Intermediate-Temperature, Proton-Conducting Membranes of Hafnium Phosphate and Zirconium Phosphate/Borate/Sulfate

Uniform, defect-free nanofilms ͑around 100 nm thick͒ of hafnium phosphate were prepared via layer-by-layer deposition of precursor solution of metal alkoxides and were shown to give practically useful proton conductivity at 300-400°C as fuel cell electrolyte membrane. Annealing of the deposited precursor film at 500°C gave a lower area specific resistance ͑R AS ͒ than that at 400°C, and this effect was coincident with the formation of the pyrophosphate unit. The best ͑lowest͒ R AS value of less than 0.1 ⍀ cm 2 for hafnium phosphate membrane ͑annealing at 500°C, 291 nm thick, conductivity of 3 ϫ 10 −4 S cm −1 at 320°C͒ was comparable to that of a nanofilm of an yttrium-doped zirconium phosphate. The R AS values of nanofilms of zirconium sulfate and zirconium borate were 2-3 orders higher than that of the corresponding zirconium phosphate. It is clear from these results that nanofilms of various solid acids are promising candidates for the electrolyte membrane of fuel cells operating at the intermediate temperatures of 300-400°C. © 2010 The Electrochemical Society. ͓DOI: 10.1149/1.3432600͔ All rights reserved. Solid acid catalysis has been widely used within the petrochemical industry and is attracting increasing attention due to its environment-friendly feature. 1 Solid acid catalysts are composed of wide-ranging acidic materials including clay minerals, zeolites, metal oxides and sulfides, metal hydroxides, and heteropoly acids. The acid sites may either be Broensted or Lewis type. Being a most popular class of solid acids, zeolites are microporous crystalline solids of aluminosilicate with well-defined cage structures of SiO 2 and Al 2 O 3 units. Such basic atomic connectivity must be maintained in the aluminosilicate compounds regardless if they are crystalline or not. The development of electrolyte membranes that are composed of inexpensive materials and are operable at the intermediate temperatures of 200-400°C is strongly desired to enable the next-generation fuel cell ͑FC͒ technology. The novel electrolyte membrane should show satisfactory material stability, effective proton conduction, efficient redox reaction and the consequent reduction of electrocatalyst loading, improvement in CO tolerance, and manageable heat recovery. 2 In addition, anhydrous proton conduction in the intermediate-temperature regime eliminates the elaborate water management required for Nafion-based polymer electrolyte fuel cells ͑PEFCs͒. The idea of using inorganic solid acid as FC electrolyte membrane was examined in our previous study. 3 Surprisingly, amorphous nanofilms of aluminosilicate in fact contained acid sites that enabled efficient proton conduction across the film. A 70 nm thick amorphous aluminosilicate film gave practically useful proton conductivity at 300-400°C as an electrolyte membrane of an FC. This was a strong indication that the local atomic connection of the zeolite structure that provides acid sites was basically preserved even in the ultrathin amorphous film. Our subsequent study 4 demonstrated that many of the silica-based double oxides gave rise to proton conductivity of different degrees in the form of amorphous nanofilms. The presence of effective acid sites was also shown in phosphate materials. 5 Amorphous nanofilms of zirconium phosphate exhibited a practically useful level of proton conductivity at 300°C. A covalent network of metal and phosphate species provided quite robust nanofilms whose morphology and proton conductivity remained unchanged for hundreds of hours during the electrochemical examination of up to 400°C. These findings suggest an exciting possibility of developing a rich variety of proton-conducting nanofilms, even more so than the case of silica-based double oxides. Uniform amorphous nanofilms may be obtainable from a large combination of metal species and multivalent oxyacids. Therefore, they provide a valuable opportunity to conduct an extensive search for materials that would give superior proton conductivity and robustness. An advantage of the amorphous texture is that the variety of combination is not restricted by the stoichiometry of the two major components. Certain metal phosphates 6 and borates 7 exhibit highly efficient proton conductivities as crystalline compounds. Unfortunately, these compounds, as such, did not produce robust uniform nanofilms probably due to their crystalline nature. Because the basic atomic framework is common, their amorphous counterparts may provide acid sites for proton conduction, if properly prepared. As the first step of materials search, we replaced the zirconium species with hafnium, in the current study, as the two metal species belong to the identical IV family in the periodic table. We assumed that they would form closely related covalent networks. The film formation and proton conductivity of hafnium phosphate were compared with those of zirconium phosphate. As for the multivalent oxyacid, we chose boric acid and sulfuric acid by considering the chemical stability of their metal oxygen bonds, and the electrochemical behavior of their zirconium compounds were compared with those of the previously reported results of zirconium phosphate. Nanofilms of zirconium sulfate and zirconium borate were prepared to examine their proton conductivities in relation to their phosphate counterpart. Experimental Materials.-2-Methoxyethanol ͑reagent grade͒, ethanol ͑reagent grade͒, diphosphorous pentoxide, boric acid, ethylenediamine, and anhydrous zirconium tetra-n-butoxide ͑85-90%͒ were obtained from Kanto Chemical. Hafnium tetra-n-butoxide ͑95%͒ and yttrium methoxyethoxide ͑15-18% in methoxyethanol͒ were obtained from Gelest Inc. Yttrium tetra-n-butoxide and indium tin oxide ͑ITO͒ coated glass substrate ͑30 nm thick ITO layer, 70 ⍀͒ were obtained from Aldrich. Sulfuric acid was bought from Junsei Chemical. Preparation of thin films of metal phosphate/borate/ sulfate.-The ITO substrate ͑2.5 ϫ 4 cm͒ was cleaned by sonication in ethanol for 2 min and then dried by flushing nitrogen gas. A thin film of amorphous hafnium phosphate was deposited in a layerby-layer fashion by the surface sol-gel process. The precursor sol was prepared as follows: 0.496 g of hafnium tetra-n-butoxide was added to 20 mL of 2-methoxyethanol. The mixture was vigorously stirred for 5 min at room temperature. P 2 O 5 ͑0.213 g͒ was added to 30 mL of 2-methoxyethanol and the mixture was sonicated in an c Present address

