membranes

Article Development of pH-sensitive Derivatives with Strong Adjuvant Function and Their Application to Delivery

Eiji Yuba * ID , Shinya Uesugi, Maiko Miyazaki, Yuna Kado, Atsushi Harada and Kenji Kono †

Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan; [email protected] (S.U.); [email protected] (M.M.); [email protected] (Y.K.); [email protected] (A.H.) * Correspondence: [email protected]; Tel.: +81-72-254-9913 † Prof. Kenji Kono has passed away.

Received: 10 June 2017; Accepted: 1 August 2017; Published: 4 August 2017

Abstract: To achieve efficient cancer immunotherapy, the induction of cytotoxic T lymphocyte-based cellular immunity is necessary. In order to induce cellular immunity, antigen carriers that can deliver antigen into cytosol of antigen presenting cells and can activate these cells are required. We previously developed 3-methyl glutarylated dextran (MGlu-Dex) for cytoplasmic delivery of antigen via membrane disruption ability at weakly acidic pH in endosome/lysosomes. MGlu-Dex-modified liposomes delivered model into cytosol of dendritic cells and induced antigen-specific cellular immunity. However, their antitumor effects were not enough to complete the regression of the tumor. In this study, antigen delivery performance of dextran derivatives was improved by the introduction of more hydrophobic spacer groups next to carboxyl groups. 2-Carboxycyclohexane-1-carboxylated dextran (CHex-Dex) was newly synthesized as pH-responsive dextran derivative. CHex-Dex formed stronger hydrophobic domains at extremely weak acidic pH and destabilized lipid membrane more efficiently than MGlu-Dex. CHex-Dex-modified liposomes were taken up by dendritic cells 10 times higher than MGlu-Dex-modified liposomes and delivered model antigen into cytosol. Furthermore, CHex-Dex achieved 600 times higher IL-12 production from dendritic cells than MGlu-Dex. Therefore, CHex-Dex is promising as multifunctional having both cytoplasmic antigen delivery function and strong activation property of dendritic cells for induction of cellular immunity.

Keywords: pH-sensitive liposome; dextran; cytokine; cancer immunotherapy; adjuvant

1. Introduction Recent success of immune checkpoint inhibitors such as CTLA-4 antibody or PD-1 antibody has increased attention to cancer immunotherapy [1]. The canceling of immunosuppression by tumor microenvironments leads to the activation of cancer-specific immune responses, especially cytotoxic T lymphocytes (CTLs), which play a crucial role in eliminating tumor cells, resulting in tumor regression. On the other hand, it has been also reported that immune checkpoint inhibitors hardly show therapeutic effects on the patients that have no cancer-specific CTLs before treatment of immune checkpoint inhibitors [2]. These facts clearly show the importance of cancer-specific CTL induction along with the treatment with immune checkpoint inhibitors. For induction of cancer-specific immune responses in vivo, the delivery of cancer antigen into antigen presenting cells (APCs) such as dendritic cells or macrophages, which start and activate antigen-specific immune responses are crucial [3,4]. Particularly, cross-presentation, which is an antigen presentation of exogenous antigen via MHC class

