The Improved Antigen Uptake and Presentation of Dendritic Cells Using Cell-Penetrating D-octaarginine-Linked PNVA-co-AA as a Novel Dendritic Cell-Based Vaccine.

Cancer immunotherapy using antigen-pulsed dendritic cells can induce strong cellular immune responses by priming cytotoxic T lymphocytes. In this study, we pulsed tumor cell lysates with VP-R8, a cell-penetrating D-octaarginine-linked co-polymer of N-vinylacetamide and acrylic acid (PNVA-co-AA), into the DC2.4 murine dendritic cell line to improve antigen uptake and then determined the anti-tumor effect in tumor-bearing mice. DC2.4 cells were pulsed with the cell lysate of EL4, a murine lymphoma cell line, and VP-R8 to generate the DC2.4 vaccine. For the in vivo study, DC2.4 cells pulsed with EL4 lysate and VP-R8 were subcutaneously injected into the inguinal lymph node to investigate the anti-tumor effect against EL4 and EL4-specific T cell immune responses. VP-R8 significantly improved antigen uptake into DC2.4 compared to conventional keyhole limpet hemocyanin (p < 0.05). The expression of MHC class I, MHC class II, and CD86 in DC2.4 cells significantly increased after pulsing tumor lysates with VP-R8 compared to other treatments (p < 0.05). The intra-lymph node injection of DC2.4 pulsed with both VP-R8 and EL4 lysate significantly decreased tumor growth compared to DC2.4 pulsed with KLH and lysates (p < 0.05) and induced tumor-infiltrating CD8T cells. The DC2.4 vaccine also remarkably increased the population of IFN-gamma-producing T cells and CTL activity against EL4 cells. In conclusion, we demonstrated that VP-R8 markedly enhances the efficiency of dendritic cell-based vaccines in priming robust anti-tumor immunity, suggesting its potential as a beneficial additive for dendritic cell-based immunotherapy.

Validation of the Absorption-Enhancing Ability of Oligoarginines Grafted onto a Backbone of Hyaluronic Acid through Animal Studies from Rodents to Primates.

The purpose of our research is to develop functional additives that enhance mucosal absorption of biologics, such as peptide/protein and antibody drugs, to provide their non-to-poor invasive dosage forms self-managed by patients. Our previous in vivo and in vitro studies demonstrated that the intranasal absorption of biologics in mice was significantly improved when coadministered with oligoarginines anchored chemically to hyaluronic acid via a glycine spacer, presumably through syndecan-4-mediated macropinocytosis under activation by oligoarginines. The present mouse experiments first revealed that diglycine-L-tetraarginine-linked hyaluronic acid significantly enhanced the intranasal absorption of sulpiride, which is a poor-absorptive organic compound with a low molecular weight. However, similar enhancement was not observed for levofloxacin, which has a similarly low molecular weight but is a well-absorptive organic compound, probably because its absorption was mostly dominated by passive diffusion. The subsequent monkey experiments revealed that there was no species difference in the absorption-enhancing ability of diglycine-L-tetraarginine-linked hyaluronic acid for not only organic compounds but also biologics. This was presumably because the expression levels of endocytosis-associated membrane proteins on the nasal mucosa in monkeys were almost equivalent to those in mice, and poorly membrane-permeable/membrane-impermeable drugs were mainly absorbed via syndecan-4-mediated macropinocytosis, regardless of animal species. Drug concentrations in the brain assessed in mice and monkeys and those in the cerebral spinal fluids (CSFs) assessed in monkeys indicated that drugs would be delivered from the systemic circulation to the central nervous system by crossing the blood-brain and the blood-CSF barriers under coadministration with the hyaluronic acid derivative. In line with our original hypothesis, this new set of data supported that our oligoarginine-linked hyaluronic acid would locally perform on the mucosal surface and enhance the membrane permeation of drugs under its colocalization.

In Vitro Digestion-In Situ Absorption Setup Employing a Physiologically Relevant Value of the Membrane Surface Area/Volume Ratio for Evaluating Performance of Lipid-Based Formulations: A Comparative Study with an In Vitro Digestion-Permeation Model.

