AbstractPassive targeting provides a simple strategy based on natural properties of the carriers to deliver DNA molecules to desired compartments. Polyethylenimine (PEI) is a potent non-viral system that has been known to deliver efficiently both plasmids and oligonucleotides (ODNs) in vitro. However, in vivo systemic administration of DNA/PEI complexes has encountered significant difficulties because these complexes are toxic and have low biodistribution in target tissues. This study evaluates PEI grafted with poly(ethylene oxide) (PEO(8K)- g -PEI(2K)) and PEI grafted with non-ionic amphiphilic block copolymer, Pluronic ® P85 (P85- g -PEI(2K)) as carriers for systemic delivery of ODNs. Following i.v. injection an antisense ODN formulated with PEO(8K)- g -PEI(2K) accumulated mainly in kidneys, while the same ODN formulated with P85- g -PEI(2K) was found almost exclusively in the liver. Furthermore, in the case of the animals injected with the P85- g -PEI(2K)-based complexes most of the ODN was found in hepatocytes, while only a minor portion of ODN was found in the lymphocyte/monocyte populations. The results of this study suggest that formulating ODN with PEO(8K)- g -PEI(2K) and P85- g -PEI(2K) carriers allows targeting of the ODN to the liver or kidneys, respectively. The variation in the tissue distribution of ODN observed with the two carriers is probably due to the different hydrophilic-lipophilic balance of the polyether chains grafted to PEI in these molecules. Therefore, polyether-grafted PEI carriers provide a simple way to enhance ODN accumulation in a desired compartment without the need of a specific targeting moiety.Keywords: : KidneyLiverGene DeliveryOligonucleotide
Abstract This study was undertaken towards enabling PET imaging of polymer‐based drug delivery. Iodinated doxorubicin conjugate (DOXIB) was characterised against un‐conjugated DOX within and outside a polymer formulation in tumour cell lines and tumour bearing animal models. The results provide the basis for developing the feasibility of [ 124 I]‐DOXIB PET to assess in vivo pharmacokinetics of drug formulation.
Polyelectrolyte complexes formed between DNA and poly(N-ethyl4-vinylpyridinium) cations were shown to effectively transfect mammalian cells [7]. This work suggests that the polycation-mediated uptake of the plasmid DNA and cell transfection are significantly enhanced when these complexes are administered simultaneously with a poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) copolymer, Pluronic P85. The uptake studies were performed using radioactively labeled pRSV CAT plasmid on NIH 3T3, MDCK, and Jurkat cell lines. The transfection was investigated by chloramphenicol acetyltransferase assay using 3T3 cells as a model. The effects reported may be useful for the enhancement of the polycation-mediated cell transfection.
Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 μm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel™ was completely inhibited in the presence of 10 μm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and β-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-β-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure. Vascular endothelial growth factor (VEGF) is known to play a predominant role in tumor angiogenesis and metastasis formation that is mediated by its interactions with two tyrosine kinase receptors, VEGFRI (Flt-1) and VEGFRII (KDR). Inhibition of VEGF-dependent events in tumor tissues is known to enhance apoptosis and to suppress tumor growth. A novel peptide, SP5.2, which selectively binds Flt-1 and inhibits a broad range of VEGF-mediated events, was identified using a phage-display library screening. The fluorescein-labeled SP5.2 specifically bound to VEGF-stimulated primary human cerebral endothelial cells (HCECs), whereas non-stimulated HCECs, as well as human neuroblastoma