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.
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 ?-d-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-?(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-?(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.
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 ?-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.
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.
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.
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.
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.
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-?(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 ?g 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.
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.
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.
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