RESUMEN
We have developed an aggregate of D-octaarginine immobilized at multiple points on a co-polymer of N-vinylacetamide and acrylic acid. Previous studies revealed that immunoglobulin G and A were induced when mice were inoculated with influenza virus antigens under coadministration with the D-octaarginine-immobilized polymers as a mucosal vaccine adjuvant. Infection experiments demonstrated that mice vaccinated with a mixture of inactivated influenza viruses and the polymers were protected from infection with mouse-adapted infectious viruses. In the present study, we investigated the mechanism on antigen delivery under mucosal vaccination using the polymers. Two-hour retention of fluorescein-labeled ovalbumin (F-OVA) on the nasal mucosa was observed when applied with the polymers; nevertheless F-OVA was eliminated less than 10 min under polymer-free conditions. F-OVA mixed with the polymers was vigorously taken up into murine dendritic cells. Electrophoresis and dynamic light scattering analysis indicated that OVA interacted with the polymers. The uptake of F-OVA was hardly ever inhibited by the addition of an excess amount of intact OVA. The results suggested that viral antigens were accumulated on the mucosa and delivered into dendritic cells under basolateral membranes via dendrites extending to the mucosal surface and/or subsequent to their permeation through epithelial cells, when they were coadministered with D-octaarginine-immobilized polymers.
Asunto(s)
Péptidos de Penetración Celular , Adyuvantes de Vacunas , Animales , Ratones , Mucosa Nasal , Polímeros , VacunaciónRESUMEN
Our previous mouse studies demonstrated that mean bioavailability of exendin-4, which is an injectable glucagon-like peptide-1 (GLP-1) analogue whose molecular weight (Mw) and isoelectric point (pI) are ca. 4.2 kDa and 4.5, respectively, administered nasally with poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing D-octaarginine, which is a typical cell-penetrating peptide, was 20% relative to subcutaneous administration even though it was less than 1% when exendin-4 alone was given nasally. The studies also revealed that the absorption-enhancing ability of D-octaarginine-linked PNVA-co-AA for exendin-4 was statistically equivalent to that of sodium salcaprozate (SNAC), which is an absorption enhancer formulated in tablets of semaglutide approved recently as an orally available GLP-1 analogue. From a perspective of clinical application of our technology, we have separately developed hyaluronic acid modified with L-octaarginine via a tetraglycine spacer which would be degraded in biological conditions. The present study revealed that tetraglycine-L-octaarginine-linked hyaluronic acid enhanced nasal absorption of exendin-4 in mice, as did D-octaarginine-linked PNVA-co-AA. There was no significant difference in absorption-enhancing abilities between the hyaluronic acid derivative and SNAC when octreotide (Mw: ca. 1.0 kDa, pI: 8.3) and lixisenatide (Mw: ca. 4.9 kDa, pI: 9.5) were used as a model protein drug. On the other hand, SNAC did not significantly enhance nasal absorption of somatropin (Mw: ca. 22.1 kDa, pI: 5.3) when compared with absorption enhancer-free conditions. Substitution of SNAC with tetraglycine-L-octaarginine-linked hyaluronic acid resulted in a 5-fold increase in absolute bioavailability of somatropin with statistical significance. It appeared that pI hardly ever influenced absorption-enhancing abilities of both enhancers. Results indicated that our polysaccharide derivative would be a promising absorption enhancer which delivers biologics applied on the nasal mucosa into systemic circulation and was of greater advantage than SNAC for enhancing nasal absorption of protein drugs with a larger Mw.