The aim of this study is to establish and test an in vitro digestion-in situ absorption model that can mimic in vivo drug flux by employing a physiologically relevant value of the membrane surface area (S)/volume (V) ratio for accurate prediction of oral drug absorption from lipid-based formulations (LBFs). Three different types of LBFs (Type IIIA-MC, Type IIIA-LC, and Type IV) loaded with cinnarizine (CNZ), a lipophilic weak base with borderline permeability, and a control suspension were prepared. Subsequently, a simultaneous in vitro digestion-permeation experiment was conducted using a side-by-side diffusion cell with a dialysis membrane having a low S/V value. During digestion, CNZ partially precipitated for Type IV, while it remained solubilized in the aqueous phase for Type IIIA-MC and Type IIIA-LC in the donor compartment. However, in vitro drug fluxes for Type IIIA-MC and Type IIIA-LC were lower than those for Type IV due to the reduced free fraction of CNZ in the donor compartment. In pharmacokinetic studies, a similar improvement in in vivo oral exposure relative to suspension was observed, regardless of the LBFs used. Consequently, a poor correlation was found between in vitro permeation and areas under the plasma concentration-time curve (AUCoral) (R2 = 0.087). A luminal concentration measurement study revealed that this discrepancy was attributed to the extremely high absorption rate of CNZ in the gastrointestinal tract compared to that across a dialysis membrane evaluated by the in vitro digestion-permeation model, i.e., the absorption of CNZ in vivo was completed regardless of the extent of the free fraction, owing to the rapid removal of CNZ from the intestine. Subsequently, we aimed to predict the oral absorption of CNZ from the same formulations using a model that demonstrated high drug flux by employing the physiologically relevant S/V value and rat jejunum segment as an absorption sink (for replicating in vivo intestinal permeability). Predigested formulations were injected into the rat intestinal loop, and AUCloop values were calculated from the plasma concentration-time profiles. A better correlation was found between AUCloop and AUCoral (R2 = 0.72), although AUCloop underestimated AUCoral for Type IV due to the precipitation of CNZ during the predigestion process. However, this result indicated the importance of mimicking the in vivo drug absorption rate in the predictive model. The method presented herein is valuable for the development of LBFs.

Mucosal absorption of antibody drugs enhanced by cell-penetrating peptides anchored to a platform of polysaccharides.

Our previous studies demonstrated that L-octaarginine grafted onto hyaluronic acid via a tetraglycine spacer significantly enhanced intranasal absorption of protein drugs with a molecular weight (Mw) of 22 kDa or less. The present study focused on its potential as an absorption enhancer for antibody drugs with a larger Mw and the enhancement mechanism. When ranibizumab (48 kDa) alone was intranasally administered in mice, its absolute bioavailability was 0.67% on average. The mean bioavailability elevated to 6.2% under coadministration with tetraglycine-L-octaarginine-linked hyaluronic acid. A similar result was observed under substitution of ranibizumab with certolizumab pegol (91 kDa), although bioavailability itself decreased with the Mw increase, irrespective of coadministration with the hyaluronic acid derivative. Rat experiments also revealed that coadministration with the polysaccharide derivative resulted in significant enhancement of intranasal absorption of trastuzumab (148 kDa). In vitro studies using gene-knocked down cells indicated that syndecan-4-induced macropinocytosis played a crucial role on acceleration of antibody uptake into epithelial cells on the nasal mucosa, irrespective of their Mw. It appeared that neither clathrin heavy chain nor caveolin-1 involved in cellular uptake of antibodies. Tetraglycine-L-octaarginine-linked hyaluronic acid was concluded to be a promising delivery tool that possessed universal absorption-enhancing abilities independent to Mw of biologics.

Performance of Cell-Penetrating Peptides Anchored to Polysaccharide Platforms Applied via Various Mucosal Routes as an Absorption Enhancer.

