RESUMEN
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
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
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
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.
Asunto(s)
Adenocarcinoma/diagnóstico , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/diagnóstico , Diagnóstico por Imagen/métodos , Adenocarcinoma/metabolismo , Animales , Colonoscopía , Neoplasias Colorrectales/metabolismo , Detección Precoz del Cáncer , Femenino , Fluorescencia , Colorantes Fluorescentes , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , Nanosferas , Ratas , Ratas Desnudas , Células Tumorales CultivadasRESUMEN
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
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.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Neoplasias Colorrectales/diagnóstico , Cumarinas , Diagnóstico por Imagen/métodos , Nanosferas , Aglutinina de Mani , Tiazoles , Animales , Antígenos de Carbohidratos Asociados a Tumores/genética , Western Blotting , Estudios de Casos y Controles , Colon/metabolismo , Neoplasias Colorrectales/metabolismo , Cumarinas/farmacocinética , Colorantes Fluorescentes , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Transgénicos , Aglutinina de Mani/farmacocinética , Poliestirenos/química , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Recto/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Propiedades de Superficie , Tiazoles/farmacocinética , Distribución Tisular , Células Tumorales CultivadasRESUMEN
We evaluated the potential of poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, as a carrier for mucosal vaccine delivery. Mice were nasally inoculated four times every seventh day with PBS containing ovalbumin with or without the d-octaarginine-linked polymer. The polymer enhanced the production of ovalbumin-specific immunoglobulin G (IgG) and secreted immunoglobulin A (IgA) in the serum and the nasal cavity, respectively. Ovalbumin internalized into nasal epithelial cells appeared to stimulate IgA production. Ovalbumin transferred to systemic circulation possibly enhanced IgG production. An equivalent dose of the cholera toxin B subunit (CTB), which was used as a positive control, was superior to the polymer in enhancing antibody production; however, dose escalation of the polymer overcame this disadvantage. A similar immunization profile was also observed when ovalbumin was replaced with influenza virus HA vaccines. The polymer induced a vaccine-specific immune response identical to that induced by CTB, irrespective of the antibody type, when its dose was 10 times that of CTB. Our cell-penetrating peptide-linked polymer is a potential candidate for antigen carriers that induce humoral immunity on the mucosal surface and in systemic circulation when nasally coadministered with antigens.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Membrana Mucosa/metabolismo , Mucosa Nasal/metabolismo , Polímeros/administración & dosificación , Vacunas/administración & dosificación , Acetamidas/administración & dosificación , Acetamidas/química , Acetamidas/inmunología , Acrilatos/administración & dosificación , Acrilatos/química , Acrilatos/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Administración Intranasal/métodos , Animales , Formación de Anticuerpos/inmunología , Antígenos/administración & dosificación , Antígenos/química , Antígenos/inmunología , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/inmunología , Toxina del Cólera/inmunología , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Inmunidad Humoral/inmunología , Inmunoglobulina A Secretora/inmunología , Inmunoglobulina G/inmunología , Vacunas contra la Influenza/administración & dosificación , Vacunas contra la Influenza/química , Vacunas contra la Influenza/inmunología , Ratones , Ratones Endogámicos BALB C , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/inmunología , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/inmunología , Ovalbúmina/inmunología , Soluciones Farmacéuticas/administración & dosificación , Soluciones Farmacéuticas/química , Polímeros/química , Polivinilos/administración & dosificación , Polivinilos/química , Vacunación/métodos , Vacunas/químicaRESUMEN
We are investigating a new class of penetration enhancers that enable poorly membrane-permeable molecules physically mixed with them to effectively penetrate cell membranes without their concomitant cellular uptake. Since we previously revealed that poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, significantly enhanced the nasal absorption of insulin, we examined the performance of the polymers on cell membranes. When Caco-2 cells were incubated with 5(6)-carboxyfluorescein (CF) for 30 min, approximately 0.1% of applied CF was internalized into the cells. This poor membrane permeability was dramatically enhanced by d-octaarginine-linked polymers; a 25-fold increase in the cellular uptake of CF was observed when the polymer concentration was adjusted to 0.2mg/mL. None of the individual components, for example, d-octaarginine, had any influence on CF uptake, demonstrating that only d-octaarginine anchored chemically to the polymeric platform enhanced the membrane permeation of CF. The polymer-induced CF uptake was consistently high even when the incubation time was extended to 120 min. Confocal laser scanning microphotographs of cells incubated with d-octaarginine-linked polymers bearing rhodamine red demonstrated that the cell outline was stained with red fluorescence. The polymer-induced CF uptake was significantly suppressed by 5-(N-ethyl-N-isopropyl)amiloride, which is an inhibitor of macropinocytosis. Results indicated that d-octaarginine-linked polymers remained on the cell membrane and poorly membrane-permeable CF was continuously internalized into cells mainly via macropinocytosis repeated for the individual peptidyl branches in the polymer backbone.
