Rod-Like Amphiphile of Diblock Polyisocyanate Leading to Cylindrical Micelle and Spherical Vesicle in Water

The self-assembling property of an amphiphilic rod–rod diblock copolymer has been demonstrated using poly­(<i>n</i>-hexyl isocyanate)-<i>block</i>-poly­(2,5,8,11-tetraoxatridecyl isocyanate) (<b>PHIC-</b><i><b>b</b></i><b>-PEOIC</b>) with different degrees of polymerization for the <b>PHIC</b> and <b>PEOIC</b> segments. The critical aggregation concentrations (CAC) of <b>PHIC-</b><i><b>b</b></i><b>-PEOIC</b> decreased with the increasing fraction of the hydrophobic <b>PHIC</b> segment. The relation between the hydrodynamic radius (<i>R</i>_h) and the <b>PHIC</b>/<b>PEOIC</b> ratio differed from that for the radius of gyration (<i>R</i>_g). The self-assembled <b>PHIC-</b><i><b>b</b></i><b>-PEOIC</b> was stable in various polymer concentrations. The ρ parameter (<i>R</i>_g/<i>R</i>_h) strongly suggested that the macromolecular architecture was a cylindrical micelle for <b>PHIC</b>_<b>13</b><b>-</b><i><b>b</b></i><b>-PEOIC</b>_<b>41</b> (ρ = 1.63) and a spherical vesicle for <b>PHIC</b>_<b>22</b><b>-</b><i><b>b</b></i><b>-PEOIC</b>_<b>36</b> (ρ = 1.09) and <b>PHIC</b>_<b>31</b><b>-</b><i><b>b</b></i><b>-PEOIC</b>_<b>31</b> (ρ = 1.09). Transmission electron microscope (TEM) images closely agreed with the structures of the cylindrical micelle and spherical vesicle, which were expected based on the ρ parameter

PS-conjugated Ags were captured by professional APCs effectively.

<p>(A) Splenocytes were classified into five subpopulations (I to V) based on the expression pattern of CD11b and CD11c. I:CD11b⁻CD11c⁻ cells, II:CD11b^intCD11c⁻ cells, III:CD11b^highCD11c⁻ cells, IV:CD11b⁺CD11c⁺ cells, V:CD11b⁻CD11c⁺ cells. (B, C) Each isolated population was co-cultured with sfGFP, sfGFP-PS or sfGFP-liposome for 60 min, and then the amount of uptake was analyzed by flow cytometry. (D, E) Each population of isolated cells was co-cultured with DQ-OVA or DQ-OVA-PS for 60 min, and then the efficiency of antigen degradation processing was analyzed by flow cytometry.</p

Emulsified Phosphatidylserine, Simple and Effective Peptide Carrier for Induction of Potent Epitope-Specific T Cell Responses

<div><p>Background</p><p>To induce potent epitope-specific T cell immunity by a peptide-based vaccine, epitope peptides must be delivered efficiently to antigen-presenting cells (APCs) <i>in vivo</i>. Therefore, selecting an appropriate peptide carrier is crucial for the development of an effective peptide vaccine. In this study, we explored new peptide carriers which show enhancement in cytotoxic T lymphocyte (CTL) induction capability.</p> <p>Methodology/Principal Findings</p><p>Data from an epitope-specific <i>in vivo</i> CTL assay revealed that phosphatidylserine (PS) has a potent adjuvant effect among candidate materials tested. Further analyses showed that PS-conjugated antigens were preferentially and efficiently captured by professional APCs, in particular, by CD11c⁺CD11b⁺MHCII⁺ conventional dendritic cells (cDCs) compared to multilamellar liposome-conjugates or unconjugated antigens. In addition, PS demonstrated the stimulatory capacity of peptide-specific helper T cells <i>in vivo</i>.</p> <p>Conclusions/Significance</p><p>This work indicates that PS is the easily preparable efficient carrier with a simple structure that delivers antigen to professional APCs effectively and induce both helper and cytotoxic T cell responses <i>in vivo</i>. Therefore, PS is a promising novel adjuvant for T cell-inducing peptide vaccines.</p> </div

Mice immunized with PS-conjugated peptide induced epitope-specific CTL effectively <i>in vivo</i>.

<p>(A) B6 mice (3 to 4 mice per group) were immunized s.c. with each carrier-conjugated NP_366–374 (A/HK483) peptide or peptide without carrier in the presence of poly(I:C). Seven days after the immunization, bright CFSE-labeled target cells pulsed with peptide used for the immunization and dim CFSE-labeled target cells pulsed with an irrelevant peptide were injected i.v. as an <i>in vivo</i> cytotoxicity assay. Viability of the target cells in the spleen was examined 20 h after injection. Reduction ratios of epitope-specific target cells were calculated using the formula described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060068#s2" target="_blank">Materials and Methods</a>. (B) A24Tg mice (3 mice per group) were inoculated with PS- or liposome-conjugated NP_257–264 (A/HK483) peptide. The <i>in vivo</i> cytotoxicity assay was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060068#pone-0060068-g001" target="_blank">Figure 1A</a>. n.s. indicates not significant. *p<0.01, **p<0.0001.</p

Confocal laser scanning microscopy analysis of splenocytes co-cultured with PS-conjugated antigens.