Role of Interleukin-6 in the Antigen-Specific Mucosal Immunoglobulin A Responses Induced by CpG Oligodeoxynucleotide-Loaded Cationic Liposomes

An advantage of mucosal vaccines over conventional parenteral vaccines is that they can induce protective immune responses not only at mucosal surfaces but also in systemic compartments. Despite this advantage, few live attenuated or inactivated mucosal vaccines have been developed and applied clinically. We recently showed that the intranasal immunization of ovalbumin (OVA) with class B synthetic oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motif (CpG ODN)-loaded cationic liposomes synergistically exerted both antigen-specific mucosal immunoglobulin A (IgA) and systemic immunoglobulin G (IgG) responses in mice. However, the mechanism underlying the mucosal adjuvant activity of CpG ODN-loaded liposomes remains unknown. In the present study, we showed that the intranasal administration of CpG ODN-loaded cationic liposomes elicited interleukin (IL)-6 release in nasal tissues. Additionally, pre-treatment with an anti-IL-6 receptor (IL-6R) antibody attenuated antigen-specific nasal IgA production but not serum IgG responses. Furthermore, the intranasal administration of OVA and CpG ODN-loaded cationic liposomes increased the number of IgA+/CD138+ plasma cells and IgA+/B220+ B cells in the nasal passages. This increase was markedly suppressed by pre-treatment with anti-IL-6R blocking antibody. In conclusion, IL-6 released by CpG ODN-loaded cationic liposomes at the site of administration may play a role in the induction of antigen-specific IgA responses by promoting differentiation into IgA+ plasma cells for IgA secretion from B cells

The Parathyroid Hormone Second Receptor PTH2R and its Ligand Tuberoinfundibular Peptide of 39 Residues TIP39 Regulate Intracellular Calcium and Influence Keratinocyte Differentiation

