: general discussion. Cellular and molecular life sciences : CMLS. 54, 825-832
11 Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L., Ackerman, S. J. and McKean, D. J. (1986) Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease. Proceedings of the National Academy of Sciences of the United States of America. 83, 3146-3150
12 Slifman, N. R., Loegering, D. A., McKean, D. J. and Gleich, G. J. (1986) Ribonuclease activity associated with human eosinophil-derived neurotoxin and eosinophil cationic protein. Journal of immunology. 137, 2913-2917
13 Irie, M. (1997) [Structures and functions of ribonucleases]. Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan. 117, 561-582
14 Venge, P., Bystrom, J., Carlson, M., Hakansson, L., Karawacjzyk, M., Peterson, C., Seveus, L. and Trulson, A. (1999) Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology. 29, 1172-1186
15 Rosenberg, H. F., Ackerman, S. J. and Tenen, D. G. (1989) Human eosinophil cationic protein. Molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity. J Exp Med. 170, 163-176
16 Eriksson, J., Woschnagg, C., Fernvik, E. and Venge, P. (2007) A SELDI-TOF MS study of the genetic and post-translational molecular heterogeneity of eosinophil cationic protein. J Leukoc Biol. 82, 1491-1500
17 Torrent, M., de la Torre, B. G., Nogues, V. M., Andreu, D. and Boix, E. (2009) Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment. The Biochemical journal. 421, 425-434
18 Torrent, M., Pulido, D., de la Torre, B. G., Garcia-Mayoral, M. F., Nogues, M. V., Bruix, M., Andreu, D. and Boix, E. (2011) Refining the eosinophil cationic protein antibacterial pharmacophore by rational structure minimization. Journal of medicinal chemistry. 54, 5237-5244
19 Torrent, M., Odorizzi, F., Nogues, M. V. and Boix, E. (2010) Eosinophil cationic protein aggregation: identification of an N-terminus amyloid prone region. Biomacromolecules. 11, 1983-1990
20 Conchillo-Sole, O., de Groot, N. S., Aviles, F. X., Vendrell, J., Daura, X. and Ventura, S. (2007) AGGRESCAN: a server for the prediction and evaluation of "hot spots" of aggregation in polypeptides. BMC bioinformatics. 8, 65
21 Pulido, D., Moussaoui, M., Andreu, D., Nogues, M. V., Torrent, M. and Boix, E. (2012) Antimicrobial action and cell agglutination by the eosinophil cationic protein are modulated by the cell wall lipopolysaccharide structure. Antimicrobial agents and chemotherapy. 56, 2378-2385
22 Boix, E., Salazar, V. A., Torrent, M., Pulido, D., Nogues, M. V. and Moussaoui, M. (2012) Structural determinants of the eosinophil cationic protein antimicrobial activity. Biological chemistry. 393, 801-815
23 Fuchs, S. M. and Raines, R. T. (2006) Internalization of cationic peptides: the road less (or more?) traveled. Cellular and molecular life sciences : CMLS. 63, 1819-1822
24 Barker, R. L., Loegering, D. A., Ten, R. M., Hamann, K. J., Pease, L. R. and Gleich, G. J. (1989) Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases. Journal of immunology. 143, 952-955
25 Fan, T. C., Fang, S. L., Hwang, C. S., Hsu, C. Y., Lu, X. A., Hung, S. C., Lin, S. C. and Chang, M. D. (2008) Characterization of molecular interactions between eosinophil cationic protein and heparin. The Journal of biological chemistry. 283, 25468-25474
26 Navarro, S., Aleu, J., Jimenez, M., Boix, E., Cuchillo, C. M. and Nogues, M. V. (2008) The cytotoxicity of eosinophil cationic protein/ribonuclease 3 on eukaryotic cell lines takes place through its aggregation on the cell membrane. Cell Mol Life Sci. 65, 324-337