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Membranes 2017, 7, 41 2 of 14 I molecules, is required to induce CTL and cellular immunity [5]. For induction of cross-presentation, theof cross control-presentation, of antigen the fate control in APCs, of antigen specific fate receptor-mediated in APCs, specific endocytosis receptor-mediated or activation endocytosis of APCs or areactivation regarded of APCs as important are regarded factors as [5important]. The control factors of [5 the]. The intracellular control of fatethe intracellular of the antigen fate can of the be controlledantigen can by be the controlled design ofby antigen the design delivery of antigen carriers. delivery Hence, carriers. various Hence, antigen various delivery antigen carriers delivery for inductioncarriers for of cross-presentation induction of cross have-presentation been reported have such been as polymericreported such , as polymeric polymeric nanoparticles, micelles, polysaccharide-basedpolymeric micelles, polysaccharidenanoparticles and-based liposomes nanoparticles [6–12]. Among and liposomes them, membrane-based [6–12]. Among antigen them, deliverymembrane systems-based have antigen advantages delivery to systems achieve thehave control advantages of intracellular to achieve antigen the fatecontrol by usingof intracellular biological processantigen suchfate by as membraneusing biological fusion. process such as membrane fusion. WeWe previouslypreviously developeddeveloped antigenantigen carrierscarriers toto achieveachieve thethe controlcontrol ofof antigenantigen fatefate withinwithin dendriticdendritic cellscells and and to to induce induce cross-presentation cross-presentation using using liposomes liposomes modified modified with with pH-responsive pH-responsive fusogenic fusogenic polymerspolymers [[1313––1616].]. ForFor thisthis purpose,purpose, carboxylcarboxyl group-introducedgroup-introduced poly(glycidol)spoly(glycidol)s oror polysaccharidespolysaccharides havehave beenbeen designeddesigned asas pH-sensitivepH-sensitive polymerspolymers [[1313––1616].]. Especially,Especially, liposomesliposomes modifiedmodified withwith 3-methyl3-methyl glutarylatedglutarylated dextransdextrans (MGlu-Dex)(MGlu-Dex) delivereddelivered modelmodel antigenicantigenic proteinsproteins ovalbuminovalbumin (OVA)(OVA) intointo cytosolcytosol ofof dendriticdendritic cells via via membrane membrane fusion fusion with with endosoma endosomall membrane membrane responding responding to weakly to weakly acidic acidic pH pHin endosomes in endosomes [15].[ 15 These]. These liposomes liposomes induced induced MHC MHC class I class-mediated I-mediated antigen antigen presentation presentation (cross- (cross-presentation),presentation), leading leading to tumor to tumor regression regression in inE.G7 E.G7-OVA-OVA tumor tumor-bearing-bearing mice mice [ [15,1615,16]].. However, antitumorantitumor immunity immunity induced induced by by MGlu-Dex-modifiedMGlu-Dex-modified liposomes liposomes was was insufficient insufficient for for complete complete regressionregression ofof tumor.tumor. InIn thisthis study,study, thethe improvementimprovement ofof antigenantigen deliverydelivery performanceperformance ofof dextrandextran derivative-modifiedderivative-modified liposomesliposomes waswas attemptedattempted byby increasingincreasing hydrophobicityhydrophobicity ofof dextrandextran derivatives.derivatives. CHexCHex unit,unit, whichwhich havehave cyclohexylcyclohexyl groupgroup asas aa spacerspacer nextnext toto carboxylcarboxyl group,group, waswas introducedintroduced toto dextrandextran insteadinstead ofof MGluMGlu unit.unit. Resultant Resultant 2-carboxycyclohexane-1-carboxylated 2-carboxycyclohexane-1-carboxylated dextran (CHex-Dex,(CHex-Dex, Figure1 1)) was was modified modified ontoonto liposomes. Hydrophobic Hydrophobic CHex CHex-Dex-Dex is isexpected expected to toincrease increase cellular cellular association association of liposomes of liposomes and andpH- pH-responsiveresponsive membrane membrane fusion fusion ability ability compared compared with with MGlu MGlu-Dex.-Dex. Here, Here, the the effect effect of side chain chain structurestructure ofof dextrandextran derivativesderivatives onon theirtheir pH-responsivepH-responsive interactioninteraction withwith lipidlipid membrane,membrane, uptakeuptake byby dendriticdendritic cellscells andand intracellularintracellular deliverydelivery performanceperformance waswas examined.examined. InIn addition,addition, wewe unexpectedlyunexpectedly revealedrevealed the the quite strong strong adjuvant adjuvant function, function, which which is is the the ability ability to to activate activate APCs APCs or induce or induce the thematuration maturation of APCs, of APCs, of CHex of CHex-Dex-Dex compared compared with with MGlu MGlu-Dex.-Dex.

Figure 1. Carboxylated dextran derivative-modified liposomes for intracellular delivery of antigen to Figure 1. Carboxylated dextran derivative-modified liposomes for intracellular delivery of antigen todendritic dendritic cells. cells. CHex CHex-Dex,-Dex, which which has has more more hydrophobic hydrophobic side side chain chain than than conventional conventional MGlu MGlu-Dex,-Dex, is isexpected expected to to enhance enhance cellular cellular association association and and pH pH-responsive-responsive disruptio disruptionn of endosomal membranemembrane ofof liposomes,liposomes, leadingleading toto inductioninduction ofof cellularcellular immunity.immunity.