<p>(A, B) CD11b⁺ or CD11c⁺ cells were cultured with sfGFP, sfGFP-PS, DQ-OVA or DQ-OVA-PS plus Hoechst33342 for 60 min at 37°C. After the incubation, cells were washed with PBS, and then analyzed under a LSM780 confocal laser scanning microscope system. Blue: cell nucleus, Green: sfGFP or DQ-OVA, Red: CD11b or CD11c.</p

Synthesis of High Molecular Weight and End-Functionalized Poly(styrene oxide) by Living Ring-Opening Polymerization of Styrene Oxide Using the Alcohol/Phosphazene Base Initiating System

The ring-opening polymerization (ROP) of styrene oxide (SO) was carried out using 3-phenyl-1-propanol (PPA) as the initiator and a phosphazene base, 1-<i>tert</i>-butyl-4,4,4-tris(dimethylamino)-2,2-bis[tris(dimethylamino)phosphoranylidenamino]-2Λ⁵,4Λ⁵-catenadi(phosphazene) (<i>t</i>-Bu-P₄), as the catalyst at room temperature. The polymerization proceeded in a living manner, which was confirmed by the kinetic and chain extension experiments, to produce the poly(styrene oxide) (PSO) with a controlled molecular weight (5200–21 800 g mol^–1) and narrow molecular weight distribution (<1.14). The ¹H NMR and MALDI-TOF MS measurements of the obtained PSO clearly indicated the presence of the PPA residue at the chain end. In addition, the <i>t</i>-Bu-P₄-catalyzed ROP of SO with functional initiators, such as 4-vinylbenzyl alcohol, 5-hexen-1-ol, 6-azide-1-hexanol, and 3-hydroxymethyl-3-methyloxetane, successfully afforded the corresponding end-functionalized PSO with precise molecular control. The <i>t</i>-Bu-P₄-catalyzed ROP of SO proceeded through the β- and α-scissions as the main and minor ring-opening manners on the basis of the microstructure of the PSOs analyzed by the ¹³C NMR measurement, which was clarified in the model reactions corresponding to the initiation and propagation. For the thermal analysis of PSO, the glass transition temperature and 5% weight loss temperature were found to be 34 and 310 °C, respectively

CD11c⁺CD11b⁺ cells are main APCs in peptide vaccines.

<p>The five kinds of sorted splenocytes were activated with CpG5002 <i>in vitro</i> and cultured with CD8⁺ T cells from NP_366–374-PS immunized mice with serial dilutions of PS-conjugated NP_366–374 peptide for 2 days. Proliferation of NP_366–374-specific CD8⁺ cells was measured by BrdU uptake. The experiment was repeated twice with similar results. *p<0.05, **p<0.01.</p

Proliferation assay of epitope-specific helper T cells.

<p>CD4⁺ T cells from mice immunized with PS-conjugated or unconjugated peptide were co-cultured with activated BMDCs for 2 days in complete RPMI medium containing the indicated concentration of NP_311–325 peptide. Proliferation of NP_311–325-specific CD4⁺ Th cells was measured by BrdU uptake. The experiment was repeated three times with similar results. *p<0.05, **p<0.005</p

Frequency of epitope-specific CD8⁺ T cells in immunized mice.

<p>(A) PS-conjugated NP_366–374 (A/PR8) peptide, PS-conjugated OVA_257–264 peptide or unconjugated peptide was inoculated into B6 mice (3 to 4 mice per group). The <i>in vivo</i> cytotoxicity assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060068#pone-0060068-g001" target="_blank">Figure 1</a>. *p<0.01, **p<0.0001. (B) Splenocytes from naïve mice and mice immunized with PS-conjugated or unconjugated peptide in the presence of poly(I:C) were stained with tetramer and anti-mouse CD8 Ab. The percentage indicates the tetramer-positive cells in total CD8⁺ cells. The experiment was repeated three times with similar results.</p

Synthesis of Well-Defined Three- and Four-Armed Cage-Shaped Polymers via “Topological Conversion” from Trefoil- and Quatrefoil-Shaped Polymers

This paper describes a novel synthetic approach for three- and four-armed cage-shaped polymers based on the topological conversion of the corresponding trefoil- and quatrefoil-shaped precursors. The trefoil- and quatrefoil-shaped polymers were synthesized by the following three reaction steps: (1) the <i>t</i>-Bu-P₄-catalyzed ring-opening polymerization of butylene oxide using multiple hydroxy- and azido-functionalized initiators to produce the three- or four-armed star-shaped polymers possessing three or four azido groups at the focal point, respectively, (2) the ω-end modification to install a propargyl group at each chain end, and (3) the intramolecular multiple click cyclization of the clickable star-shaped precursors. The topological conversion from the trefoil- and quatrefoil-shaped polymers to the cage-shaped polymers was achieved by the catalytic hydrogenolysis of the benzyl ether linkages that had been installed at the focal point. The amphiphilic cage-shaped block copolymers together with the corresponding trefoil- and quatrefoil-shaped counterparts were synthesized in a similar way using 2-(2-(2-methoxyethoxy)­ethoxy)­ethyl glycidyl ether as a hydrophilic monomer and decyl glycidyl ether as a hydrophobic monomer. Interestingly, significant changes in the critical micelle concentration and micellar morphology were observed for the amphiphilic block copolymers upon the topological conversion from the trefoil- and quatrefoil-shaped to cage-shaped architectures