27 Sasisekharan, R., Shriver, Z., Venkataraman, G. and Narayanasami, U. (2002) Roles of heparan-sulphate glycosaminoglycans in cancer. Nat Rev Cancer. 2, 521-528
28 Hacker, U., Nybakken, K. and Perrimon, N. (2005) Heparan sulphate proteoglycans: the sweet side of development. Nat Rev Mol Cell Biol. 6, 530-541
29 Lien, P. C., Kuo, P. H., Chen, C. J., Chang, H. H., Fang, S. L., Wu, W. S., Lai, Y. K., Pai, T. W. and Chang, M. D. (2013) In silico prediction and in vitro characterization of multifunctional human RNase3. BioMed research international. 2013, 170398
30 Cardin, A. D. and Weintraub, H. J. (1989) Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis. 9, 21-32
31 Lin, S. C., Fan, T. C. and Chang, M. D. T. (2007) Characterization of heparan sulfate as a cell surface attachment molecule for human eosinophil ribonucleases. Febs Journal. 274, 98-98
32 Fan, T. C., Fang, S. L., Hwang, C. S., Hsu, C. Y., Lu, X. A., Hung, S. C., Lin, S. C. and Chang, M. D. T. (2008) Characterization of molecular interactions between eosinophil cationic protein and heparin. Journal of Biological Chemistry. 283, 25468-25474
33 Garcia-Mayoral, M. F., Moussaoui, M., de la Torre, B. G., Andreu, D., Boix, E., Nogues, M. V., Rico, M., Laurents, D. V. and Bruix, M. (2010) NMR structural determinants of eosinophil cationic protein binding to membrane and heparin mimetics. Biophysical journal. 98, 2702-2711
34 Fang, S. L., Fan, T. C., Fu, H. W., Chen, C. J., Hwang, C. S., Hung, T. J., Lin, L. Y. and Chang, M. D. (2013) A novel cell-penetrating peptide derived from human eosinophil cationic protein. Plos One. 8, e57318
35 Verdurmen, W. P. and Brock, R. (2011) Biological responses towards cationic peptides and drug carriers. Trends in pharmacological sciences. 32, 116-124
36 Ziegler, A. (2008) Thermodynamic studies and binding mechanisms of cell-penetrating peptides with lipids and glycosaminoglycans. Advanced drug delivery reviews. 60, 580-597
37 El-Sayed, A., Futaki, S. and Harashima, H. (2009) Delivery of macromolecules using arginine-rich cell-penetrating peptides: ways to overcome endosomal entrapment. AAPS J. 11, 13-22
38 Abes, R., Arzumanov, A. A., Moulton, H. M., Abes, S., Ivanova, G. D., Iversen, P. L., Gait, M. J. and Lebleu, B. (2007) Cell-penetrating-peptide-based delivery of oligonucleotides: an overview. Biochem Soc Trans. 35, 775-779
39 Koren, E. and Torchilin, V. P. (2012) Cell-penetrating peptides: breaking through to the other side. Trends in molecular medicine. 18, 385-393
40 Lao, J., Madani, J., Puertolas, T., Alvarez, M., Hernandez, A., Pazo-Cid, R., Artal, A. and Anton Torres, A. (2013) Liposomal Doxorubicin in the treatment of breast cancer patients: a review. Journal of drug delivery. 2013, 456409
41 Hagtvet, E., Roe, K. and Olsen, D. R. (2011) Liposomal doxorubicin improves radiotherapy response in hypoxic prostate cancer xenografts. Radiation oncology. 6, 135
42 Koukourakis, M. I., Koukouraki, S., Giatromanolaki, A., Archimandritis, S. C., Skarlatos, J., Beroukas, K., Bizakis, J. G., Retalis, G., Karkavitsas, N. and Helidonis, E. S. (1999) Liposomal doxorubicin and conventionally fractionated radiotherapy in the treatment of locally advanced non-small-cell lung cancer and head and neck cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 17, 3512-3521