2. Results and Discussion

2.1. Characterization of Dextran Derivatives Carboxylated dextran derivatives (CHex-Dex) with different contents of CHex groups were synthesized by reacting hydroxyl groups of dextran with various amounts of 1,2-

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2. Results and Discussion

2.1. Characterization of Dextran Derivatives Carboxylated dextran derivatives (CHex-Dex) with different contents of CHex groups Membranes 2017, 7, 41 3 of 14 were synthesized by reacting hydroxyl groups of dextran with various amounts of 1 1,2-cyclohexanedicarboxyliccyclohexanedicarboxylic anhydride anhydride (Figure (Figure 2). Figures2). Figures S1–S5 depict S1–S5 1 depictH NMR spectraH NMR of CHex spectra-Dex. of CHex-Dex.Introduction Introduction of CHex groups of CHex to dextran groups was to confirmed dextran wasfrom confirmed the existence from of peaks the existence corresponding of peaks to correspondingCHex groups (1.2 to CHex–2.0 ppm, groups 2.6 (1.2–2.0ppm). From ppm, the 2.6 integration ppm). From ratio the of integration peaks of CHex ratio ofresidues peaks ofto CHexthose residuesof dextran to thosebackbone of dextran (3.4–4.2 backbone ppm, 5.0 (3.4–4.2 ppm), ppm,the amounts 5.0 ppm), of theCHex amounts groups of combined CHex groups with combined hydroxyl withgroups hydroxyl of dextran groups were of estimated dextran as were shown estimated in Table as 1. shownDecyl chai in Tablens were1. Decylfurther chains introduced were to further CHex- introducedDex by reaction to CHex-Dex of decylamine by reaction with of carboxyl decylamine groups with of carboxyl CHex groupsunits to of fix CHex polymers units toonto fix polymersliposome onto liposome surface (Figure2). Resultant polymers were designated as CHex-Dex-C and 1H NMR surface (Figure 2). Resultant polymers were designated as CHex-Dex-C10 and 1H NMR10 spectra for spectrathese polymers for these were polymers presented were presented in Figures in S Figures6–S9. From S6–S9. theFrom integration the integration ratio between ratiobetween dextran dextranbackbone, backbone, CHex residues,CHex residues, and decyl and decyl groups groups (0.9– (0.9–1.51.5 ppm), ppm), decyl decyl residues residues and and CHex CHex residues residues combinedcombined withwith hydroxylhydroxyl groupsgroups ofof dextrandextran werewere determined determined as as shown shown in in Table Table2 .2.

Figure 2. Synthesis of CHex-Dex and CHex-Dex-C10. Figure 2. Synthesis of CHex-Dex and CHex-Dex-C10.

Table 1. Synthesis of CHex-Dex. Table 1. Synthesis of CHex-Dex. CHex- Reaction Dextran DMF Yield CHex Polymer LiCl (g) CHex-AnhydrideAnhydride ReactionTemperature Yield (g) 1 Polymer Dextran (g)(g) LiCl (g) DMF (mL)(mL) ◦ Yield (g) Yield (%)(%) CHex(%) (%) 1 (g) (g) Temperature(°C ( )C) CHex40-DexCHex40-Dex 0.5080.508 0.5050.505 1818 2.5072.507 7575 0.9810.981 9090 4040 CHex57-Dex 0.998 0.985 12 5.016 90 1.129 43 57 CHex73-DexCHex57-Dex 1.5000.998 1.5010.985 2912 14.9995.016 7590 3.2501.129 7043 7357 CHex86-DexCHex73-Dex 1.1701.500 0.9861.501 1629 10.29014.999 10075 3.5303.250 8770 8673 CHex98-Dex 0.900 0.908 16 9.007 90 2.317 67 98 CHex86-Dex 1.170 0.986 16 10.290 100 3.530 87 86 1 1 CHex98-Dex 0.900 0.908 Determined16 9.007 by H NMR. 90 2.317 67 98 1 Determined by 1H NMR. Table 2. Synthesis of CHex-Dex-C10.