Asunto(s)
Acetamidas/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Fluoresceínas/farmacocinética , Oligopéptidos/química , Polivinilos/química , Acrilatos/química , Amilorida/análogos & derivados , Amilorida/farmacología , Células CACO-2 , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Colorantes Fluorescentes/química , Humanos , Microscopía Confocal , Pinocitosis/efectos de los fármacos , Rodaminas/química , Factores de TiempoRESUMEN
The goal of this research is to develop an imaging agent that enables real-time and accurate diagnosis of small-sized colorectal cancer. Since colorectal cancer initially develops in the mucous membrane of the large intestine, a nonabsorbable colonoscopic imaging agent capable of being administered intracolonically was designed. The imaging agent is peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) chains encapsulating coumarin 6. PNA is a targeting moiety that binds to ß-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA is immobilized with the aim of reducing nonspecific interactions between the imaging agent and normal tissues, because the initial tumor-derived change is very small throughout the entire large intestine. Coumarin 6 is encapsulated into nanosphere cores to provide endoscopically-detectable fluorescence intensity. It is anticipated that the intracolonically-administered imaging agent recognizes tumor-derived changes in the large intestinal mucosa with high affinity and specificity. Real-time and accurate diagnosis of small-sized early colorectal cancer can be achieved through an imaging agent providing clear fluorescence contrast between normal and cancer tissues observed with a florescence endoscope. This review describes the design concept of this nanoprobe from a physicochemical perspective.
Asunto(s)
Neoplasias Colorrectales/diagnóstico , Colorantes Fluorescentes , Nanosferas , Aglutinina de Mani , Acetamidas/química , Animales , Colonoscopía/métodos , Neoplasias Colorrectales/patología , Cumarinas/química , Sistemas de Liberación de Medicamentos , Colorantes Fluorescentes/química , Humanos , Aglutinina de Mani/química , Poliestirenos/química , Polivinilos/química , Tiazoles/químicaRESUMEN
We have designed a novel colonoscopic imaging agent that is composed of submicron-sized fluorescent polystyrene nanospheres with two functional groups - peanut agglutinin (PNA) and poly(N-vinylaceamide) (PNVA) - on their surfaces. PNA is a targeting moiety that binds to ß-d-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-ß(1-3)GalNAc), which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells; it is anchored on the nanosphere surface via a poly(methacrylic) acid (PMAA) linker. PNVA is immobilized to enhance the specificity of PNA by reducing nonspecific interactions between the imaging agent and normal tissues. The essential nature of both functional groups was evaluated through in vivo experiments using PNA-free and PNVA-free nanospheres. The imaging agent recognized specifically tumors on the cecal mucosa of immune-deficient mice in which human colorectal cancer cells had been implanted; however, the recognition capability disappeared when PNA was replaced with wheat germ agglutinin, which has no affinity for Gal-ß(1-3)GalNAc. PNA-free nanospheres with exclusively surface PNVA chains rarely adhered to the cecal mucosa of normal mice that did not undergo the cancer cell implantation. In contrast, there were strong nonspecific interactions between normal tissues and PNA-free nanospheres with exclusively surface PMAA chains. In vivo data proved that PNA and PNVA were essential for biorecognition for tumor tissues and a reduction of nonspecific interactions with normal tissues, respectively.