43 Hanada, M., Mizuno, S., Fukushima, A., Saito, Y., Noguchi, T. and Yamaoka, T. (1998) A new antitumor agent amrubicin induces cell growth inhibition by stabilizing topoisomerase II-DNA complex. Jpn J Cancer Res. 89, 1229-1238
44 Biswas, S., Deshpande, P. P., Perche, F., Dodwadkar, N. S., Sane, S. D. and Torchilin, V. P. (2013) Octa-arginine-modified pegylated liposomal doxorubicin: An effective treatment strategy for non-small cell lung cancer. Cancer letters. 335, 191-200
45 Koren, E., Apte, A., Jani, A. and Torchilin, V. P. (2012) Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity. Journal of Controlled Release. 160, 264-273
46 Chen, Z. Y., Deng, J. X., Zhao, Y. and Tao, T. (2012) Cyclic RGD peptide-modified liposomal drug delivery system: enhanced cellular uptake in vitro and improved pharmacokinetics in rats. International journal of nanomedicine. 7, 3803-3811
47 Koch, A. M., Reynolds, F., Merkle, H. P., Weissleder, R. and Josephson, L. (2005) Transport of surface-modified nanoparticles through cell monolayers. Chembiochem. 6, 337-345
48 Torrent, M., Cuyas, E., Carreras, E., Navarro, S., Lopez, O., de la Maza, A., Nogues, M. V., Reshetnyak, Y. K. and Boix, E. (2007) Topography studies on the membrane interaction mechanism of the eosinophil cationic protein. Biochemistry. 46, 720-733
49 Boix, E., Nikolovski, Z., Moiseyev, G. P., Rosenberg, H. F., Cuchillo, C. M. and Nogues, M. V. (1999) Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity. J Biol Chem. 274, 15605-15614
50 Leonidas, D. D., Boix, E., Prill, R., Suzuki, M., Turton, R., Minson, K., Swaminathan, G. J., Youle, R. J. and Acharya, K. R. (2001) Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN). High resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors. J Biol Chem. 276, 15009-15017
51 Dews, I., Wiseman, W. T., al-Khawaja, I., Stephens, J. and VandenBurg, M. (1989) A comparison of single doses of lisinopril and enalapril in hypertension. J Hum Hypertens. 3 Suppl 1, 35-39
52 Domachowske, J. B., Dyer, K. D., Adams, A. G., Leto, T. L. and Rosenberg, H. F. (1998) Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. Nucleic acids research. 26, 3358-3363
53 Rosenberg, H. F. and Dyer, K. D. (1997) Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity. Nucleic acids research. 25, 3532-3536
54 Trau, D., Yang, W. J., Seydack, M., Caruso, F., Yu, N. T. and Renneberg, R. (2002) Nanoencapsulated microcrystalline particles for superamplified biochemical assays. Anal Chem. 74, 5480-5486
55 Ho, J. A. A., Hsu, H. W. and Huang, M. R. (2004) Liposome-based microcapillary immunosensor for detection of Escherichia coli O157 : H7. Analytical biochemistry. 330, 342-349
56 NEASFan, T. C., Chang, H. T., Chen, I. W., Wang, H. Y. and Chang, M. D. (2007) A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein. Traffic. 8, 1778-1795
57 Garcia-Mayoral, M. F., Moussaoui, M., de la Torre, B. G., Andreu, D., Boix, E., Nogues, M. V., Rico, M., Laurents, D. V. and Bruix, M. NMR structural determinants of eosinophil cationic protein binding to membrane and heparin mimetics. Biophys J. 98, 2702-2711
58 Torrent, M., Nogues, M. V. and Boix, E. Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site. J Mol Recognit
59 Garcia-Mayoral, M. F., Canales, A., Diaz, D., Lopez-Prados, J., Moussaoui, M., de Paz, J. L., Angulo, J., Nieto, P. M., Jimenez-Barbero, J., Boix, E. and Bruix, M. (2013) Insights into the Glycosaminoglycan-Mediated Cytotoxic Mechanism of Eosinophil Cationic Protein Revealed by NMR. ACS chemical biology. 8, 144-151