n-DecylTable 2. SynthesisDMT-MM of CHex-Dex-C10. Polymer CHex-Dex (g) Yield (g) Yield (%) CHex (%) 1 Anchor (%) 1 Amine (mg) (mg) CHex-Dex n-Decyl Amine DMT-MM Yield Yield CHex Anchor CHex28-Dex-CPolymer 0.507 54 107 0.430 90 28 4 10 (g) (mg) (mg) (g) (%) (%) 1 (%) 1 CHex42-Dex-C10 0.816 86 174 0.794 95 51 6 CHex53-Dex-CCHex28-Dex10-C10 0.5000.507 4654 81107 0.426 0.430 96 90 5328 4 4 CHex72-Dex-C 1.214 95 68 0.532 43 76 5 CHex42-Dex10-C10 0.816 86 174 0.794 95 51 6 1 1 CHex53-Dex-C10 0.500 46Determined by H81 NMR. 0.426 96 53 4 CHex72-Dex-C10 1.214 95 68 0.532 43 76 5 To investigate the protonation state1 Determined of carboxyl by groups1H NMR. in CHex-Dex at varying pH, acid-base titration of CHex-Dex was conducted (Figure3). CHex-Dex changed their protonation states depending To investigate the protonation state of carboxyl groups in CHex-Dex at varying pH, acid-base on pH in alkali and weakly acidic pH regions. Compared with CHex40-Dex, CHex-Dex having 57%, titration of CHex-Dex was conducted (Figure 3). CHex-Dex changed their protonation states 73% and 86% of CHex groups showed higher pKa values (Table3). This might result from the proximity depending on pH in alkali and weakly acidic pH regions. Compared with CHex40-Dex, CHex-Dex effects between carboxyl groups on CHex-Dex as reported in previous literature [15]. As a comparison, having 57%, 73% and 86% of CHex groups showed higher pKa values (Table 3). This might result pKa values of MGlu group-introduced dextran were listed in Table3. In comparison with dextran from the proximity effects between carboxyl groups on CHex-Dex as reported in previous literature derivatives having almost same carboxyl groups, CHex-Dex exhibited higher pKa than that of [15]. As a comparison, pKa values of MGlu group-introduced dextran were listed in Table 3. In comparison with dextran derivatives having almost same carboxyl groups, CHex-Dex exhibited higher pKa than that of MGlu-Dex. In general, pKa of carboxyl groups in poly(carboxylic acid)s increased with increasing their hydrophobicity. Especially, hydrophobic structure next to carboxylate promotes the protonation of carboxyl groups, resulting in an increase of pKa [17–22]. Therefore, hydrophobic spacer unit in CHex groups promoted protonation of carboxyl groups, resulting in higher pKa than MGlu-Dex. These results are consistent with our previous literature using polyallylamine or poly(glycidol)s [18,21].

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MGlu-Dex. In general, pKa of carboxyl groups in poly(carboxylic acid)s increased with increasing their hydrophobicity. Especially, hydrophobic structure next to carboxylate promotes the protonation of carboxyl groups, resulting in an increase of pKa [17–22]. Therefore, hydrophobic spacer unit in CHex groups promoted protonation of carboxyl groups, resulting in higher pKa than MGlu-Dex. These resultsMembranes are 2017 consistent, 7, 41 with our previous literature using polyallylamine or poly(glycidol)s [18,214]. of 14

Figure 3. 3. TitrationTitration curves curves of dextran of dextran derivatives derivatives measured measured by acid-base by titration acid-base using titration a conductivity using a conductivitymeter. meter.