cells (ShyY) did not show any interaction with the peptide. SP5.2 prevented proliferation of cultured primary human umbilical vein endothelial cells induced by recombinant human VEGF165 with an IC50 of 5 μm. SP5.2 was also shown to antagonize VEGF- and PLGF-induced, but not basic fibroblast growth factor-induced proliferation of HCECs. In contrast to "scrambled" peptide, SP5.2 was also found to selectively inhibit VEGF-stimulated migration of HCECs. The in vitro analysis of antiangiogenic activity of SP5.2 using a capillary-like tube formation assay showed that VEGF-induced angiogenesis of HCECs grown on Matrigel™ was completely inhibited in the presence of 10 μm SP5.2. Further studies demonstrated that SP5.2 prevented VEGF-induced permeability increase in HCECs monolayers. To explore whether SP5.2 can be used as a targeting agent, chemical and recombinant conjugates of SP5.2 with reporter proteins (peroxidase and β-galactosidase) were produced. The resulting products showed significant increases (200-fold for SP5.2-β-gal and 400-fold for SP5.2-peroxidase) in binding affinity to recombinant Flt-1 compared with the original synthetic SP5.2, suggesting that conjugate with therapeutic activity in nanomolar range could potentially be developed based on SP5.2 structure. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGFvascular endothelial growth factorFlt-1tyrosine kinase receptor VEGFRIKDRtyrosine kinase receptor VEGFRIINRP-1neuropillin-1HRPhorseradish peroxidaseHCEChuman cerebral endothelial cellHUVEChuman umbilical vein endothelial cellBSAbovine serum albuminPBSphosphate-buffered salineFlt-MPFlt-1-coated magnetic particleELISAenzyme-linked immunosorbent assaycfucolony-forming unit(s)bFGFbasic fibroblast growth factorCMVcytomegalovirusSPDPN-succinimidy 1,3-(2-pyridylthio)propionateECendothelial cellABantibodyABTS2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)PLGFplacenta growth factor. and its receptors are the focus of intense interest because of their role in blood vessel formation (angiogenesis and vasculogenesis) in a variety of physiological and pathophysiological processes, including embryogenesis, development of the fetal cardiovascular system, wound healing, tumor growth, proliferative retinopathies, and chronic inflammatory diseases such as rheumatoid arthritis (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7215) Google Scholar, 2Ferrara N. Bunting S. Curr. Opin. Nephrol. Hypertens. 1996; 5: 35-44Crossref PubMed Scopus (140) Google Scholar, 3Zachary I. Int. J. Biochem. Cell Biol. 1998; 30: 1169-1174Crossref PubMed Scopus (109) Google Scholar). VEGF is unique among the growth factors in being an endothelial cell-specific mitogen that promotes the proliferation and migration of endothelial cells, remodeling of the extracellular matrix, formation of capillary tubules and vascular leakage (4Wang D. Donner D.B. Warren R.S. J. Biol. Chem. 2000; 275: 15905-15911Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). vascular endothelial growth factor tyrosine kinase receptor VEGFRI tyrosine kinase receptor VEGFRII neuropillin-1 horseradish peroxidase human cerebral endothelial cell human umbilical vein endothelial cell bovine serum albumin phosphate-buffered saline Flt-1-coated magnetic particle enzyme-linked immunosorbent assay colony-forming unit(s) basic fibroblast growth factor cytomegalovirus N-succinimidy 1,3-(2-pyridylthio)propionate endothelial cell antibody 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) placenta growth factor. VEGF activities are mediated through binding to two high affinity receptors, human kinase domain receptor (KDR) and Fms-like tyrosine kinase receptor (Flt-1) (5Shibuya M. Yamaguchi S. Yamane A. Ikeda T. Tojo A. Matsushime H. Sato M. Oncogene. 