60 Ran, S. and Thorpe, P. E. (2002) Phosphatidylserine is a marker of tumor vasculature and a potential target for cancer imaging and therapy. Int J Radiat Oncol. 54, 1479-1484
61 Schutters, K. and Reutelingsperger, C. (2010) Phosphatidylserine targeting for diagnosis and treatment of human diseases. Apoptosis. 15, 1072-1082
62 Ishizuka, I. (1997) Chemistry and functional distribution of sulfoglycolipids. Prog Lipid Res. 36, 245-319
63 Liu, Y., Chen, Y. F., Momin, A., Shaner, R., Wang, E., Bowen, N. J., Matyunina, L. V., Walker, L. D., McDonald, J. F., Sullards, M. C. and Merrill, A. H. (2010) Elevation of sulfatides in ovarian cancer: An integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry. Mol Cancer. 9
64 Kamisago, S., Iwamori, M., Tai, T., Mitamura, K., Yazaki, Y. and Sugano, K. (1996) Role of sulfatides in adhesion of Helicobacter pylori to gastric cancer cells. Infect Immun. 64, 624-628
65 Di Paolo, G. and De Camilli, P. (2006) Phosphoinositides in cell regulation and membrane dynamics. Nature. 443, 651-657
66 Jean, S. and Kiger, A. A. (2012) Coordination between RAB GTPase and phosphoinositide regulation and functions. Nat Rev Mol Cell Bio. 13, 463-470
67 Chaloin, L., Vidal, P., Heitz, A., Van Mau, N., Mery, J., Divita, G. and Heitz, F. (1997) Conformations of primary amphipathic carrier peptides in membrane mimicking environments. Biochemistry. 36, 11179-11187
68 Morris, M. C., Vidal, P., Chaloin, L., Heitz, F. and Divita, G. (1997) A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic acids research. 25, 2730-2736
69 Pooga, M., Hallbrink, M., Zorko, M. and Langel, U. (1998) Cell penetration by transportan. Faseb J. 12, 67-77
70 Fisher, L., Soomets, U., Cortes Toro, V., Chilton, L., Jiang, Y., Langel, U. and Iverfeldt, K. (2004) Cellular delivery of a double-stranded oligonucleotide NFkappaB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide. Gene Ther. 11, 1264-1272
71 Morris, M. C., Depollier, J., Mery, J., Heitz, F. and Divita, G. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat Biotechnol. 19, 1173-1176
72 Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G. and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. The Journal of biological chemistry. 271, 18188-18193
73 Dathe, M., Schumann, M., Wieprecht, T., Winkler, A., Beyermann, M., Krause, E., Matsuzaki, K., Murase, O. and Bienert, M. (1996) Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry. 35, 12612-12622
74 Lamaziere, A., Burlina, F., Wolf, C., Chassaing, G., Trugnan, G. and Ayala-Sanmartin, J. (2007) Non-metabolic membrane tubulation and permeability induced by bioactive peptides. Plos One. 2, e201
75 Goncalves, E., Kitas, E. and Seelig, J. (2005) Binding of oligoarginine to membrane lipids and heparan sulfate: structural and thermodynamic characterization of a cell-penetrating peptide. Biochemistry. 44, 2692-2702
76 Ziegler, A., Blatter, X. L., Seelig, A. and Seelig, J. (2003) Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis. Biochemistry. 42, 9185-9194
77 Lis, L. J., Mcalister, M., Fuller, N., Rand, R. P. and Parsegian, V. A. (1982) Interactions between Neutral Phospholipid-Bilayer Membranes. Biophysical journal. 37, 657-665
78 White, S. H. and Wimley, W. C. (1994) Peptides in Lipid Bilayers - Structural and Thermodynamic Basis for Partitioning and Folding. Curr Opin Struc Biol. 4, 79-86