Table 3. pKapKa and protonation degree atat pHpH 7.47.4 ofof dextrandextran derivatives.derivatives. Polymer pKa Protonation Degree at pH 7.4 PolymerCHex40-Dex 6.42 pKa Protonation0.27 Degree at pH 7.4 CHex40-DexCHex57-Dex 7.24 6.420.46 0.27 CHex57-DexCHex73-Dex 7.25 7.240.46 0.46 CHex73-DexCHex86-Dex 7.34 7.250.49 0.46 CHex86-DexMGlu13-Dex 5.30 1 7.340.08 0.49 1 MGlu13-DexMGlu48-Dex 5.81 1 5.30 0.07 0.08 MGlu48-Dex 5.81 1 0.07 MGlu76-Dex 6.63 1 0.22 MGlu76-Dex 6.63 1 0.22 1 Reproduced from Ref [15]. 1 Reproduced from Ref. [15]. Protonation of carboxyl groups in poly(carboxylic acid)s induced coil-globule transition of polymerProtonation chains with of carboxylformation groups of hydrophobic in poly(carboxylic domains [19 acid)s,20]. Next, induced the formation coil-globule of hydrophobic transition of polymerdomain of chains CHex with-Dex formationwas investigated of hydrophobic using pyrene domains as a fluorescence [19,20]. Next, probe. the formation The pyrene of hydrophobicfluorescence domainintensity of ratio CHex-Dex of the first was investigated(373 nm) to the using third pyrene (384as nm) a fluorescence peaks, I1/I3, probe.has been The used pyrene to evaluate fluorescence the intensitymicro-environmen ratio of thetal firstpolarity (373 surrounding nm) to the thirdthe pyrene (384 nm) molecule peaks, [18,2I1/I13–,23 has]. Figure been used 4 represents to evaluate the theI1/I3 micro-environmentalratio of pyrene fluorescence polarity of surrounding CHex-Dex as the a pyrenefunction molecule of pH. At [18 pH,21 8––237,]. the Figure I1/I3 4ratios represents of pyrene the Iwere1/I3 ratioaround of pyrene1.8 for fluorescenceall dextran derivatives, of CHex-Dex indicating as a function that ofthese pH. polymers At pH 8–7, formed the I1/ noI3 ratios domain of pyrenewith a werehydrophobic around 1.8nature. for all In dextrancontrast, derivatives, the I1/I3 ratio indicating suddenly that decreased these polymers below at formedpH 6.5 nofor domainCHex40 with-Dex aor hydrophobic 7.0 for other nature.CHex-Dex In contrast, and reached the I1 to/I 3aroundratio suddenly 1.4. Cons decreasedidering the below protonation at pH 6.5 curves for CHex40-Dex (Figure 3), oraround 7.0 for 60% other of carboxyl CHex-Dex groups and reached in CHex to-Dex around were 1.4. protonated Considering at these the protonationpH region. This curves indicates (Figure that3), aroundCHex-Dex 60% could of carboxyl form strong groups hydrophobic in CHex-Dex domains were protonated even though at these 40% pHof carboxyl region. This groups indicates in CHex that- CHex-DexDex still have could anionic form strongcharges. hydrophobic According domainsto the previous even though literature 40% [15], of carboxyl the I1/I3groups ratio for in MGlu CHex-Dex-Dex stillis around have anionic1.7 even charges. after most According of carboxyl to the groups previous are protonated, literature [15 which], the Imight1/I3 ratio reflect for hydrophilic MGlu-Dex isnature around of dextran1.7 even backbone. after most Therefore, of carboxyl hydrophobic groups are protonated, interaction between which might protonate reflectd hydrophilic CHex units naturemight of mainly dextran contribute backbone. the Therefore, formation hydrophobic of hydrophobic interaction domains between because protonated of CHex their units bulky might and mainlyhydrophobic contribute structure the formationcompared ofwith hydrophobic MGlu units. domains because of their bulky and hydrophobic structure compared with MGlu units.

