title: The effect of linker type and recognition peptide conjugation chemistry on tissue affinity and cytotoxicity of charged polyacrylamide.
abstract: To increase colonoscopy competence in ambiguous situations (e.g. the existence of flat polyps), an explicit in situ (at real time) diagnosis at the molecular level is required. We have previously shown that the affinity of fluorescent cationic polyacrylamide (Flu-CPAA) to malignant regions in the colon mucosa can be improved by conjugating the recognition peptide EPPT1 to the polymer backbone (to form Flu-CPAA-Pep). Using another recognition peptide, namely VRPMPLQ, we elucidated in the present study the effect of linker type and conjugating methods on Flu-CPAA-VRPMPLQ cytotoxicity and on its affinity to cell lines as well as human colorectal cancer (CRC) biopsies. In order to derive the relationship between the response variable and the experimental factors in a minimal set of experiments, a computerized statistical design of experiment (DoE) strategy was implemented. Data were collected in a six-factor factorial design to study the effect of experimental factors (independent variables) on the ability of the Flu-CPAA polymers to bind specifically to the colon cancer cell lines or the human biopsies (the response). It was found that the presence of VRPMPLQ on the Flu-CPAA improved the polymer's affinity to the human CRC biopsies and to the colon cancer cell lines representing stage B in the Duke severity staging system. The cytotoxicity of Flu-CPAA with high charge density was reduced after conjugated with VRPMPLQ. The replacement of Ahx linker by PEG linker of similar length did not affect the affinity to the human biopsies, nor did it affect cytotoxicity. However, elongating the PEG linker reduced the in vitro affinity to the colon cancer cell lines and to human CRC biopsies. Changing the conjugation method from condensation (amide bond formation) to the click conjugation method did not affect the affinity properties of the polymers. It did reduce, however, the polymer cytotoxicity. We suggest that Flu-CPAA-Pep, with the VRPMPLQ peptide as a recognition moiety, could serve for early diagnosis and screening of CRC patients during endoscopic procedures.
authors.affiliation: The Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy Institute for Drug Research, P.O.Box 12065, Jerusalem 91120, Israel.
authors.forename: Meital B D
authors.initials: MBD
authors.lastname: Bloch
authors.affiliation: The Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy Institute for Drug Research, P.O.Box 12065, Jerusalem 91120, Israel.
authors.forename: Eylon
authors.initials: E
authors.lastname: Yavin
authors.affiliation: Department of Surgical Oncology (Surgery C), Sheba Medical Center, Tel Hashomer 52621, Israel.
authors.forename: Aviram
authors.initials: A
authors.lastname: Nissan
authors.affiliation: Surgical Oncology Laboratory, Department of Surgery, Hadassah-Hebrew University Medical Center, Mount Scopus, P.O.Box 24035, Jerusalem 91240, Israel.
authors.forename: Ilana
authors.initials: I
authors.lastname: Ariel
authors.affiliation: The Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy Institute for Drug Research, P.O.Box 12065, Jerusalem 91120, Israel; The University of Turin, Italy.
authors.forename: Ron
authors.initials: R
authors.lastname: Kenett
authors.affiliation: Alomone Labs Ltd., Protein Group, P.O. Box 4287, Jerusalem 91042, Israel.
authors.forename: Dovrat
authors.initials: D
authors.lastname: Brass
authors.affiliation: The Hebrew University of Jerusalem, Faculty of Medicine, School of Pharmacy Institute for Drug Research, P.O.Box 12065, Jerusalem 91120, Israel. Electronic address: avrir@ekmd.huji.ac.il.
authors.forename: Abraham
authors.initials: A
authors.lastname: Rubinstein
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D003106:Colon / Q000473:pathology; D003113:Colonoscopy / Q000379:methods; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000473:pathology; D042241:Early Diagnosis; D006801:Humans; D008543:Melanins / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D012007:Rectum / Q000473:pathology; D000099166:Polyacrylamides
chemical_list: D000180:Acrylic Resins; D008543:Melanins; D010455:Peptides; D011092:Polyethylene Glycols; D000099166:Polyacrylamides; C030524:fluorescent age pigment
keywords: Cationic polyacrylamide; Colorectal cancer; Design of experiment; Early diagnosis; Imaging; VRPMPLQ recognition peptide