79 Deshayes, S., Plenat, T., Aldrian-Herrada, G., Divita, G., Le Grimellec, C. and Heitz, F. (2004) Primary amphipathic cell-penetrating peptides: Structural requirements and interactions with model membranes. Biochemistry. 43, 7698-7706
80 Magzoub, M., Kilk, K., Eriksson, L. E. G., Langel, U. and Graslund, A. (2001) Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles. Bba-Biomembranes. 1512, 77-89
81 Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K. and Sugiura, Y. (2001) Arginine-rich peptides - An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. Journal of Biological Chemistry. 276, 5836-5840
82 Dathe, M., Schumann, M., Wieprecht, T., Winkler, A., Beyermann, M., Krause, E., Matsuzaki, K., Murase, O. and Bienert, M. (1996) Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry. 35, 12612-12622
83 Oehlke, J., Krause, E., Wiesner, B., Beyermann, M. and Bienert, M. (1997) Extensive cellular-uptake into endothelial cells of an amphipathic beta-sheet forming peptide. FEBS letters. 415, 196-199
84 Thoren, P. E. G., Persson, D., Lincoln, P. and Norden, B. (2005) Membrane destabilizing properties of cell-penetrating peptides. Biophys Chem. 114, 169-179
85 Tiriveedhi, V. and Butko, P. (2007) A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes. Biochemistry. 46, 3888-3895
86 Hitz, T., Iten, R., Gardiner, J., Namoto, K., Walde, P. and Seebach, D. (2006) Interaction of alpha- and beta-oligoarginine-acids and amides with anionic lipid vesicles: A mechanistic and thermodynamic study. Biochemistry. 45, 5817-5829
87 Carreras, E., Boix, E., Rosenberg, H. F., Cuchillo, C. M. and Nogues, M. V. (2003) Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein. Biochemistry. 42, 6636-6644
88 Carreras, E., Boix, E., Navarro, S., Rosenberg, H. F., Cuchillo, C. M. and Nogues, M. V. (2005) Surface-exposed amino acids of eosinophil cationic protein play a critical role in the inhibition of mammalian cell proliferation. Molecular and cellular biochemistry. 272, 1-7
89 Fuchs, S. M. and Raines, R. T. (2006) Internalization of cationic peptides: the road less (or more?) traveled. Cellular and Molecular Life Sciences. 63, 1819-1822
90 Chen, L. and Harrison, S. D. (2007) Cell-penetrating peptides in drug development: enabling intracellular targets. Biochemical Society transactions. 35, 821-825
91 Rothbard, J. B., Garlington, S., Lin, Q., Kirschberg, T., Kreider, E., McGrane, P. L., Wender, P. A. and Khavari, P. A. (2000) Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation. Nature medicine. 6, 1253-1257
92 Muller, I., Niethammer, D. and Bruchelt, G. (1998) Anthracycline-derived chemotherapeutics in apoptosis and free radical cytotoxicity (Review). Int J Mol Med. 1, 491-494
93 Gewirtz, D. A. (1999) A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics Adriamycin and daunorubicin. Biochem Pharmacol. 57, 727-741
94 Boucek, R. J., Dodd, D. A., Atkinson, J. B., Oquist, N. and Olson, R. D. (1997) Contractile failure in chronic doxorubicin-induced cardiomyopathy. J Mol Cell Cardiol. 29, 2631-2640
95 Tong, N. N., Zhang, J., Chen, Y. R., Li, Z. B., Luo, Y. H., Zuo, H. and Zhao, X. Y. (2012) Berberine sensitizes mutliple human cancer cells to the anticancer effects of doxorubicin in vitro. Oncology letters. 3, 1263-1267
96 Shao, C., Lu, C., Chen, L., Koty, P. P., Cobos, E. and Gao, W. (2011) p53-Dependent anticancer effects of leptomycin B on lung adenocarcinoma. Cancer chemotherapy and pharmacology. 67, 1369-1380