Asunto(s)
Acetamidas/química , Neoplasias Colorrectales/patología , Medios de Contraste/química , Colorantes Fluorescentes/química , Nanosferas/química , Aglutinina de Mani/química , Polivinilos/química , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Ciego/metabolismo , Ciego/patología , Química Farmacéutica , Colonoscopía , Neoplasias Colorrectales/metabolismo , Femenino , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones SCID , Microscopía Fluorescente , Trasplante de Neoplasias , Aglutinina de Mani/metabolismo , Propiedades de SuperficieRESUMEN
We designed peanut agglutinin (PNA)-immobilized fluorescent nanospheres as a non-absorbable endoscopic imaging agent capable of being administered intracolonically. Following our previous researches with evidence that the imaging agent recognized small-sized colorectal tumors on the mucosal surface with high affinity and specificity in animal experiments, a potential of this nanoprobe as a drug candidate was evaluated from a safety perspective. The imaging agent detects colorectal tumors through recognition of the tumor-specific antigen by PNA immobilized on the nanosphere surface, and the detection is made via the fluorescent signal derived from coumarin 6 encapsulated into the nanosphere core. The stability studies revealed that the high activity of PNA was maintained and there was no significant leakage of coumarin 6 after intracolonic administration of the imaging agent. Cytotoxicity studies indicated that no local damage to the large intestinal membrane was induced by the imaging agent. Further, in vitro and in vivo permeation studies demonstrated that there was no significant permeation of the imaging agent through the monolayer of cultured cells and that the imaging agent administered locally to the luminal side of the large intestine was almost completely recovered from the administration site. Therefore, we concluded that the imaging agent is a safe and stable probe which remains in the large intestine without systemic exposure.
Asunto(s)
Colonoscopía/métodos , Cumarinas , Detección Precoz del Cáncer/métodos , Nanosferas , Aglutinina de Mani , Tiazoles , Animales , Células CACO-2 , Colon/patología , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Cumarinas/química , Femenino , Colorantes Fluorescentes/química , Humanos , Indicadores y Reactivos/química , Mucosa Intestinal/química , Ratones , Nanosferas/química , Aglutinina de Mani/química , Tiazoles/químicaRESUMEN
Oligoarginines, which are known as cell-penetrating peptides, enhance the cellular uptake of poorly membrane-permeable bioactive molecules that are chemically conjugated to them. We designed a novel polymer: oligoarginine-linked poly(N-vinylacetamide-co-acrylic acid), with the expectation that the polymers will enhance the cellular uptake of the bioactive molecules that are physically mixed with them. Oligoarginines were grafted onto the polymer backbone through the chemical reaction with acrylic acid functional groups. The changes in the blood glucose concentration after nasal administration of insulin with and without the polymer were monitored in mice. The blood glucose concentration was slightly reduced when insulin was given solely at a dose of 10IU/kg. A D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) with a grafting degree of 2% significantly enhanced the insulin-induced hypoglycemic effect. A similar enhancement was not observed when the polymer was substituted with intact D-octaarginine. The penetration-enhancing function of D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) increased dramatically with an increase in the grafting degree of D-octaarginine. Substitution of D-octaarginine with the corresponding optical isomer and an increase in the number of arginine residues rather reduced the penetration-enhancing function. In vitro cell studies also indicated that a D-octaarginine-linked poly(N-vinylacetamide-co-acrylic acid) with a grafting degree of 17% enabled fluorescein isothiocyanate-dextran to effectively penetrate the cell membrane. Results demonstrated that our oligoarginine-linked polymer has a potential to provide a new class of penetration enhancers.
Asunto(s)
Acetamidas/administración & dosificación , Acrilatos/administración & dosificación , Permeabilidad de la Membrana Celular/efectos de los fármacos , Portadores de Fármacos , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Oligopéptidos/administración & dosificación , Péptidos/administración & dosificación , Polivinilos/administración & dosificación , Ponzoñas/administración & dosificación , Acetamidas/química , Acrilatos/química , Administración Intranasal , Animales , Glucemia/efectos de los fármacos , Células CACO-2 , Química Farmacéutica , Composición de Medicamentos , Exenatida , Femenino , Humanos , Hipoglucemiantes/química , Insulina/química , Ratones , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Oligopéptidos/química , Péptidos/química , Polivinilos/química , Factores de Tiempo , Ponzoñas/químicaRESUMEN
Peanut agglutinin (PNA)-immobilized fluorescent nanospheres were designed as a novel imaging agent for colonoscopy. PNA is a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The in vivo performance of the imaging agent was evaluated using a human colorectal cancer orthotopic animal model. Human colorectal adenocarcinoma cell lines, HT-29, HCT-116, and LS174T, were implanted on the cecal serosa of immune-deficient mice. A loop of the tumor-bearing cecum was made, and the luminal side was treated with the imaging agent. Strong fluorescence was observed at several sites of the cecal mucosa, irrespective of cancer cell type. Microscopic histological evaluation of the cecal mucosa revealed that bright areas with fluorescence derived from the imaging agent and dark areas without the fluorescence well denoted the presence and absence, respectively, of the invasion of implanted cancer cells on the mucosal side. This good correlation showed that PNA-immobilized fluorescent nanospheres recognized millimeter-sized tumors on the cecal mucosa with high affinity and specificity.