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Figure 4. pH-Dependence of I1/I3 of pyrene fluorescence in the presence of dextran derivatives Figure 4. pH-Dependence of I1/I3 of pyrene fluorescence in the presence of dextran derivatives dissolving in 30 mM sodium acetate and 120 mM NaCl solution of varying pH. Concentrations of dissolvingFigure 4. in pH 30-Dependence mM sodium of acetate I1/I3 of and pyrene 120 mMfluorescence NaCl solution in the of presence varying of pH. dextran Concentrations derivatives of polymers and pyrene were 0.25 mg/mL and 0.6 μM, respectively. I1/I3 was defined as the fluorescence polymers and pyrene were 0.25 mg/mL and 0.6 µM, respectively. I /I was defined as the fluorescence dissolvingintensity ratio in 30 of mMthe first sodium band acetate at 373 nmand to 120 the mM third NaCl band solution at 384 nm.1 of 3 varying pH. Concentrations of intensitypolymers ratio and of pyrene the first were band 0.25 at mg/mL 373 nm and to the 0.6 thirdμM, respectively. band at 384 I nm.1/I3 was defined as the fluorescence intensityNext, interaction ratio of the of first dextran band at derivatives 373 nm to the with third lipid band membraneat 384 nm. was investigated. Fluorescence dye,Next, pyranine interaction-loaded of egg dextran yolk derivativesphosphatidylcholine with lipid (EYPC) membrane liposomes was investigated. were incubated Fluorescence with CHex dye,- pyranine-loadedDex atNext, varying interaction pH egg and yolk ofrelease dextran phosphatidylcholine of pyranine derivatives was with monitored. (EYPC) lipid liposomesmembrane Figure S10 werewas shows investigated. incubated time courses with Fluorescence of pyranine CHex-Dex dye, pyranine-loaded egg yolk phosphatidylcholine (EYPC) liposomes were incubated with CHex- at varyingrelease from pH andliposomes release after of pyranineaddition of was CHex monitored.-Dex. At alkali Figure or S10 neutral shows pH, time addition courses of CHex of pyranine-Dex Dex at varying pH and release of pyranine was monitored. Figure S10 shows time courses of pyranine releasehardly from affected liposomes the leakage after additionof pyranine of CHex-Dex.from liposomes, At alkali whereas or neutral remarkable pH, addition content release of CHex-Dex was release from liposomes after addition of CHex-Dex. At alkali or neutral pH, addition of CHex-Dex hardlyobserved affected within the a leakagefew minutes of pyranine at low pH. from Figure liposomes, 5 represents whereas pH-dependence remarkable of content pyranine release release was hardly affected the leakage of pyranine from liposomes, whereas remarkable content release was observedafter 30 withinmin-incubation. a few minutes All CHex at low-Dex pH. induced Figure content5 represents release pH-dependenceresponding to pH of decrease pyranine and release pH observed within a few minutes at low pH. Figure 5 represents pH-dependence of pyranine release afterregion 30 min-incubation. where content release All CHex-Dex occurred induced was quite content narrow release and totally responding corresponded to pH decrease to pH region and pH afterwhere 30 CHexmin-incubation.-Dex formed All CHex hydrophobic-Dex induced domains content (Figure release 4). responding These results to pH indicate decrease thatand pH the regionregion where where content content release release occurred occurred was wa quites quite narrow narrow and and totally totally corresponded corresponded to pH to region pH region where CHex-Dexdestabilization formed of hydrophobicliposomal membrane domains by (Figure CHex-4Dex). These with resultshydrophobic indicate domains that the induced destabilization content whererelease CHex from - liposomes.Dex formed Th us, hydrophobic pH-responsive domains dextran (Figure derivatives 4). These which results could indicate destabilize that lipid the of liposomal membrane by CHex-Dex with hydrophobic domains induced content release from destabilizationmembrane effectively of liposomal were successfully membrane developed.by CHex-Dex with hydrophobic domains induced content liposomes.release from Thus, liposomes. pH-responsive Thus, pH dextran-responsive derivatives dextran derivatives which could which destabilize could destabilize lipid membrane lipid effectivelymembrane were effectively successfully were developed.successfully developed.

Figure 5. pH-Dependence of pyranine release from liposomes after addition of various dextran

derivatives at various pH and 37 °C . Content release after 30 min-incubation is shown. Lipid Figure 5. pH-Dependence of pyranine release from liposomes after addition of various dextran Figureconcentration 5. pH-Dependence was 2.0 × 10−5 of M. pyranine The ratio releaseby weight from of lipid liposomes: polymer after is 9: addition1. of various dextran derivatives at various pH and 37 °C . Content release after 30 min-incubation is shown. Lipid derivatives at various pH and 37 ◦C. Content release after 30 min-incubation is shown. Lipid concentration was 2.0 × 10−5 M. The ratio by weight of lipid: polymer is 9:1. concentration was 2.0 × 10−5 M. The ratio by weight of lipid: polymer is 9:1.