Asunto(s)
Ácido Hialurónico/administración & dosificación , Absorción Nasal/efectos de los fármacos , Oligopéptidos/administración & dosificación , Péptidos/administración & dosificación , Administración Intranasal , Animales , Exenatida/administración & dosificación , Exenatida/química , Exenatida/farmacocinética , Hormona de Crecimiento Humana/administración & dosificación , Hormona de Crecimiento Humana/química , Hormona de Crecimiento Humana/farmacocinética , Ácido Hialurónico/química , Ácido Hialurónico/farmacocinética , Ratones , Absorción Nasal/fisiología , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Octreótido/administración & dosificación , Octreótido/química , Octreótido/farmacocinética , Oligopéptidos/química , Oligopéptidos/farmacocinética , Péptidos/química , Péptidos/farmacocinéticaRESUMEN
Mucosal vaccination, which secretion of immunoglobulin A (IgA) on the mucosa is accompanied by induction of immunoglobulin G (IgG) in the blood, is one of the most effective ways to circumvent influenza epidemics caused by incorrect prediction of epidemic viral strains or viral mutation. Secreted IgA is expected to prevent hosts from being infected with heterologous viruses because this antibody cross-reacts to strains other than those used for immunization. Our previous mouse experiments revealed that intranasal IgA with cross-reactivity was induced through nasal inoculation with inactivated whole viral particles of the H1N1 A/New Caledonia/20/99 IVR116 (NCL) strain in the presence of hyaluronic acid modified with tetraglycine-l-octaarginine. In the present study, heterologous influenza virus challenge was performed to validate a potential of the hyaluronic acid derivative as a mucosal adjuvant with cross-protective abilities. Serious weight loss was observed when mice were nasally inoculated with inactivated NCL viruses alone and subsequently exposed to mouse-adapted infectious viruses of the H1N1 A/Puerto Rico/8/34 (PR8) strain. The symptom associated with virus infection was hardly ever observed for mice inoculated with a mixture of the viral antigens and tetraglycine-l-octaarginine-linked hyaluronic acid, presumably due to high induction of IgG and IgA capable of cross-reacting to PR8 viruses. Less proliferation of PR8 viruses in those mice was also supported by an insignificant elevation of antibody levels through virus exposure. Our polysaccharide derivative enabled hosts to acquire adaptive immunity with cross-protective abilities against heterologous virus infection.
Asunto(s)
Adyuvantes Inmunológicos/química , Alphainfluenzavirus/inmunología , Reacciones Cruzadas/inmunología , Ácido Hialurónico/farmacología , Vacunas contra la Influenza/química , Gripe Humana/prevención & control , Adyuvantes Inmunológicos/farmacología , Administración Intranasal , Animales , Humanos , Ácido Hialurónico/química , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Subtipo H1N1 del Virus de la Influenza A/inmunología , Ratones , Oligopéptidos/químicaRESUMEN
We have been investigating the potential use of polymers modified with cell-penetrating peptides as an adjuvant for mucosal vaccination and have already developed nondegradable poly( N-vinylacetamide- co-acrylic acid) (PNVA- co-AA) with which d-octaarginine, a typical cell-penetrating peptide, was grafted. Our previous murine infection experiments demonstrated that immunoglobulin G (IgG) and immunoglobulin A (IgA) were induced in systemic circulation and secreted on nasal mucosa, respectively, through 4-time nasal inoculations with a mixture of influenza viral antigens and d-octaarginine-linked PNVA- co-AA at 7-day intervals, and that immunized mice were perfectly protected from homologous virus infection. In the present study, we designed novel biodegradable polymers bearing cell-penetrating peptides from a perspective of clinical application. Hyaluronic acid whose glucuronic acid was modified with tetraglycine-l-octaarginine at a monosaccharide unit ratio of 30% was successfully developed. The hyaluronic acid derivative exhibited adjuvant activities identical to PNVA- co-AA bearing either d-octaarginine or tetraglycine-d-octaarginine under the above-mentioned inoculation schedule. We further found that there was no difference in humoral immunity between the 4-time inoculations at 7-day intervals and the 2-time inoculations at 28-day intervals. Intranasal IgA induced through the latter schedule with a smaller number of inoculations, which is clinically practical, exhibited cross-reactivity beyond the subtype of viral strains. In vitro toxicity studies demonstrated that the hyaluronic acid derivative was much less toxic than the corresponding PNVA- co-AA derivatives, and that both the polymers and their metabolites did not exhibit genotoxicity. Our results suggested that tetraglycine-l-octaarginine-linked hyaluronic acid would be a clinically valuable and safe adjuvant for mucosal vaccination.