We have been investigating the potential of cell-penetrating peptides anchored to polymeric platforms as a novel absorption enhancer which delivers biologics into systemic circulation via mucosal routes. Our previous mouse experiments demonstrated that hyaluronic acid modified with l-octaarginine, a typical cell-penetrating peptide, via a tetraglycine spacer significantly enhanced the mucosal absorption of protein drugs applied into the nasal cavities, irrespective of the molecular weights (Mw) of the drugs. The present study evaluated the performance of tetraglycine-l-octaarginine-linked hyaluronic acid applied via various mucosal routes. Somatropin (Mw: ca. 22.1 kDa) was moderately absorbed from the lung mucosa, and the mean absolute bioavailability (BA) reached 19% under enhancer-free conditions; nevertheless, its BA under intranasal administration was approximately 1% or less. Its BA significantly elevated to 46% on average through intrapulmonary coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid. When the administration site was replaced with the oral cavities, an extreme reduction in somatropin absorption was observed with a mean BA of 0.056% under enhancer-free conditions. Intraoral coadministration with tetraglycine-l-octaarginine-linked hyaluronic acid resulted in a 6.3-fold elevation of somatropin absorption with statistical significance. A similar enhancement was observed under intrarectal administration with a further reduction in BA. On the other hand, the hyaluronic acid derivative did not exhibit the absorption-enhancing ability under intragastric administration, probably due to the lack of stabilization effects against enzyme-susceptible biologics. The results indicated that the intrapulmonary route was suitable for maximizing the mucosal absorption of biologics, and that there was a likelihood of the intraoral route with user convenience. When somatropin was substituted with fluorescein isothiocyanate-conjugated dextran with an average Mw range of 4-70 kDa, similar phenomena were observed under intrapulmonary and intranasal administration. BA decreased with an increase in the Mw of dextran; however, the ratio of BA under enhancer-present conditions to that under enhancer-free conditions was consistently around 3, indicating that the performance of the hyaluronic acid derivative was Mw-independent, irrespective of the administration route.

Evaluation of a D-Octaarginine-linked polymer as a transfection tool for transient and stable transgene expression in human and murine cell lines.

Poly(N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) promotes the cellular uptake of peptides/proteins in vitro; however, details of the transfection efficacy of VP-R8, such as the cell types possessing high gene transfer, are not known. Herein, we compared the ability of VP-R8 to induce the cellular uptake of plasmid DNA in mouse and human cell lines from different tissues and organs. A green fluorescent protein (GFP)-expression plasmid was used as model genetic material, and fluorescence as an indicator of uptake and plasmid-derived protein expression. Three mouse and three human cell lines were incubated with a mixture of plasmid and VP-R8, and fluorescence analysis were performed two days after transfection. To confirm stable transgene expression, we performed drug selection three days after transfection. A commercially available polymer-based DNA transfection reagent (PTR) was used as the transfection control and standard for comparing transgene expression efficiency. In the case of transient transgene expression, slight-to-moderate GFP expression was observed in all cell lines transfected with plasmid via VP-R8; however, transfection efficiency was lower than using the PTR for gene delivery. In the case of stable transgene expression, VP-R8 promoted drug-resistance acquisition more efficiently than the PTR did. Cells that developed drug resistance after VP-R8-mediated gene transfection expressed GFP more efficiently than cells that developed drug resistance after transfection with the PTR. Thus, VP-R8 shows potential as an in vitro or ex vivo nonviral transfection tool for generating cell lines with stable transgene expression.

Acquisition of absorption-enhancing abilities of cationic oligopeptides with short chain arginine residues through conjugation to hyaluronic acid.