Genes related to the parathyroid hormone (PTH) influence cutaneous immune defense and development, but the full functions of the PTH family in cutaneous biology remain incompletely understood. In this study, we examined the expression and potential functions of the PTH second receptor (PTH2R) and its ligand, the tuberoinfundibular peptide of 39 residues (TIP39), in the skin. TIP39 and PTH2R mRNA and protein were detectable in both human and mouse skin, and in cultured keratinocytes and adipocytes. TIP39 was observed in the basal layer of human skin, whereas PTH2R was detected in the spinous to granular layer. The subcellular localization of TIP39 in keratinocytes changed during calcium-induced differentiation and shifted to colocalize with PTH2R at the membrane. The addition of recombinant TIP39 to normal human keratinocytes in culture induced an increase in intercellular calcium and triggered aspects of terminal differentiation including decreased keratin-14 and increased involucrin expression. Consistent with these observations, PTH2R(-/-) mice were observed to have increased epidermal thickness. In summary, identification of TIP39 and its receptor in the epidermis reveals an additional PTH family member that is expressed in the skin and may influence keratinocyte function

Role of liposome surface charge on the mucosal adjuvant effect.

<p>BALB/c female mice were immunized intranasally with PBS, OVA (5 μg/mouse) alone, OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes (0.4 μmol/mouse), anionic PS liposomes (0.4 μmol/mouse), or neutral PC liposomes (0.4 μmol/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay. The data are obtained from at least three independent experiments. The box-plot shows the median value with the 25th-75th percentiles and the error bars indicate the 5th-95th percentiles. Significance was assessed using the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i><0.01.</p

<i>In vitro</i> antigen-specific production of IFN-γ and IL–4 in splenocytes and nasal passages from BALB/c mice immunized intranasally with OVA and DOTAP/DC-chol liposomes.

<p>Splenocytes from vaccinated BALB/c mice were cultured for 72 h in the presence of OVA (0, 1, 10, or 100 μg/ml). After culture, the supernatants were collected, and concentrations of IFN-γ and IL–4 in the culture supernatants were determined by ELISA assay. The data are representative of at least three independent experiments and are expressed as the mean ± standard deviation for samples assayed in triplicate. Significance was assessed with the <i>t</i>-test with Welch correction: *<i>p</i><0.05.</p

Kinetics of the appearance of OVA-specific serum IgG in BALB/c female mice immunized intranasally with OVA and DOTAP/DC-chol liposomes.

<p>BALB/c female mice were immunized intranasally with PBS alone, DOTAP/DC-chol liposomes (0.4 μmol/mouse) alone, OVA (5 μg/mouse) alone, or OVA (5 μg/mouse) plus DOTAP/DC-chol liposomes (0.4 μmol/mouse) once weekly (days 0, 7, 14, 21, and 28). Serum was collected every week immediately prior to immunization (days 0, 7, 14, 21, 28, and 35). Anti-OVA IgG, IgG1, and IgG2a levels in serum were determined by ELISA assay. The data are obtained from at least three independent experiments and are expressed as the mean ± the standard error. Significance was assessed with the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i><0.05, **<i>p</i><0.0001.</p

Cationic DOTAP/DC-chol liposome elicits dose-dependent OVA-specific antibody production depending on antigen (A) and liposome (B) concentrations.

<p>BALB/c female mice were immunized intranasally with various doses of OVA plus DOTAP/DC-chol liposomes on days 0 and 7. Serum was collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay. The data are obtained from at least three independent experiments and are expressed as the mean ± the standard error. Significance was assessed with the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i><0.05, **<i>p</i><0.01.</p

Induction of OVA-specific serum IgG and nasal tissue IgA responses in BALB/c mice immunized intranasally with OVA and cationic liposomes.

<p>BALB/c female mice were immunized intranasally with PBS, OVA (5 μg/mouse) alone, or OVA (5 μg/mouse) plus various cationic liposomes (0.4 μmol/mouse) on days 0 and 7. Serum and nasal washes were collected on day 14. The anti-OVA IgG, IgG1, and IgG2a levels in serum and anti-OVA IgA level in nasal washes were detected by ELISA assay as described in the “Materials and Methods” section. The data were obtained from at least three independent experiments. The box-plot shows the median value with the 25th-75th percentiles and the error bars indicate the 5th-95th percentiles. Significance was assessed using the Kruskal–Wallis with Dunn’s post–hoc test: *<i>p</i> < 0.05, **<i>p</i> < 0.01, NS: not significant.</p