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2.2. Preparation of Dextran Derivative-Modified Liposomes So far, basic characteristics of dextran derivatives were evaluated using dextran derivatives without C10 groups. Next, anchor-introduced polymers were used for surface modification of liposomes. Dextran derivative-modified liposomes were prepared by lipid-thin film hydration methods as previously reported [15]. Mixed thin film of EYPC and dextran derivatives having anchor moieties was dispersed in PBS containing OVA as a model antigenic protein. Then, resultant lipid/polymer suspension was passed through polycarbonate membranes with pore size of 100 nm and unloaded OVA was removed by repeated ultracentrifugation. Table4 shows the size and zeta potential of resultant liposomes. All liposomes exhibited a size of about 100 nm, which corresponds to pore size of polycarbonate membrane for extrusion. Dextran derivative-modified liposomes possessed more negative zeta potentials than that of unmodified liposomes. This might reflect the modification of dextran derivatives having carboxyl groups on liposomal surface.

Table 4. Characteristics of liposomes.

Liposome Size (nm) Zeta Potential (mV) Unmodified 135 ± 3 −10 ± 5 CHex28-Dex-C10 118 ± 4 −36 ± 0.2 CHex42-Dex-C10 120 ± 7 −31 ± 1 CHex53-Dex-C10 112 ± 1 −27 ± 1 CHex72-Dex-C10 109 ± 1 −37 ± 1 MGlu67-Dex-C10 104 ± 1 −33 ± 1

Next, pH-responsive content release from dextran derivative-modified liposomes was investigated. Pyranine were encapsulated in liposomes instead of OVA and pH-responsive release behaviors were evaluated (Figure S11 and Figure6). In our previous literature, weight ratio of lipid: polymer was optimized as 7:3 for their pH-sensitivity. Actually, MGlu67-Dex-C10-modified liposomes with weight ratio of 7:3 exhibited pH-responsive release behavior at weakly acidic pH region (Figure6). However, we could not obtain stable liposomes modified with CHex-Dex-C10 at weight ratio of 7:3. After optimization of lipid: polymer ratio in stability of liposomes, encapsulation efficiency of contents and evaluation of pH-sensitivity (Figure S12), lipid: polymer ratio was set to 9:1 for preparation of CHex-Dex-C10-modified liposomes. Resultant liposomes induced content release within a few minutes at extremely weakly acidic pH (Figure S11), suggesting that CHex-Dex became hydrophobic on liposome surface after protonation of carboxyl groups and liposomal membrane was immediately destabilized. CHex-Dex-C10-modified liposomes showed content release at higher pH region than MGlu-Dex-C10 modified liposomes. In addition, CHex-Dex-C10 having high amount of CHex groups tends to induce content release at high pH region (Figure6). These results might reflect the difference of pKa and hydrophobicity of CHex-Dex having various amounts of CHex groups and MGlu-Dex (Table3 and Figure4). Membranes 2017, 7, 41 7 of 15 Membranes 2017, 7, 41 7 of 14

Figure 6. pH-Dependence of pyranine release from EYPC liposomes modified with or without 10 Figure 6. pH-Dependence of pyranine release from EYPC liposomes modified with or without 10 wt % wt % CHex-Dex-C10 or 30 wt % MGlu-Dex-C10 ◦at 37 °C after 30 min-incubation. Lipid concentrations CHex-Dex-C10 or 30 wt % MGlu-Dex-C10 at 37 C after 30 min-incubation. Lipid concentrations were were 2.0 × 10−5 M. 2.0 × 10−5 M.

