ABSTRACT
We are investigating an imaging agent that detects early-stage primary colorectal cancer on the mucosal surface in real time under colonoscopic observation. The imaging agent, which is named the nanobeacon, is fluorescent nanospheres conjugated with peanut agglutinin and poly(N-vinylacetamide). Its potential use as an imaging tool for colorectal cancer has been thoroughly validated in numerous studies. Here, toxicities of the nanobeacon were assessed in rats. The nanobeacon was prepared according to the synthetic manner which is being established as the Good Manufacturing Practice-guided production. The rat study was performed in accordance with Good Laboratory Practice regulations. No nanobeacon treatment-related toxicity was observed. The no observable adverse effect levels (NOAEL) of the nanobeacon in 7-day consecutive oral administration and single intrarectal administration were estimated to be more than 1000mg/kg/day and 50mg/kg/day, respectively. We concluded that the nanobeacon could be developed as a safe diagnostic agent for colonoscopy applications. FROM THE CLINICAL EDITOR: Colon cancer remains a major cause of death. Early detection can result in early treatment and thus survival. In this article, the authors tested potential systemic toxicity of coumarin 6-encapsulated polystyrene nanospheres conjugated with peanut agglutinin (PNA) and poly(N-vinylacetamide) (PNVA), which had been shown to bind specifically to colonic cancer cells and thus very promising in colonoscopic detection of cancer cells.
Subject(s)
Acetamides/toxicity , Colonoscopy , Coumarins/toxicity , Fluorescent Dyes/toxicity , Nanospheres/toxicity , Peanut Agglutinin/toxicity , Polystyrenes/toxicity , Polyvinyls/toxicity , Thiazoles/toxicity , Acetamides/administration & dosage , Acetamides/chemistry , Animals , Body Weight/drug effects , CHO Cells , Caco-2 Cells , Colon/drug effects , Colon/pathology , Colorectal Neoplasms/diagnosis , Coumarins/administration & dosage , Coumarins/chemistry , Cricetulus , Drinking/drug effects , Eating/drug effects , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Humans , Male , Nanospheres/administration & dosage , Nanospheres/chemistry , Peanut Agglutinin/administration & dosage , Peanut Agglutinin/chemistry , Polystyrenes/administration & dosage , Polystyrenes/chemistry , Polyvinyls/administration & dosage , Polyvinyls/chemistry , Rats , Rectum/drug effects , Rectum/pathology , Thiazoles/administration & dosage , Thiazoles/chemistryABSTRACT
PURPOSE: To investigate whether intravitreally injected peanut agglutinin (PNA) conjugated with a fluorochrome can specifically label retinal cones in vivo and to evaluate its clinical potential. METHODS: Fluorescein- or rhodamine-conjugated PNA (0.005%-0.5%) was intravitreally injected into anesthetized mouse, guinea pig, or monkey and retinas were removed at various intervals for fluorescence microscopy. Immunofluorescence and TUNEL assay were carried out to investigate whether PNA injection adversely affected other retinal neurons. Gross visual function was studied in a visual cliff test. The retina of an N-methyl, N-nitrosourea (MNU)-induced mouse model of retinal degeneration was stained with PNA to evaluate how spatiotemporal pattern of the staining would reflect the progression of degeneration. RESULTS: Intravitreally injected PNA resulted in specific labeling of cone outer and inner segments and cone pedicles within 30 minutes over the entire retina and in all tested species. The labeling was reversible; cones did not show any labeling 3 weeks after the injection but could be restained with PNA. TUNEL signal and expression pattern of several retinal proteins in PNA-injected mouse retina were indistinguishable from normal. Similarly, visual behavior of mouse 10 hours after the injection was normal. The pattern of PNA labeling in mice with MNU-induced retinal degeneration showed progressive disappearance of cones from the center to the periphery. CONCLUSIONS: Intravitreal injection of fluorochrome-conjugated PNA results in specific and reversible labeling of mammalian cones in vivo without causing any gross adverse effects. This novel method may eventually provide a clinical tool to examine diseased retina.
Subject(s)
Fluoresceins/metabolism , Peanut Agglutinin/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/metabolism , Rhodamines/metabolism , Alkylating Agents/toxicity , Animals , Biomarkers/metabolism , Disease Models, Animal , Fluoresceins/toxicity , Guinea Pigs , In Situ Nick-End Labeling , Injections , Macaca mulatta , Methylnitrosourea/toxicity , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Peanut Agglutinin/toxicity , Photoreceptor Cells, Vertebrate/drug effects , Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/pathology , Retinal Cone Photoreceptor Cells/drug effects , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/chemically induced , Retinal Degeneration/diagnosis , Staining and Labeling , Vitreous BodyABSTRACT
Peanut agglutinin lectin (PNA) binds the Thomsen-Friedenreich (TF) oncofetal carbohydrate antigen (galactose beta1-3N-acetylgalactosamine alpha) that shows increased expression in colon cancer, adenomas, and inflammatory bowel disease. PNA is mitogenic, both in vitro and in vivo, for colon epithelial cells. In these cells, PNA binds predominantly to cell-surface TF antigen expressed by high molecular weight isoforms of the transmembrane glycoprotein CD44 that are generated in inflamed and neoplastic colonic epithelia by altered RNA splicing. Our aim was to identify the signaling mechanism underlying the proliferative response to PNA. This was investigated in HT29, T84, and Caco2 colon cancer cells. Parallel lectin and immunoblotting of PNA affinity-purified HT29 cell membrane extracts showed PNA binding to high molecular weight CD44v6 isoforms. Within 5 min, PNA (25 microg/mL) caused a 6-fold increase in phosphorylation of hepatocyte growth factor receptor c-Met, known to co-associate with CD44v6. This was followed by the downstream activation of p44/p42 mitogen-activated protein kinase (MAPK) over 15-20 min. The presence of 100 microg/mL asialofetuin, a TF antigen-expressing glycoprotein, blocked both PNA-induced c-Met and MAPK activation. A similar PNA-induced c-Met and MAPK phosphorylation was also seen in T84 cells that express CD44v6 but not in Caco2 cells that lack CD44v6. PNA-induced cell proliferation was completely blocked by 1 microM PD98059, an inhibitor of MAPK activation (p < 0.0001). The expression of TF antigen by CD44 isoforms in colonic epithelial cells allows lectin-induced mitogenesis that is mediated by phosphorylation of c-Met and MAPK. It provides a mechanism by which dietary, microbial, or endogenous galactose-binding lectins could affect epithelial proliferation in the cancerous and precancerous colon.