Asunto(s)
Adyuvantes Inmunológicos/efectos adversos , Adyuvantes Farmacéuticos/efectos adversos , Ácido Hialurónico/análogos & derivados , Ácido Hialurónico/efectos adversos , Oligopéptidos/química , Vacunación/métodos , Administración Intranasal , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/metabolismo , Reacciones Cruzadas/inmunología , Femenino , Humanos , Ácido Hialurónico/farmacología , Inmunidad Humoral , Inmunidad Mucosa , Inmunoglobulina A/inmunología , Inmunoglobulina A/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos BALB C , Mucosa Nasal/metabolismoRESUMEN
We are investigating an imaging agent for early detection of colorectal cancer. The agent, named the nanobeacon, is coumarin 6-encapsulated polystyrene nanospheres whose surfaces are covered with poly(N-vinylacetamide) and peanut agglutinin that reduces non-specific interactions with the normal mucosa and exhibits high affinity for terminal sugars of the Thomsen-Friedenreich antigen, which is expressed cancer-specifically on the mucosa, respectively. We expect that cancer can be diagnosed by detecting illumination of intracolonically administered nanobeacon on the mucosal surface. In the present study, biopsied human tissues were used to evaluate the potential use of the nanobeacon in the clinic. Prior to the clinical study, diagnostic capabilities of the nanobeacon for detection of colorectal cancer were validated using 20 production batches whose characteristics were fine-tuned chemically for the purpose. Ex vivo imaging studies on 66 normal and 69 cancer tissues removed from the colons of normal and orthotopic mouse models of human colorectal cancer, respectively, demonstrated that the nanobeacon detected colorectal cancer with excellent capabilities whose rates of true and false positives were 91% and 5%, respectively. In the clinical study, normal and tumor tissues on the large intestinal mucosa were biopsied endoscopically from 11 patients with colorectal tumors. Histological evaluation revealed that 9 patients suffered from cancer and the rest had adenoma. Mean fluorescence intensities of tumor tissues treated with the nanobeacon were significantly higher than those of the corresponding normal tissues. Correlation of magnitude relation of the intensity in individuals was observed in cancer patients with a high probability (89%); however, the probability reduced to 50% in adenoma patients. There was a reasonable likelihood for diagnosis of colorectal cancer by the nanobeacon applied to the mucosa of the large intestine.
Asunto(s)
Neoplasias Colorrectales/patología , Cumarinas/análisis , Colorantes Fluorescentes/análisis , Nanosferas/análisis , Aglutinina de Mani/análisis , Tiazoles/análisis , Animales , Colon/química , Colon/patología , Femenino , Células HT29 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Peptide and protein drugs, which are categorized as biologics, exhibit poor membrane permeability. This pharmacokinetic disadvantage has largely restricted the development of noninvasive dosage forms of biologics that deliver into systemic circulation. We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel absorption enhancer to overcome this challenge. Since our previous study revealed that biocompatible poly( N-vinylacetamide- co-acrylic acid) modified with d-octaarginine, a typical cell-penetrating peptide, enhanced in vitro permeation of biomolecules such as plasmid DNA and bovine serum albumin through cell membranes, the present study evaluated whether the polymers enhanced in vivo absorption of biologics applied on the mucosa. Mouse experiments demonstrated that d-octaarginine-linked polymers drastically enhanced nasal absorption of exendin-4, whose injection is clinically used. The mean bioavailability was 20% relative to subcutaneous administration, even though it fell short of 1% when exendin-4 alone was administered nasally. The absorption-enhancing function of the polymers was superior to that of sodium caprate and sodium N-(8-(2-hydroxybenzoyl)amino) caprylate, which have been used for humans as an absorption enhancer. In vitro experiments using several biologics with different characteristics revealed that biologics interacted with d-octaarginine-linked polymers and were taken up into cells when incubated with the polymers. The interaction and cellular uptake were enhanced as molecular weights of the biologics increased; however, their charge-dependent in vitro performance was not clearly observed. The current data suggested that biologics formulated with our polymers became an alternative to their conventional invasive parenteral formulations.