Cell-penetrating peptides such as oligoarginines are one of promising tools that improve mucosal absorption of poorly membrane-permeable biologics. We have already demonstrated that conjugation of L-octaarginine to hyaluronic acid via a tetraglycine spacer resulted in a 3-fold enhancement of nasal absorption of somatropin (Mw: ca. 22.1 kDa) in mice when compared with the unmodified peptide. Here, we evaluated absorption-enhancing abilities and safety profiles of oligopeptides with short chain arginine residues conjugated to hyaluronic acid. Somatropin absorption was hardly ever enhanced by diglycine-L-tetraarginine. The peptide acquired the absorption-enhancing ability through the conjugation; however, it disappeared when arginine residues were halved. In vivo data were consistent to in vitro cellular uptake of somatropin. When somatropin was substituted with exendin-4 (Mw: ca. 4.2 kDa), cellular uptake was significantly enhanced by diglycine-L-diarginine conjugated to hyaluronic acid under comparison with the unmodified peptide. The conjugate also exhibited the enhancement ability in mice, as observed for hyaluronic acid derivatives with four and more arginine residues. Another cell studies revealed that oligoarginine-linked hyaluronic acid tended to be less toxic as arginine residues were reduced. Results indicated that diglycine-L-tetraarginine-linked hyaluronic acid was the most suitable candidate as an absorption enhancer whose Mw-independent enhancement ability and safety were well-balanced.

Mechanism on antigen delivery under mucosal vaccination using cell-penetrating peptides immobilized at multiple points on polymeric platforms.

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.

Polypod-like structured guanine-rich oligonucleotide aptamer as a selective and cytotoxic nanostructured DNA to cancer cells.

Guanine-rich oligonucleotide (GRO) can be developed as an effective anticancer agent owing to its high selectivity, affinity and antiproliferative activity in cancer cells. In this study, to increase the potency of GRO29A, a 29-mer GRO aptamer against nucleolin, an overexpressed protein in cancer cells, GRO29A was incorporated into three or six pods of polypod-like structured DNA (polypodna), tripodna or hexapodna, respectively. The polypod-like structured GROs, tri-G3, consisting of one tripodna and three GRO29A, or hexa-G1, hexa-G3 or hexa-G6, each of which comprises one hexapodna and one, three or six GRO29A, respectively, were designed. Tri-G3, hexa-G1 and hexa-G3 were prepared in high yield, except for hexa-G6. Polypod-like structured GROs had quadruplex structures under physiological salt conditions, and degraded at a slower rate in buffer containing serum. Cellular interaction experiments using fluorescently labelled DNA samples showed that the uptake of hexa-G3 by nucleolin-positive MCF-7 cells was more than 2-fold higher than GRO29A, and the interaction was increasingly dependent on the number of GRO29A in the structures. Hexa-G3 inhibited the proliferation of MCF-7 cells in more than 40%, but not of CHO cells. These results indicate that polypod-like structured GROs are useful DNA aptamers with high selectivity and cytotoxicity against nucleolin-positive cancer cells.

Nasal absorption enhancement of protein drugs independent to their chemical properties in the presence of hyaluronic acid modified with tetraglycine-L-octaarginine.

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.

Cross-Protective Abilities of Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Mucosal Adjuvant against Infection with Heterologous Influenza Viruses.

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.

Biodegradable Hyaluronic Acid Modified with Tetraglycine-l-octaarginine as a Safe Adjuvant for Mucosal Vaccination.

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.

Tumor recognition of peanut agglutinin-immobilized fluorescent nanospheres in biopsied human tissues.

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.

Phase separation of supersaturated solution created from amorphous solid dispersions: Relevance to oral absorption.

Dissolution of amorphous solid dispersions (ASDs) is a complicated process, which may involve phase separation from the supersaturated state and formation of a colloidal phase. However, relevance of the phase separation behavior to oral absorption from ASDs is still not well understood. We investigated phase separation of a supersaturated fenofibrate (FEN) solution in the presence of polymers, in vitro dissolution of FEN ASDs, and their in vivo absorption. The supersaturation behavior was assessed based on turbidity measurement in an artificial supersaturation system, where FEN ethanol solutions were added to aqueous polymer solutions. The phase separation concentration of FEN was ca. 1 µg/mL regardless of the presence/absence of the polymer, which was approximately 10-fold the equilibrium solubility. In the presence of 0.1% Tween 80 in the media, the phase separation concentration depended on the polymer species, presumably due to differences in their inhibitory effect of crystallization. The degrees of supersaturation achieved by the ASDs were similar to those found in the artificial system, suggesting that the artificial system works for comprehending the effect of polymer species on supersaturation ability for designing ASDs. A robust in vitro-in vivo correlation was achieved using the paddle and the flow-through cell methods by employing non-sink and pH-shift conditions. However, the phase separation concentration may rather be a good and simple indicator to estimate the absorption-enhancing ability of the polymeric excipients for ASDs, if the absorption is limited by solubility.