1990; 5: 519-524PubMed Google Scholar), both of which are selectively expressed on endothelial cells during embryogenesis and VEGF-related pathologies (6Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1760) Google Scholar). Both of these receptors are class III tyrosine kinases (7Vaisman N. Gospodarowicz D. Neufeld G. J. Biol. Chem. 1990; 265: 19461-19466Abstract Full Text PDF PubMed Google Scholar, 8Kaipainen A. Korhonen J. Pajusola K. Aprelikova O. Persico M.G. Terman B.I. Alitalo K. J. Exp. Med. 1993; 178: 2077-2088Crossref PubMed Scopus (216) Google Scholar) that undergo ligand-induced dimerization that triggers signal transduction. Studies in mice have shown that the expression of KDR reaches the highest levels during embryonic vasculogenesis and angiogenesis (6Millauer B. Wizigmann-Voos S. Schnurch H. Martinez R. Moller N.P. Risau W. Ullrich A. Cell. 1993; 72: 835-846Abstract Full Text PDF PubMed Scopus (1760) Google Scholar). In contrast, low Flt-1 mRNA levels were found during fetal growth, moderate during organogenesis, and high in newborn mice (9Peters K.G. De Vries C. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8915-8919Crossref PubMed Scopus (412) Google Scholar). The third VEGF receptor from the tyrosine kinase family, Flt-4, is largely confined to the lymphatic vasculature and has a role in lymph angiogenesis (10Dumont D.J. Jussila L. Taipale J. Lymboussaki A. Mustonen T. Pajusola K. Breitman M. Alitalo K. Science. 1998; 282: 946-949Crossref PubMed Scopus (697) Google Scholar). Flt-4 binds VEGF-C and -D, but not VEGF (11Achen M.G. Stacker S.A. Int. J. Exp. Pathol. 1998; 79: 255-265Crossref PubMed Scopus (106) Google Scholar). Neuropillin-1 (NRP-1) was recently identified as a new receptor for VEGF (12Partanen T.A. Makinen T. Arola J. Suda T. Weich H.A. Alitalo K. Circulation. 1999; 100: 583-586Crossref PubMed Scopus (43) Google Scholar). It is expressed in the enocardium, coronary vessels, myocardial capillaries, and epicardial blood vessels of human fetal heart (12Partanen T.A. Makinen T. Arola J. Suda T. Weich H.A. Alitalo K. Circulation. 1999; 100: 583-586Crossref PubMed Scopus (43) Google Scholar). Experiments with knockout mice deficient in Flt-1 or KDR receptor revealed that KDR is essential for the development of endothelial cells, whereas Flt-1 is necessary for the organization of embryonic vasculature (13Fong G.H. Rossant J. Gertsenstein M. Breitman M.L. Nature. 1995; 376: 66-70Crossref PubMed Scopus (2216) Google Scholar, 14Shalaby F. Rossant J. Yamaguchi T.P. Gertsenstein M. Wu X.F. Breitman M.L. Schuh A.C. Nature. 1995; 376: 62-66Crossref PubMed Scopus (3358) Google Scholar). The VEGF-Flt-1 receptor system also plays an important role in the simulation of tumor angiogenesis, which makes Flt-1 an interesting target for antiangiogenic drugs (15Shibuya M. Adv. Cancer Res. 1995; 67: 281-316Crossref PubMed Google Scholar). Human Flt-1 receptor is composed of seven extracellular Ig-like domains containing the ligand binding region, a single short membrane-spanning sequence, and an intracellular region containing the tyrosine kinase domain. The amino acid sequences of Flt-1 and KDR show ∼45% identity; however, Flt-1 has a higher affinity for VEGF (KD = 10–20 pm) compared with KDR (KD = 75–125 pm). The activation of Flt-1 receptor by VEGF regulates interactions of endothelial cells with each other or the basement membrane on which they reside (16Quinn T.P. Peters K.G. De Vries C. Ferrara N. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7533-7537Crossref PubMed Scopus (671) Google Scholar, 17Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar). Alternative splicing of the Flt-1 receptor results in two forms, the full-length membrane-spanning receptor and a soluble form, denoted sFlt-1. Isolated sFlt-1 retains specific high affinity binding for VEGF and fully inhibits VEGF-stimulated endothelial cell mitogenesis by dominant negative mechanism (18He Y. Smith S.K. Day K.A. Clark D.E. Licence D.R. Charnock-Jones D.S. Mol. Endocrinol. 