97 Lu, C. W., Shao, C. X., Cobos, E., Singh, K. P. and Gao, W. M. (2012) Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin. Plos One. 7
98 Ibsen, S., Zahavy, E., Wrasdilo, W., Berns, M., Chan, M. and Esener, S. (2010) A Novel Doxorubicin Prodrug with Controllable Photolysis Activation for Cancer Chemotherapy. Pharm Res-Dordr. 27, 1848-1860
99 Park, J. W. (2002) Liposome-based drug delivery in breast cancer treatment. Breast Cancer Res. 4, 93-97
100 Davis, M. E., Chen, Z. and Shin, D. M. (2008) Nanoparticle therapeutics: an emerging treatment modality for cancer. Nature Reviews Drug Discovery. 7, 771-782
101 Speth, P. A. J., Vanhoesel, Q. G. C. M. and Haanen, C. (1988) Clinical Pharmacokinetics of Doxorubicin. Clin Pharmacokinet. 15, 15-31
102 Drummond, D. C., Meyer, O., Hong, K. L., Kirpotin, D. B. and Papahadjopoulos, D. (1999) Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol Rev. 51, 691-743
103 Barenholz, Y. (2012) Doxil (R) - The first FDA-approved nano-drug: Lessons learned. Journal of Controlled Release. 160, 117-134
104 Muggia, F. and Hamilton, A. (2001) Phase III data on Caelyx (R) in ovarian cancer. European journal of cancer. 37, S15-S18
105 Hsiao, S. M., Chen, C. A., Lin, H. H., Hsieh, C. Y. and Wei, L. H. (2009) Phase II trial of carboplatin and distearoylphosphatidylcholine pegylated liposomal doxorubicin (Lipo-Dox (R)) in recurrent platinum-sensitive ovarian cancer following front-line therapy with paclitaxel and platinum. Gynecologic oncology. 112, 35-39
106 Harrington, K. J., Mohammadtaghi, S., Uster, P. S., Glass, D., Peters, A. M., Vile, R. G. and Stewart, J. S. W. (2001) Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes. Clinical Cancer Research. 7, 243-254
107 Duncan, R. (2006) Polymer conjugates as anticancer nanomedicines. Nature Reviews Cancer. 6, 688-701
108 He, X., Na, M. H., Kim, J. S., Lee, G. Y., Park, J. Y., Hoffman, A. S., Nam, J. O., Han, S. E., Sim, G. Y., Oh, Y. K., Kim, I. S. and Lee, B. H. (2011) A novel peptide probe for imaging and targeted delivery of liposomal doxorubicin to lung tumor. Molecular pharmaceutics. 8, 430-438
109 Lowery, A., Onishko, H., Hallahan, D. E. and Han, Z. Z. (2011) Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors. Journal of Controlled Release. 150, 117-124
110 Ruoslahti, E. (1996) RGD and other recognition sequences for integrins. Annual review of cell and developmental biology. 12, 697-715
111 Mardilovich, A. and Kokkoli, E. (2004) Biomimetic peptide-amphiphiles for functional biomaterials: The role of GRGDSP and PHSRN. Biomacromolecules. 5, 950-957
112 Shroff, K. and Kokkoli, E. (2012) PEGylated liposomal doxorubicin targeted to alpha5beta1-expressing MDA-MB-231 breast cancer cells. Langmuir : the ACS journal of surfaces and colloids. 28, 4729-4736
113 Yang, W. H., Luo, D. F., Wang, S. X., Wang, R., Chen, R., Liu, Y., Zhu, T., Ma, X. Y., Liu, R. H., Xu, G., Meng, L., Lu, Y. P., Zhou, J. F. and Ma, D. (2008) TMTP1, a novel tumor-homing peptide specifically targeting metastasis. Clinical Cancer Research. 14, 5494-5502
114 Wang, Z. H., Yu, Y., Dai, W. B., Lu, J. K., Cui, J. R., Wu, H. N., Yuan, L., Zhang, H., Wang, X. Q., Wang, J. C., Zhang, X. and Zhan, Q. (2012) The use of a tumor metastasis targeting peptide to deliver doxorubicin-containing liposomes to highly metastatic cancer. Biomaterials. 33, 8451-8460
115 Shahin, M., Soudy, R., Aliabadi, H. M., Kneteman, N., Kaur, K. and Lavasanifar, A. (2013) Engineered breast tumor targeting peptide ligand modified liposomal doxorubicin and the effect of peptide density on anticancer activity. Biomaterials. 34, 4089-4097
