Ken-ichiro Hiwatari

Oral peptide delivery using nanoparticles composed of novel graft copolymers having hydrophobic backbone and hydrophilic branches

Nanoparticles composed of new graft copolymers having a hydrophobic backbone and hydrophilic branches were prepared by the dispersion copolymerization of hydrophilic polyvinyl macromonomers with styrene in a polar solvent. The potential of these nanoparticles as carriers for oral peptide delivery, was investigated using salmon calcitonin (sCT) in rats. The rate of sCT incorporated in nanoparticles was high and was affected by the macromonomer structure. Anionic nanoparticles having poly(methacrylic acid) macromonomer chains on their surfaces showed the highest incorporating activity. When the mixture of sCT and nanoparticles was administered orally, the decrease in the blood ionized calcium concentration was greater than that after oral administration of sCT aqueous solution. This hypocalcemic effect was also affected by the macromonomer structure, and the absorption of sCT was enhanced most strongly by nanoparticles having poly(N-isopropylacrylamide) macromonomer chains. However, the calcium concentration changed less when the nanoparticle concentration was low. On the other hand, the hypocalcemic effect was independent of the nanoparticle size and molecular weight of the macromonomers. The absorption enhancement of sCT by the nanoparticles probably results from both bioadhesion to the gastrointestinal (GI) mucosa and the increase of the stability of sCT in the GI tract. These nanoparticles were demonstrated to be useful carriers for incorporating highly water-soluble peptides and for enhancing peptide absorption via the GI tract.

Optimized chemical structure of nanoparticles as carriers for oral delivery of salmon calcitonin

Nanoparticles having two kinds of surface hydrophilic polymeric chains were prepared by the free radical copolymerization between styrene and hydrophilic macromonomers terminating in vinylbenzyl groups. Their potential as carriers for oral peptide delivery was investigated using salmon calcitonin (sCT) in rats. After oral administration of mixtures of sCT and nanoparticles, the ionized calcium concentration in blood was measured. The absorption of sCT was significantly enhanced by nanoparticles having poly-N-isopropylacrylamide (PNIPAAm) chains on their surfaces. This enhancement effect was considerably increased by introducing cationic poly-vinylamine (PVAm) groups to the surface of PNIPAAm nanoparticles. The absorption enhancement depended on the ratio of NIPAAm and VAm macromonomers to styrene in the nanoparticle preparation. In contrast, the introduction of nonionic poly-vinylacetamide (PNVA) groups eliminated completely the absorption-enhancing function of PNIPAAm nanoparticles. It was suggested that this disappearance was due to the shielding of PNIPAAm groups by PNVA groups. The enhancement effect of sCT absorption by nanoparticles was greatly dominated by their chemical structure that was closely related to surface characteristics. Optimization of the chemical structure on the basis of the mechanism of the absorption enhancement resulted in the further improvement of sCT absorption.

Absorption enhancement of orally administered salmon calcitonin by polystyrene nanoparticles having poly(N-isopropylacrylamide) branches on their surfaces

Abstract Polystyrene nanoparticles having poly(N-isopropylacrylamide) branches on their surfaces (PNIPAAm nanoparticles) were synthesized and various attempts were made in rats to increase the absorption enhancement of orally administered salmon calcitonin (sCT) by these nanoparticles. The hypocalcemic effect after oral administration of a mixture of sCT and PNIPAAm nanoparticles depended greatly on the administration schedule. When one half of a dose of the mixture was given orally 40 min after the other half, sCT-induced hypocalcemic effect was markedly enhanced by PNIPAAm nanoparticles and the area of the reduction of the blood ionized calcium concentration was about 3 times that after administration of a single full dose of the same mixture. However, there was no further enhancement of the pharmacological activity of sCT when the half-doses were administered 120 min apart, sCT absorption was also affected by the hydrophobicity of the PNIPAAm nanoparticles. The hydrophobic PNIPAAm nanoparticles dispersed in hydrochloric acid-sodium chloride solution of pH 1.2, increased in sCT-induced hypocalcemic effect considerably. When two half-doses of the mixture containing these hydrophobic nanoparticles were given orally 40 min apart, the hypocalcemic effect remained strong, even though the dose was reduced to less than half. These changes probably depended on the bioadhesion of PNIPAAm nanoparticles to the gastric mucosa. It was demonstrated that PNIPAAm nanoparticles are good drug carriers that substantially enhance sCT absorption via the gastrointestinal tract.

Stabilization of salmon calcitonin by polystyrene nanoparticles having surface hydrophilic polymeric chains, against enzymatic degradation

Abstract The effect of polystyrene nanoparticles having surface hydrophilic polymeric chains on the stability of salmon calcitonin (sCT) in the presence of digestive enzymes was investigated in vitro. sCT was protected against pepsin- or trypsin-catalyzed degradation by nanoparticles other than those with surface poly( N -vinylacetamide) chains, which do not enhance sCT absorption via the gastrointestinal tract in vivo. This stabilizing effect was affected by the structure of the polymeric chains. Nanoparticles whose surface was covered by poly( N -isopropylacrylamide) inhibited completely sCT degradation by pepsin. However, they did not increase sCT stability in the presence of trypsin. The degradation of sCT by trypsin was inhibited totally by nanoparticles with surface poly(methacrylic acid) chains, even though sCT stability in the presence of pepsin was only slightly improved by them. Nanoparticles having poly(vinylamine) chains on their surfaces stabilized sCT in the presence of either enzyme. It is probable that the stabilizing effect results mainly from the physicochemical interaction between the enzyme and the nanoparticles. These results demonstrated that nanoparticles have the property of stabilizing peptide drugs in the gastrointestinal tract, and that this property affects the absorption enhancement of orally administered sCT.

In vitro/in vivo biorecognition of lectin-immobilized fluorescent nanospheres for human colorectal cancer cells.

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.

Oligoarginine-linked polymers as a new class of penetration enhancers

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.

Detection of early colorectal cancer imaged with peanut agglutinin-immobilized fluorescent nanospheres having surface poly(N-vinylacetamide) chains.

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.

Performance of cell-penetrating peptide-linked polymers physically mixed with poorly membrane-permeable molecules on cell membranes

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.

A potential of peanut agglutinin-immobilized fluorescent nanospheres as a safe candidate of diagnostic drugs for colonoscopy

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.

Poly(N-vinylacetamide) chains enhance lectin-induced biorecognition through the reduction of nonspecific interactions with nontargets

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.