1999; 13: 537-545Crossref PubMed Google Scholar). Furthermore, it was suggested that sFlt-1 might form heterodimeric complexes with KDR with potentially negative effect on KDR signal transduction (19Kendall R.L. Wang G. Thomas K.A. Biochem. Biophys. Res. Commun. 1996; 226: 324-328Crossref PubMed Scopus (623) Google Scholar). Because VEGF receptors are implicated in several pathologies, pharmacological interference with the VEGF/VEGF receptor system antagonists is clinically attractive. Humanized neutralizing antibodies that interact with VEGF near the KDR and Flt-1 binding sites (20Kim K.J. Li B. Winer J. Armanini M. Gillett N. Phillips H.S. Ferrara N. Nature. 1993; 362: 841-844Crossref PubMed Scopus (3352) Google Scholar, 21Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. Structure. 1997; 5: 1325-1338Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 22Muller Y.A. Chen Y. Christinger H.W. Li B. Cunningham B.C. Lowman H.B. de Vos A.M. Structure. 1998; 6: 1153-1167Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) and systemic evolution of ligands by exponential enrichment (SELEX)-derived RNA molecules (23Jellinek D. Green L.S. Bell C. Janjic N. Biochemistry. 1994; 33: 10450-10456Crossref PubMed Scopus (162) Google Scholar) that selectively bind to Flt-1 have been shown to block tumor growth dependent on vascularization of adjacent normal tissue (24Plate K.H. Breier G. Risau W. Brain Pathol. 1994; 4: 207-218Crossref PubMed Scopus (224) Google Scholar). Similarly, anti-KDR monoclonal antibodies inhibited VEGF-induced signaling and demonstrated a high antitumor activity (25Witte L. Hicklin D.J. Zhu Z. Pytowski B. Kotanides H. Rockwell P. Bohlen P. Cancer Metastasis. Rev. 1998; 17: 155-161Crossref PubMed Scopus (303) Google Scholar). Soluble Flt receptor (26Gehlbach P. Demetriades A.M. Yamamoto S. Deering T. Xiao W.H. Duh E.J. Yang H.S. Lai H. Kovesdi I. Carrion M. Wei L. Campochiaro P.A. Hum. Gene. Ther. 2003; 14: 129-141Crossref PubMed Scopus (88) Google Scholar), fragments of VEGF, as well as small molecule inhibitors of the VEGF receptors tyrosine kinase activity, such as PTK787/ZK222584 (27Drevs J. Hofmann I. Hugenschmidt H. Wittig C. Madjar H. Muller M. Wood J. Martiny-Baron G. Unger C. Marme D. Cancer Res. 2000; 60: 4819-4824PubMed Google Scholar) and ZD4190 (28Wedge S.R. Ogilvie D.J. Adv. Exp. Med. Biol. 2000; 476: 307-310Crossref PubMed Scopus (7) Google Scholar), have been shown to inhibit angiogenesis in vivo. Anti-VEGF antisense oligonucleotide has been shown to inhibit VEGF expression, VEGF-induced neovascularization, and tumor implantation and growth (29Melnyk O. Shuman M.A. Kim K.J. Cancer Res. 1996; 56: 921-924PubMed Google Scholar, 30Benjamin L.E. Keshet E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8761-8766Crossref PubMed Scopus (444) Google Scholar, 31Cheng N. Brantley D.M. Liu H. Lin Q. Enriquez M. Gale N. Yancopoulos G. Cerretti D.P. Daniel T.O. Chen J. Mol. Cancer Res. 2002; 1: 2-11Crossref PubMed Scopus (36) Google Scholar). However, no truly antagonistic compounds that would selectively discriminate between Flt-1 and KDR receptors are available yet. In the present study, a new peptide motif that inhibits VEGF binding to Flt-1 has been identified using a phage-displayed peptide library. A random 16-mer peptide library displayed on the surface of the filamentous phage M13 was screened against the extracellular domain of Flt-1. This screening resulted in a peptide (SP5.2) that competed with VEGF for the Flt-1 binding and inhibited a broad range of VEGF-induced events in cultured endothelial cells. Potential use of SP5.2 as a targeting agent was evaluated using both synthetic and genetically constructed conjugates of the peptide and reporter proteins. Materials—A recombinant Flt-1 receptor chimera consisting