116 Koren, E., Apte, A., Jani, A. and Torchilin, V. P. (2012) Multifunctional PEGylated 2C5-immunoliposomes containing pH-sensitive bonds and TAT peptide for enhanced tumor cell internalization and cytotoxicity. Journal of controlled release : official journal of the Controlled Release Society. 160, 264-273
117 Biswas, S., Deshpande, P. P., Perche, F., Dodwadkar, N. S., Sane, S. D. and Torchilin, V. P. (2013) Octa-arginine-modified pegylated liposomal doxorubicin: An effective treatment strategy for non-small cell lung cancer. Cancer letters
118 Lu, R. M., Chang, Y. L., Chen, M. S. and Wu, H. C. (2011) Single chain anti-c-Met antibody conjugated nanoparticles for in vivo tumor-targeted imaging and drug delivery. Biomaterials. 32, 3265-3274
119 Rosenberg, H. F., Dyer, K. D., Tiffany, H. L. and Gonzalez, M. (1995) Rapid evolution of a unique family of primate ribonuclease genes. Nature genetics. 10, 219-223
120 Torrent, M., Nogues, M. V. and Boix, E. (2011) Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site. Journal of molecular recognition : JMR. 24, 90-100
121 Singh, A. and Batra, J. K. (2011) Role of unique basic residues in cytotoxic, antibacterial and antiparasitic activities of human eosinophil cationic protein. Biological chemistry. 392, 337-346
122 Boix, E., Pulido, D., Moussaoui, M., Nogues, M. V. and Russi, S. (2012) The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form. Journal of structural biology. 179, 1-9
123 Yandek, L. E., Pokorny, A., Floren, A., Knoelke, K., Langel, U. and Almeida, P. F. (2007) Mechanism of the cell-penetrating peptide transportan 10 permeation of lipid bilayers. Biophysical journal. 92, 2434-2444
124 Christiaens, B., Symoens, S., Verheyden, S., Engelborghs, Y., Joliot, A., Prochiantz, A., Vandekerckhove, J., Rosseneu, M. and Vanloo, B. (2002) Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes. European journal of biochemistry / FEBS. 269, 2918-2926
125 Persson, D., Thoren, P. E., Esbjorner, E. K., Goksor, M., Lincoln, P. and Norden, B. (2004) Vesicle size-dependent translocation of penetratin analogs across lipid membranes. Biochimica et biophysica acta. 1665, 142-155
126 Persson, D., Thoren, P. E., Herner, M., Lincoln, P. and Norden, B. (2003) Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes. Biochemistry. 42, 421-429
127 Tiriveedhi, V. and Butko, P. (2007) A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes. Biochemistry. 46, 3888-3895
128 Ziegler, A. and Seelig, J. (2004) Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: Binding mechanism and thermodynamic parameters. Biophysical journal. 86, 254-263
129 Goncalves, E., Kitas, E. and Seelig, J. (2005) Binding of oligoarginine to membrane lipids and heparan sulfate: Structural and thermodynamic characterization of a cell-penetrating peptide. Biochemistry. 44, 2692-2702
130 Wadia, J. S., Stan, R. V. and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature medicine. 10, 310-315
131 Liu, B. R., Lo, S. Y., Liu, C. C., Chyan, C. L., Huang, Y. W., Aronstam, R. S. and Lee, H. J. (2013) Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence. Plos One. 8
132 Chen, Z., Deng, J., Zhao, Y. and Tao, T. (2012) Cyclic RGD peptide-modified liposomal drug delivery system: enhanced cellular uptake in vitro and improved pharmacokinetics in rats. International journal of nanomedicine. 7, 3803-3811