RESUMO
Colon cancer is one of the most common death-related cancers in the world. For treating colon cancer, it is crucial to detect and remove malignant lesions early. Here, we developed hyaluronate (HA)-peanut agglutinin (PNA) conjugates for the bioimaging of colon cancer. The HA-PNA conjugates were successfully synthesized by the coupling reaction between aldehyde-modified HA and the N-terminal amine group of PNA. For diagnostic imaging, rhodamine B (RhoB) was chemically conjugated onto PNA in HA-PNA conjugates. After intraluminal injection of HA-PNA-RhoB conjugates into tumor-bearing mice, small-sized colon cancers could be effectively visualized by ex vivo imaging with an in vivo imaging system (IVIS) and a two-photon microscope. With these results taken together, we could confirm the feasibility of HA-PNA-RhoB conjugates as a bioimaging agent for detecting colon cancers.
Assuntos
Neoplasias do Colo/patologia , Ácido Hialurônico/química , Microscopia de Fluorescência/métodos , Aglutinina de Amendoim/química , Animais , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/metabolismo , Sulfato de Dextrana/toxicidade , Humanos , Ácido Hialurônico/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Aglutinina de Amendoim/farmacocinética , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This research aimed to validate the specificity of the newly developed nanobeacon for imaging the Thomsen-Friedenreich (TF) antigen, a potential biomarker of colorectal cancer. The imaging agent is comprised of a submicron-sized polystyrene nanosphere encapsulated with a Coumarin 6 dye. The surface of the nanosphere was modified with peanut agglutinin (PNA) and poly(N-vinylacetamide (PNVA) moieties. The former binds to Gal-ß(1-3)GalNAc with high affinity while the latter enhances the specificity of PNA for the carbohydrates. The specificity of the nanobeacon was evaluated in human colorectal cancer cells and specimens, and the data were compared with immunohistochemical staining and flow cytometric analysis. Additionally, distribution of the nanobeacon in vivo was assessed using an "intestinal loop" mouse model. Quantitative analysis of the data indicated that approximately 2 µg of PNA were detected for each milligram of the nanobeacon. The nanobeacon specifically reported colorectal tumors by recognizing the tumor-specific antigen through the surface-immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a loss of fluorescence signal, which suggests the specificity of the probe. Most importantly, the probe was not absorbed systematically in the large intestine upon topical application. As a result, no registered toxicity was associated with the probe. These data demonstrate the potential use of this novel nanobeacon for imaging the TF antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer at the molecular level.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Neoplasias Colorretais/diagnóstico , Cumarínicos , Diagnóstico por Imagem/métodos , Nanosferas , Aglutinina de Amendoim , Tiazóis , Animais , Antígenos Glicosídicos Associados a Tumores/genética , Western Blotting , Estudos de Casos e Controles , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Cumarínicos/farmacocinética , Corantes Fluorescentes , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Transgênicos , Aglutinina de Amendoim/farmacocinética , Poliestirenos/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reto/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Propriedades de Superfície , Tiazóis/farmacocinética , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Germ-free (GF) animals lack a colonic microflora like that seen in conventional (CV) animals. Bacterial presence plays a role in the development of glycoproteins in the gastrointestinal (GI) tract; the absence of a microbiota has been seen to suppress the production of certain glycoproteins and glycolipids. Binding patterns of lectins are modified when glycoprotein structures are altered (e.g., during development or disease). Little information on lectin binding patterns in mature GF animals is available. We examined the binding of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) [P(HPMA)-(WGA-FITC)] and FITC-labeled peanut agglutinin (PNA) [P(HPMA)-(PNA-FITC)] in CV and GF mouse colon with and without neuraminidase pretreatment. Anti-Thomsen-Friedenreich (TF) antigen (a development and disease-related glycoprotein) antibody binding was also examined in these tissues. Subtle differences were seen in the binding patterns between CV and GF animals. CV animals showed strong P(HPMA)-(WGA-FITC) binding in goblet cells, but minimal P(HPMA)-(PNA-FITC) binding was visible. In GF animals, luminal surface binding of P(HPMA)-(WGA-FITC) was visible, and goblet cell binding of P(HPMA)-(PNA-FITC) was seen. These subtle changes suggest that altered glycoprotein expression occurred under GF conditions.