of the six N-terminal extracellular domains of human Flt receptor and Fc fragment of human IgG was obtained from R&D Systems (Minneapolis, MN). The goat polyclonal anti-hFlt-1 antibody, rhVEGF165, hKDR/Fc chimera, mNRP-1/Fc chimera, hsICAM-1, mFlt-1/Fc chimera, and mFlk-1/Fc chimera were obtained from R&D Systems. Biotin-labeled anti-human IgG (Fc-specific) rabbit polyclonal antibody was purchased from ICN (Costa Mesa, CA). The mouse monoclonal anti-β-galactosidase clone Gal-13 and anti-mouse IgG peroxidase conjugate developed in goat and absorbed with rat serum proteins were obtained from Sigma (Oakville, Ontario, Canada). The anti-histidine HRP conjugate antibody was purchased from Invitrogen (Burlington, Ontario, Canada). Scrambled peptide was synthesized at Supratek (Laval, Quebec, Canada). Cell Culture—Human umbilical vein endothelial cells (HUVECs) were purchased from BioWhittaker Inc. (Walkersville, MD). The cells were cultured in the endothelial growth medium, Clonetics media EGM & EGM BulletKit™ (BioWhittaker Inc.). The cells were used for the experiments at their 3rd or 4th growth passage. HCECs were isolated using previously described protocols (32Stanimirovic D. Morley P. Ball R. Hamel E. Mealing G. Durkin J.P. J. Cell. Physiol. 1996; 169: 455-467Crossref PubMed Scopus (44) Google Scholar). Purity of HCEC cultures generated by these procedures was routinely assessed by the immunocytochemical staining for Factor VIII-related antigen and the lack of staining for smooth muscle β-actin and was estimated to be >95%. The morphological, phenotypic, biochemical, and functional characteristics of these HCEC cultures have been described in detail previously (32Stanimirovic D. Morley P. Ball R. Hamel E. Mealing G. Durkin J.P. J. Cell. Physiol. 1996; 169: 455-467Crossref PubMed Scopus (44) Google Scholar, 33Muruganandam A. Herx L.M. Monette R. Durkin J.P. Stanimirovic D.B. FASEB J. 1997; 11: 1187-1197Crossref PubMed Scopus (130) Google Scholar). For endothelial permeability studies, HCECs were used as an in vitro blood-brain barrier model described previously (33Muruganandam A. Herx L.M. Monette R. Durkin J.P. Stanimirovic D.B. FASEB J. 1997; 11: 1187-1197Crossref PubMed Scopus (130) Google Scholar). Bacterial Strains and Bacteriophages—K91Kan cells obtained from G. Smith were grown on LB agar supplemented with kanamycin (100 μg/ml). Cells were made competent using the protocol described previously by Smith (34Smith G.P. Scott J.K. Methods Enzymol. 1993; 217: 228-257Crossref PubMed Scopus (698) Google Scholar). M15 was purchased from Qiagen (Mississauga, Ontario, Canada) cells made competent following the protocol from Qiagen. pQE16 vector purchased from Qiagen and pCMVb vector from Clontech (Palo Alto, CA). The phage library containing the 16-amino acid peptide was constructed essentially as previously described, using fUSE5 as the phage vector (35Popkov M. Lussier I. Medvedkine V. Esteve P.O. Alakhov V. Mandeville R. Eur. J. Biochem. 1998; 251: 155-163Crossref PubMed Scopus (24) Google Scholar). This linear library consisted of 109 independent recombinant phages recovered as tetracycline-resistant colonies. Sequencing of randomly selected clones indicated that the majority of these phages (>90%) contain inserts. Screening the Phage Display Library with Flt-1-coated Magnetic Particles—The receptor, rhFlt-1 chimera (10 μg in 50 μl of 0.1% BSA/PBS), was immobilized on streptavidin-coated magnetic particles (Roche Applied Science, Laval, Canada) using biotinylated anti-hIgG(Fc) antibody. The Flt-1-coated magnetic particles (Flt-MPs) were blocked with 3% BSA/PBS for 2 h at room temperature. For selection, phage (1 × 1011 cfu) from the linear 16-amino acid random peptide phage display library diluted in 0.1% BSA/PBS were added to Flt-MP and incubated overnight at 4 °C. After extensive washing with 0.1% BSA/PBS, the bound phage were either eluted with a low pH buffer (0.2 m glycine-HCl, pH 2.2) or displaced from Flt-MP with 10 μg/ml rhVEGF165 for 1 h at 22 °C. Recovered phages were amplified using competent K91Kan Escherichia coli cells and then subjected to four subsequent rounds of selection on Flt-MP. Phage binding was quantified by counting the phage titer in eluted aliquots from Flt-MP as described earlier (34Smith G.P. Scott J.K. Methods Enzymol. 