Biocompatible Polymers Modified with d-Octaarginine as an Absorption Enhancer for Nasal Peptide Delivery.

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.

Effects of the Chemical Structures of Oligoarginines Conjugated to Biocompatible Polymers as a Mucosal Adjuvant on Antibody Induction in Nasal Cavities.

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.

SPECIFIC MOLECULAR RECOGNITION AS A STRATEGY TO DELINEATE TUMOR MARGIN USING TOPICALLY APPLIED FLUORESCENCE EMBEDDED NANOPARTICLES.

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.

Elucidation of the Mechanism of Increased Activity of Immunostimulatory DNA by the Formation of Polypod-like Structure.

PURPOSE: We previously demonstrated that the immunostimulatory activity of CpG DNA is increased by the formation of polypod-like structures. The present study was designed to elucidate the mechanism underlying this increase. METHODS: Tripodna (three pods) and hexapodna (six pods) were prepared. The cellular uptake of Alexa Fluor 488-labeled DNA samples was examined in several cell lines by measuring the MFI of cells. TNF-α release from RAW264.7 cells was measured after addition of polypodna containing CpG motifs. Dissociation of double stranded DNA was evaluated using FRET. RESULTS: Tripodna and hexapodna were efficiently taken up by macrophage-like RAW264.7 cells and dendritic DC2.4 cells, but not by fibroblast or endothelial cell lines. The uptake by RAW264.7 cells was highest for hexapodna, followed by tripodna, dsDNA, and ssDNA. The release of TNF-α from RAW264.7 cells was also highest for hexapodna. The ratio of TNF-α release to cellular uptake was highest for ssDNA, and lowest for dsDNA. Tripodna and hexapodna were more easily dissociated into single strands after cellular uptake than was dsDNA. CONCLUSIONS: The efficient cellular uptake and prompt dissociation into single strands can be directly related to the high immunostimulatory activity of polypod-like structured DNAs containing CpG motifs.

Evaluation of a novel fluorescent nanobeacon for targeted imaging of Thomsen-Friedenreich associated colorectal cancer.

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.

Physicochemical Properties of Solid Phospholipid Particles as a Drug Delivery Platform for Improving Oral Absorption of Poorly Soluble Drugs.

PURPOSE: A novel drug delivery platform, mesoporous phospholipid particle (MPP), is introduced. Its physicochemical properties and ability as a carrier for enhancing oral absorption of poorly soluble drugs are discussed. METHODS: MPP was prepared through freeze-drying a cyclohexane/t-butyl alcohol solution of phosphatidylcholine. Its basic properties were revealed using scanning electron microscopy, x-ray diffraction, thermal analysis, hygroscopicity measurement, and so on. Fenofibrate was loaded to MPP as a poorly soluble model drug, and effect of MPP on the oral absorption behavior was observed. RESULTS: MPP is spherical in shape with a diameter typically in the range of 10-15 µm and a wide surface area that exceeds 10 m2/g. It has a bilayer structure that may accommodate hydrophobic drugs in the acyl chain region. When fenofibrate was loaded in MPP as a model drug, it existed partially in a crystalline state and improvement in the dissolution behavior was achieved in the presence of a surfactant, because of the formation of mixed micelles composed of phospholipids and surfactants in the dissolution media. MPP greatly improved the oral absorption of fenofibrate compared to that of the crystalline drug and its efficacy was almost equivalent to that of an amorphous drug dispersion. CONCLUSION: MPP is a promising option for improving the oral absorption of poorly soluble drugs based on the novel mechanism of dissolution improvement.