Asunto(s)
Exenatida/administración & dosificación , Exenatida/farmacocinética , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Oligopéptidos/metabolismo , Vehículos Farmacéuticos/metabolismo , Polímeros/metabolismo , Administración Intranasal , Animales , Línea Celular , Femenino , Ratones , Membrana Mucosa/metabolismo , Oligopéptidos/química , Vehículos Farmacéuticos/química , Polímeros/químicaRESUMEN
We have been investigating the potential of oligoarginine-linked polymers as an adjuvant for mucosal vaccination that induces immunoglobulin G (IgG) in systemic circulation and immunoglobulin A (IgA) secreted on the mucosa. Our latest infection experiments demonstrated that mice immunized nasally with a mixture of inactivated influenza viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) modified with D-octaarginine were perfectly protected from homologous virus infection. On the contrary, virus infection was observed in mice immunized with the antigen alone. This difference was presumably due to insignificant induction of secreted IgA on the nasal mucosa in the latter mice. Since it was unclear whether the current induction level was sufficient for heterologous virus infection, we evaluated the effects of the chemical structures of oligoarginines conjugated to PNVA-co-AA on induction of intranasal IgA. The number and optical activity of the arginine residues and the degree of modification with oligoarginines in the polymer backbone were listed as a factor that would influence IgA induction. Mouse experiments revealed that maximization of the modification resulted in an increase in adjuvant activities of oligoarginine-linked polymers most effectively. Glycine segments inserted between oligoarginines and the polymer backbone were a prerequisite for the maximization. The highest IgA level was observed when antigens were coadministered with diglycine-D-octaarginine-linked PNVA-co-AA.
Asunto(s)
Adyuvantes Inmunológicos/química , Anticuerpos/inmunología , Arginina/química , Materiales Biocompatibles/química , Membrana Mucosa/inmunología , Cavidad Nasal/inmunología , Polímeros/química , Animales , Anticuerpos/química , Arginina/análogos & derivados , Femenino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Membrana Mucosa/químicaRESUMEN
The Thomsen-Friedenreich (TF) antigen is a tumor-associated antigen consistently expressed on the apical surface of epithelial-based cancer cells, including pancreatic cancer. In this work, we report the development of multimodal imaging probe, the tripolymer fluorescent nanospheres, whose surface was fabricated with peanut agglutinin (PNA) moieties as TF molecular recognition molecules. Here, we demonstrate that the probe is able to detect TF antigen in human pancreatic cancer tissues and differentiate from normal tissue. What is most noteworthy regarding the probe is its ability to visualize tumor margins defined by epithelial TF antigen expression. Further, in vivo preclinical studies using an orthotopic mouse model of pancreatic cancer suggest the potential use of the nanospheres for laparoscopic imaging of pancreatic cancer tumor margins to enhance surgical resection and improve clinical outcomes.
RESUMEN
The Thomsen-Friedenreich (TF) antigen represents a prognostic biomarker of colorectal carcinoma. Here, using a nanobeacon, the surface of which was fabricated with peanut agglutinin as TF-binding molecules, we demonstrate that the nanobeacon is able to detect TF antigen in frozen and freshly biopsied polyps using fluorescence microscopy. Our results provide important clues about how to detect aberrant colonic tissues in the most timely fashion. Given the versatile application method for this topical nanobeacon, the protocol used in this work is amenable to clinical colonoscopy. Moreover, the prospects of clinical translation of this technology are evident.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Neoplasias Colorrectales/diagnóstico , Colorantes Fluorescentes/química , Sondas Moleculares/química , Nanopartículas/química , Adenocarcinoma/diagnóstico , Adenocarcinoma/patología , Adenoma/diagnóstico , Adenoma/patología , Neoplasias Colorrectales/patología , Humanos , Microscopía Fluorescente , Imagen Óptica , Aglutinina de Mani/químicaRESUMEN
Mucosal vaccination is one of the most effective ways to reduce the risk of pandemics as a result of incorrect prediction of epidemic strains of influenza viruses or virus mutation. However, adjuvants and antigen carriers with potent immunostimulatory activities are a prerequisite for significant induction of mucosal immunity because most antigens are poorly immunogenic when solely applied to the mucosa. Our previous studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing d-octaarginine induced the secretion of antigen-specific immunoglobulin A (IgA) on the mucosa when nasally administered with virus antigens and that intranasal IgA reacts to viral strains other than the one used for immunization. Therefore, the present study evaluated capabilities of secreted IgA for protection against virus infection. When mice were inoculated with a mixture of inactivated H1N1 A/Puerto Rico/8/34 influenza viruses and d-octaarginine-linked polymers, antigen-specific secreted IgA was induced on the nasal mucosa. Immunized mice were completely protected from virus infection of the inoculated strain. To the contrary, mice nasally inoculated with inactivated viruses alone were infected with the homologous viruses presumably because of insignificant induction of secreted IgA. Results demonstrated that our polymer would be a promising adjuvant for mucosal vaccination.