1993; 217: 228-257Crossref PubMed Scopus (698) Google Scholar). Phages from selected clones were sequenced at Sheldon Biotechnology center (McGill, Montreal). ELISA for the Displacement of Phage Binding—The rhFlt-1 receptor chimera were immobilized on Streptavidin-coated microtiter plates (total binding capacity for biotin-labeled AB = 1.5 μg/well, Roche Applied Science). Biotin-labeled anti-human FC antibody (ICN), at a concentration of 10 μg/ml in 0.1% BSA/PBS, were added into each well of the plates. The plates with assay mixtures were incubated at 4 °C for 8 h in a humidified container and then washed four times with 0.1% BSA/PBS. Recombinant human Flt-1/Fc chimera (1 μg/well in 0.1% BSA/PBS, R&D Systems, Minneapolis, MN) were added into each well, and the plates were incubated overnight at 4 °C in a sealed container to allow the receptor to attach to the ligand. Unbound receptor was washed away with 2 ml of 0.1% BSA/PBS. Each well was then filled with 250 μl of blocking buffer (2% nonfat dry milk in PBS) and incubated at room temperature for 2–3 h. As a negative control, three of the wells were blocked with the blocking buffer (2% nonfat milk in PBS). Phage particles (5 × 109 to 1 × 1010 cfu/well) were added to each well and incubated for 2 h at room temperature. In competition experiments, phage suspension was premixed with various concentrations of competing agent (SP5.2 peptide, rhVEGF165) and then added to immobilized receptor. Wells were washed with 0.1% BSA/PBS, and the amount of bound phages was detected with peroxidase-conjugated anti-M13 antibody (Amersham Biosciences). After the addition of the substrate, ABTS, and H2O2, antibody reaction was analyzed in a microtiter plate reader at 405 nm. Production of Mutant Variants of SP5.2 Phage—Several mutations in SP5.2 coding oligonucleotide inserts were carried out to identify the amino acids involved in the receptor binding site domain. The single point mutations in V5.2 phage insert coding sequence were produced as described previously (36Hoess R. Brinkmann U. Hanel T. Pastan I. Gene (Amst.). 1993; 128: 43-49Crossref PubMed Scopus (171) Google Scholar). Briefly, a series of SP5.2-coding oligonucleotides, with a particular amino acid coding triplet replaced with GCT (alanine-coding triplet), was synthesized by Invitrogen (Ontario, Canada). The mutant oligonucleotides were cloned into fUSE5 phage vector as described (35Popkov M. Lussier I. Medvedkine V. Esteve P.O. Alakhov V. Mandeville R. Eur. J. Biochem. 1998; 251: 155-163Crossref PubMed Scopus (24) Google Scholar). All mutants were purified and verified by DNA sequencing. Peptide Synthesis—Peptide amides were synthesized manually on solid Rink amide resin (Nova Biochem) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) protocol and (benzotriazol-1-yloxy)tripyrro-lidinophosphonium hexafluorophosphate (PyBOP) activation. The fluorescein residue was introduced into the peptide by standard coupling of N-terminal-deprotected peptide with fluorescein-5-carboxylic acid. Peptide amides were cleaved from the resin with trifluoroacetic acid:water: ethanedithiol:triisopropylsilane (95:5:2.5:2.5, v/v) and were recovered by precipitation with ice-cold diethyl ether. Crude products were purified by high performance liquid chromatography on a Vydac C18 column using a linear gradient of 30% to 70% acetonitrile/water (0.1% trifluoroacetic acid), for 60 min at a 