Asunto(s)
Acetamidas/química , Neoplasias Colorrectales/diagnóstico , Diagnóstico por Imagen/métodos , Colorantes Fluorescentes/química , Nanosferas/química , Aglutinina de Mani/química , Polivinilos/química , Animales , Ciego/patología , Línea Celular Tumoral , Colitis/diagnóstico , Colitis/patología , Colon/patología , Neoplasias Colorrectales/patología , Detección Precoz del Cáncer/métodos , Femenino , Colorantes Fluorescentes/síntesis química , Mucosa Intestinal/patología , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Ratones , Ratones Desnudos , Ratones SCID , Microscopía Fluorescente , Estructura Molecular , Trasplante de Neoplasias , Tamaño de la Partícula , Propiedades de Superficie , Proteína Fluorescente RojaRESUMEN
Peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) chains encapsulating coumarin 6 were designed as a novel colonoscopic imaging agent. PNA was a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA was immobilized with the aim of reducing nonspecific interactions between imaging agents and normal tissues. Coumarin 6 was encapsulated into nanosphere cores to provide endoscopically detectable fluorescence intensity. After incubation of imaging agents with human cells, the fluorescence intensity of imaging agent-bound cells was estimated quantitatively. The average fluorescence intensity of any type of colorectal cancer cell used in this study was higher than that of small intestinal epithelial cells that had not exposed the carbohydrate. The in vivo performance of imaging agents was subsequently evaluated using a human colorectal cancer orthotopic animal model. Imaging agent-derived strong fluorescence was observed at several sites of the large intestinal mucosa in the tumor-implanted nude mice after the luminal side of the colonic loop was contacted with imaging agents. In contrast, when mice that did not undergo tumor implantation were used, the fluorescence intensity on the mucosal surface was extremely low. Data indicated that imaging agents bound to colorectal cancer cells and the cancer cell-derived tumors with high affinity and specificity.
Asunto(s)
Neoplasias Colorrectales/diagnóstico , Diagnóstico por Imagen/métodos , Colorantes Fluorescentes/química , Nanosferas/química , Aglutinina de Mani/química , Acetamidas/química , Animales , Antígenos de Carbohidratos Asociados a Tumores/química , Línea Celular , Cumarinas/química , Humanos , Ratones , Ratones Desnudos , Modelos Animales , Polivinilos/química , Tiazoles/químicaRESUMEN
Lectin-immobilized fluorescent nanospheres were designed with the aim of developing a novel endoscopic imaging agent for the detection of early colorectal cancer. Submicron-sized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) and poly(methacrylic acid) (PMAA) chains encapsulating fluorescein-labeled cholesterol were prepared as a platform of the imaging agent. Peanut agglutinin (PNA) was immobilized on the surface of fluorescent nanospheres through a chemical reaction with PMAA in order to recognize beta-D-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-beta(1-3)GalNAc), which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The effect of surface structure of nanospheres on the affinity and specificity of immobilized PNA for Gal-beta(1-3)GalNAc was examined. Agglutination of normal and Gal-beta(1-3)GalNAc-expressed erythrocytes in the presence of nanospheres showed that PNA was immobilized actively on the nanosphere surface. Molecular weights of PNVA and PMAA affected the PNA activity most strongly. When the weight-average molecular weight of PNVA was nearly equal to that of PMAA, the affinity of PNA immobilized on the nanosphere surface for Gal-beta(1-3)GalNAc was as strong as that of intact PNA; the specificity for the carbohydrate residue was higher than that of the PNA. Results indicated that PNVA enhanced the specificity of PNA through the reduction of nonspecific interactions between PNA and carbohydrates other than Gal-beta(1-3)GalNAc on the erythrocyte surface without a significant decrease in the affinity.
Asunto(s)
Colonoscopía/métodos , Neoplasias Colorrectales/diagnóstico , Lectinas/química , Nanosferas , Polímeros/química , Colesterol/química , Fluorescencia , Humanos , Aglutinina de ManiRESUMEN
Chitosan is functionalized with phthalic anhydride at the amino group and poly(ethylene glycol) methyl ether at the hydroxyl group via homogeneous reaction. The product regenerated forms particles in either aqueous solution or organic solvents. The particles are spheres with the size of few micrometers as observed from scanning electronmicroscopy. The present work demonstrates that by simply adjusting the hydrophilicity and hydrophobicity, we can directly obtain spheres without a specific processing technique.