Asunto(s)
Resinas Acrílicas/química , Subtipo H1N1 del Virus de la Influenza A/inmunología , Membrana Mucosa/inmunología , Oligopéptidos/química , Polímeros/química , Vacunación , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Animales , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/inmunologíaRESUMEN
We have been investigating the potential use of cell-penetrating peptide-linked polymers as a novel penetration enhancer. Since previous in vivo studies demonstrated that poly(N-vinylacetamide-co-acrylic acid) bearing D-octaarginine, a typical cell-penetrating peptide, enhanced membrane permeation of biomolecules, its potential as an in vitro transfection tool was evaluated in this study. A plasmid DNA encoding green fluorescent protein (pGFP-C1), ß-galactosidase, and bovine serum albumin (BSA) were used as model biomolecules. Anionic pGFP-C1 interacted electrostatically with cationic d-octaarginine-linked polymers. When the ratio of mass concentration of polymers to that of pGFP-C1 reached 2.5, complexes whose size and zeta potential were approximately 200 nm and 15 mV, respectively, were obtained. GFP expression was observed in cells incubated with complexes prepared under conditions in which the polymer/pDNA concentration ratio exceeded 2.5. The expression level elevated with an increase in the concentration ratio, but physicochemical properties of the complexes remained unchanged. Results suggested that free polymers contributed to pGFP-C1 internalization. Another cell study demonstrated that ß-galactosidase premixed with polymers was taken up into cells in its active tetrameric form. Similar electrostatic interaction-driven complex formation was observed for BSA charged negatively in neutral solution. However, it appeared that the internalization processes of BSA differed from those of pGFP-C1. A mass concentration-dependent increase in internalized BSA was observed, irrespective of the polymer/protein concentration ratio. Due to frail interactions, polymers that were released from the complexes and subsequently immobilized on cell membranes might also contribute to membrane permeation of BSA.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Oligopéptidos/química , Plásmidos/administración & dosificación , Polímeros/química , Albúmina Sérica Bovina/metabolismo , beta-Galactosidasa/metabolismo , Animales , Bovinos , Permeabilidad de la Membrana Celular , Portadores de Fármacos/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Albúmina Sérica Bovina/genética , Transfección , beta-Galactosidasa/genéticaRESUMEN
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.
Asunto(s)
Acetamidas/toxicidad , Colonoscopía , Cumarinas/toxicidad , Colorantes Fluorescentes/toxicidad , Nanosferas/toxicidad , Aglutinina de Mani/toxicidad , Poliestirenos/toxicidad , Polivinilos/toxicidad , Tiazoles/toxicidad , Acetamidas/administración & dosificación , Acetamidas/química , Animales , Peso Corporal/efectos de los fármacos , Células CHO , Células CACO-2 , Colon/efectos de los fármacos , Colon/patología , Neoplasias Colorrectales/diagnóstico , Cumarinas/administración & dosificación , Cumarinas/química , Cricetulus , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Humanos , Masculino , Nanosferas/administración & dosificación , Nanosferas/química , Aglutinina de Mani/administración & dosificación , Aglutinina de Mani/química , Poliestirenos/administración & dosificación , Poliestirenos/química , Polivinilos/administración & dosificación , Polivinilos/química , Ratas , Recto/efectos de los fármacos , Recto/patología , Tiazoles/administración & dosificación , Tiazoles/químicaRESUMEN
We evaluated cross-reactivity of immunoglobulin A (IgA) secreted on the nasal mucosa in mice that were nasally inoculated 4 times with a mixture of inactivated H1N1 influenza A viruses and poly(N-vinylacetamide-co-acrylic acid) (PNVA-co-AA) bearing d-octaarginine at 7-day intervals. Three viral strains (A/Puerto Rico/8/34, A/New Caledonia/20/99 IVR116, and A/Solomon Islands/03/2006) and D-octaarginine-linked polymers with different molecular weights were used as antigens and their carriers, respectively. Secretion of intranasal IgA was barely observed when the inactivated virus alone was administered. The polymer induced the production of intranasal IgA specific to the inoculated viruses, irrespective of the viral strain and molecular weight of the polymer. The respective antibodies cross-reacted to recombinant hemagglutinin proteins of not only the viral strain used for immunization but also other H1N1 strains, including A/Puerto Rico/8/34 strain whose hemagglutinin proteins are diverse from those of other strains. Mice with high reactivity of IgA to the inoculated viruses tended to acquire clear cross-reactivity to other viral strains. Notably, IgA induced by inactivated H1N1 A/New Caledonia/20/99 IVR116 strain with the strongest immunogenicity between 3 antigens in the presence of the polymer cross-reacted to recombinant hemagglutinin proteins of the A/Brisbane/10/2007 and A/Viet Nam/1194/2004 strains, which are categorized into H3N2 and H5N1, respectively. Our polymer is a potential candidate for an efficient antigen carrier that induces mucosal IgA having cross-reactivity to antigenically drifted variants, irrespective of the subtype of viral strains.