5-ml/minute flow rate. The identity of peptides was verified by electrospray mass spectrometry (PE Sciex API III Biomolecular Mass Analyzer, Applied Biosystems, CA). Binding Competition for Flt-1 Receptor—Fluorescein-labeled peptide in 0.1% BSA/PBS buffer, pH 8.5, was added at various concentrations (0, 0.1, 1, 10, 50, and 100 μm) into wells of an ELISA plate coated with immobilized Flt-1 receptor. In competition experiments, the various concentrations of phage or VEGF165 were mixed and added into wells. After 2-h incubation, microtiter plates were washed 10 times with 0.1% BSA/PBS buffer, pH 8.5, and the bound peptide was measured using a microplate fluorescence reader (FL600, BioTek) (λex = 485 nm; λem = 530 nm). Flt-1 Receptor Immunochemistry—HCECs were grown on glass coverslips in a 24-well dish for 3 days until sub-confluent. Media was aspirated out; cells were washed two times in PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were subsequently washed three times in PBS, incubated in blocking solution containing 5% goat serum in PBS for 1 h at room temperature, and exposed to the primary antibody (5 μg/ml goat anti-human Flt-1 in blocking solution; R&D Systems Inc) for 1 h at room temperature. After washing three times in blocking solution, cells were exposed to the secondary antibody (1:20 dilution of donkey anti-goat IgG-immunogold in blocking solution; Accurate Chemical and Scientific Corp.) for 30 min at room temperature. Cells were then extensively washed (three times with blocking solution and three times with distilled water) and incubated in silver enhancing solution for 30 min (1:1 dilution of reagents A and B). After washing with distilled water (two times for 5 min), slides were viewed under a microscope. SP5.2 Binding to Cells—Fluorescein-labeled SP5.2 peptide was added to HCECs cells at different concentrations, (1, 10, and 50 μm) and incubated for 30 min. The neuroblastoma ShyY cell line was used as a negative control. Media were aspirated, and cells were washed in phenol red-free M199 and observed under a fluorescence microscope. Endothelial Cell Proliferation Assay—HUVECs were placed in 96-well plates (Costar) at 104 cells per well in 200 μl of EGM-2 medium (Clonetics) supplemented with 0.5% heat-inactivated fetal bovine serum. Cells were incubated for 24 h at 37 °C in 5% CO2. HCECs were placed in 12-well plates and allowed to grow for 3 days until sub-confluent. Cells were then washed once by PBS and incubated in a serum- and glucose-free DMEM for 24 h to suppress cell growth. Both HCECs and HUVECs were then exposed to various treatments (i.e. 10% fetal bovine serum, VEGF165 (20 ng/ml), PLGF (100 ng/ml), bFGF (10 ng/ml) in the absence or presence of SP5.2 peptide). Scrambled peptide NGSAIAASSAVTHGMS at the same concentration was used as control. After 48 h of incubation, 1 μCi of [methyl-3H]thymidine (20 Ci/mmol; ICN) was added in each well, and plates were incubated for an additional 24 h. The cells were then placed on ice, washed twice with EGM-2 medium containing 10% fetal bovine serum, and fixed for 10 min in ice-cold 10% trichloroacetic acid. After washing with ice-cold water, cells were lysed and DNA was solubilized in 50 μl of 2% SDS. [3H]Thymidine incorporation was determined by scintillation counting. Endothelial Cell Migration—Migration of calcein-AM labeled (Molecular Probes, Eugene, OR, 10 μm for 20 min at 37 °C) HCECs stimulated by VEGF165 were tested in the absence or presence of SP5.2 peptide or scrambled peptide using a ChemoTx #101-5 assembly (Neuro Probe, Inc., Gaithersburg, MD) consisting of a 96-well plate and a polycarbonate filter membrane, as described in a previous study (37Junger W.G. Cardoza T.A. Liu F.C. Hoyt D.B. Goodwin R. J. Immunol. Methods. 