Asunto(s)
Inmunoglobulina A/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Mucosa Nasal/inmunología , Oligopéptidos/química , Acetamidas/química , Acrilatos/química , Animales , Antígenos/inmunología , Reacciones Cruzadas , Femenino , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Mucosa Nasal/virología , Polímeros/química , Polivinilos/químicaRESUMEN
We have been investigating an imaging agent that enables real-time and accurate diagnosis of early colorectal cancer at the intestinal mucosa by colonoscopy. The imaging agent is peanut agglutinin-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) chains encapsulating coumarin 6. Intracolonically-administered lectin-immobilized fluorescent nanospheres detect tumor-derived changes through molecular recognition of lectin for the terminal sugar of cancer-specific antigens on the mucosal surface. The focus of the present study was to evaluate imaging abilities of the nanospheres in animal models that reflect clinical environments. We previously developed an orthotopic mouse model with human colorectal tumors growing on the mucosa of the descending colon to better resemble the clinical disease. The entire colon of the mice in the exposed abdomen was monitored in real time with an in vivo imaging apparatus. Fluorescence from the nanospheres was observed along the entire descending colon after intracolonical administration from the anus. When the luminal side of the colon was washed with phosphate-buffered saline, most of the nanospheres were flushed. However, fluorescence persisted in areas where cancer cells were implanted. Histological evaluation demonstrated that tumors were present in the mucosal epithelia where the nanospheres fluoresced. In contrast, no fluorescence was observed when control mice, without tumors were tested. The lectin-immobilized fluorescent nanospheres were tumor-specific and remained bound to tumors even after vigorous washing. The nanospheres nonspecifically bound to normal mucosa were easily removed through mild washing. These results indicate that the nanospheres combined with colonoscopy, will be a clinically-valuable diagnostic tool for early-stage primary colon carcinoma.
Asunto(s)
Neoplasias Colorrectales/patología , Colorantes Fluorescentes , Mucosa Intestinal/patología , Nanosferas/química , Neoplasias Experimentales/patología , Imagen Óptica/métodos , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Mucosa Intestinal/metabolismo , Lectinas/química , Lectinas/farmacología , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismoRESUMEN
Thomsen-Friedenreich (TF) antigen belongs to the mucin-type tumor-associated carbohydrate antigen. Notably, TF antigen is overexpressed in colorectal cancer (CRC) but is rarely expressed in normal colonic tissue. Increased TF antigen expression is associated with tumor invasion and metastasis. In this study, we sought to validate a novel nanobeacon for imaging TF-associated CRC in a preclinical animal model. We developed and characterized the nanobeacon for use with fluorescence colonoscopy. In vivo imaging was performed on an orthotopic rat model of CRC. Both white light and fluorescence colonoscopy methods were utilized to establish the ratio-imaging index for the probe. The nanobeacon exhibited specificity for TF-associated cancer. Fluorescence colonoscopy using the probe can detect lesions at the stage which is not readily confirmed by conventional visualization methods. Further, the probe can report the dynamic change of TF expression as tumor regresses during chemotherapy. Data from this study suggests that fluorescence colonoscopy can improve early CRC detection. Supplemented by the established ratio-imaging index, the probe can be used not only for early detection, but also for reporting tumor response during chemotherapy. Furthermore, since the data obtained through in vivo imaging confirmed that the probe was not absorbed by the colonic mucosa, no registered toxicity is associated with this nanobeacon. Taken together, these data demonstrate the potential of this novel probe for imaging TF antigen as a biomarker for the early detection and prediction of the progression of CRC at the molecular level.