1993; 160: 73-79Crossref PubMed Scopus (46) Google Scholar). Briefly, the wells of the plates were loaded with buffer containing various chemoattractants or phenol red- and serum-free media conditioned by cancer cells. The framed filter membrane was positioned on top, and 50,000 calcein-AM-labeled HCECs were suspended in 20 μl of matching buffer, or media was applied on the top of each membrane/well. The assembly was incubated for 120 min at 37 °C, and the number of endothelial cells transmigrated into the wells was quantified by measuring the intensity of fluorescence (excitation/emission: 485/530 nm) in a CytoFluor 2350 reader (Millipore, Bedford, MA). Capillary Tube Formation in Matrigel™—HCECs were seeded (8 × 104 cells) on a semi-solid basement membrane matrix, Matrigel™ (Collaborative Biomedical Products, Bedford, MA)-coated 24-well plates and exposed to serum-free media containing various treatments. Cells were treated with 10 nm VEGF165 or glioblastoma cell (U-87MG, ATCC, Rockville, MD)-conditioned media (serum-free Dulbecco's modified Eagle's medium conditioned for 48–72 h) in the absence or presence of SP5.2 or control peptide for 24 h. HCECs were then labeled with a vital dye, calcein-AM (2 μm) for 30 min. Cells were then washed in PBS and capillary-like tube formation, a measure of in vitro angiogenesis, was observed using a Zeiss LSM 410 inverted laser scanning microscope (Thornwood, NY) with an argon/krypton ion laser and a Zeiss LD achroplan 5×, 0.6-numerical aperture objective. Confocal apertures for each recorded wavelength were adjusted to a full-width half maximum of 20 μm. The emitted light was collected through a 515- to 540-nm band-pass filter using an excitation of 488 nm. 8–32 individual optical sections were collected, and the average was calculated. Standard image processing was performed to enhance brightness and contrast using ImageSpace software (Molecular Dynamics Inc., Sunnyvale, CA). SP5.2-β-gal Fusion Construction—The prokaryotic expression vector, pQE-16, purchased from Qiagen, was used to construct the fusion protein SP5.2-β-gal, containing SP5.2 peptide on the N terminus and His6 on the C terminus of β-galactosidase. For this the vector pCMV-β from Clontech (Palo Alto, CA) was digested with BamH1 (New England Biolabs), the resulting β-galactosidase gene was purified from agarose gel and modified by PCR as so to introduce BglII at the 3′-end for cloning into pQE16 instead of dihydrofolate reductase, which has been removed by digestion of plasmid with BamHI and BglII. The SP5.2 insert, containing the EcoRI site at the 5′-end and the BamHI site at the 3′-end, was prepared by annealing the two single-stranded oligonucleotides synthesized by INRS-IAF (Laval, Quebec, Canada) and inserting them into the pQE16-β-gal vector. pQE16-SP5.2-β-gal was used to transform competent E. coli M15 cells. The resulting clones were sequenced to verify the fusion gene. Fusion Protein Expression and Purification—An overnight culture of a single colony was grown in 400 ml of LB medium containing 100 μg/ml ampicillin and 25 μg/ml kanamycin to an optical density of ∼0.6 at 600 nm. The culture was induced with 1 mm isopropyl-1-thio-β-d-galacto-pyranoside for 4 h at 37 °C, centrifuged, and the resulting pellet was stored at –80 °C. The expression of β-galactosidase and the presence of His tag were verified by Western blot using anti-β-galactosidase (Sigma, Ontario, Canada) and anti-His-HRP antibody (Invitrogen, Ontario, Canada). The pellet was thawed and resuspended in lysis buffer (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole, pH 8.0), and lysozyme was added to a concentration of 1 mg/ml. After incubation for 30 min at 4 °C, 400 μl of 50% nickel-nitrilotriacetic acid resin (Qiagen, Ontario, Canada) was added to the supernatant and incubated 1 h shaking at 4 °C. The lysate/nickel-nitrilotriacetic acid mixture was loaded into a column (Qiagen),
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