2.3. Interaction of Liposomes with Dendritic Cells 2.3. Interaction of Liposomes with Dendritic Cells Next, cellular association of liposomes with dendritic cells was examined to evaluate their Next, cellular association of liposomes with dendritic cells was examined to evaluate their antigen antigen delivery performance. OVA-loaded liposomes were fluorescently labeled with fluorescence delivery performance. OVA-loaded liposomes were fluorescently labeled with fluorescence dye dye with hydrophobic moieties, DiI. These liposomes were applied to DC2.4 cells, a murine dendritic with hydrophobic moieties, DiI. These liposomes were applied to DC2.4 cells, a murine line, and fluorescence intensity of cells were measured using a flow cytometer (Figure 7). cell line, and fluorescence intensity of cells were measured using a flow cytometer (Figure7). MGlu67-Dex-C10-modified liposomes showed five times higher cellular association than that of MGlu67-Dex-C -modified liposomes showed five times higher cellular association than that of unmodified liposomes.10 Dendritic cells have scavenger receptors that recognize anionic molecules on unmodified liposomes. Dendritic cells have scavenger receptors that recognize anionic molecules aged cells or apoptotic cells [24–27]. Modification of liposomes with poly(carboxylic acid)s is known on aged cells or apoptotic cells [24–27]. Modification of liposomes with poly(carboxylic acid)s to increase cellular association of liposomes by APCs [28]. Our previous literatures also revealed that is known to increase cellular association of liposomes by APCs [28]. Our previous literatures carboxylated poly(glycidol)- or carboxylated polysaccharide-modified liposomes were recognized by also revealed that carboxylated poly(glycidol)- or carboxylated polysaccharide-modified liposomes scavenger receptors and cellular association of liposomes increased [16,29]. Therefore, MGlu67-Dex- were recognized by scavenger receptors and cellular association of liposomes increased [16,29]. C10-modified liposomes might be also recognized by scavenger receptors on DC2.4 cells. Compared Therefore, MGlu67-Dex-C10-modified liposomes might be also recognized by scavenger receptors on with MGlu67-Dex-C10, modification of CHex-Dex-C10 strongly increased cellular association of DC2.4 cells. Compared with MGlu67-Dex-C10, modification of CHex-Dex-C10 strongly increased liposomes (Figure 7). Cellular association of CHex-Dex-C10-modified liposomes was 40–70 times cellular association of liposomes (Figure7). Cellular association of CHex-Dex-C 10-modified higher than unmodified liposomes and over 10 times higher than MGlu67-Dex-C10-modified liposomes was 40–70 times higher than unmodified liposomes and over 10 times higher than liposomes. As shown in Table 3, protonation state of MGlu-Dex at physiological pH is around 7%– MGlu67-Dex-C -modified liposomes. As shown in Table3, protonation state of MGlu-Dex at 22%, whereas those10 of CHex-Dex are 27%–49%. This might suggest that CHex-Dex on liposome physiological pH is around 7%–22%, whereas those of CHex-Dex are 27%–49%. This might suggest surface possess more hydrophobic nature than MGlu-Dex, which increased the interactions with that CHex-Dex on liposome surface possess more hydrophobic nature than MGlu-Dex, which cells. Among CHex-Dex-C10 having various amounts of CHex groups, CHex42-Dex-C10 showed the increased the interactions with cells. Among CHex-Dex-C10 having various amounts of CHex groups, highest cellular association (Figure 7). Compared with CHex42-Dex-C10, CHex28-Dex-C10 might have CHex42-Dex-C10 showed the highest cellular association (Figure7). Compared with CHex42-Dex-C 10, low protonation state, which might decrease the hydrophobic interaction with cells. CHex72-Dex-C10 CHex28-Dex-C might have low protonation state, which might decrease the hydrophobic interaction has almost twice10 carboxylates, which might cause electrostatic repulsion with cellular surface and with cells. CHex72-Dex-C has almost twice carboxylates, which might cause electrostatic repulsion decrease cellular associatio10n of liposomes. Therefore, medium amounts of CHex group-introduced with cellular surface and decrease cellular association of liposomes. Therefore, medium amounts dextran derivatives exhibited the highest interaction with cells, which is consistent with our previous of CHex group-introduced dextran derivatives exhibited the highest interaction with cells, which is results in MGlu group-introduced polysaccharide derivatives [15,16]. consistent with our previous results in MGlu group-introduced polysaccharide derivatives [15,16].

Membranes 2017, 7, 41 8 of 15 Membranes 2017, 7, 41 8 of 14

Figure 7. Relative fluorescence intensity for DC2.4 cells treated with DiI-labeled liposomes modified Figure 7. Relative fluorescence intensity for DC2.4 cells treated with DiI-labeled liposomes modified with or without 30 wt % MGlu67-Dex-C10 or 10 wt % CHex-Dex-C10 having various amounts of CHex with or without 30 wt % MGlu67-Dex-C10 or 10 wt % CHex-Dex-C10 having various amounts of CHex groups. DC2.4 cells were incubated with liposomes for 4 h at 37 °C in serum-free medium. * p < 0.01. groups. DC2.4 cells were incubated with liposomes for 4 h at 37 ◦C in serum-free medium. * p < 0.01.

Intracellular delivery performance of antigenic proteins was further investigated using DiI- labeledIntracellular and FITC delivery-OVA-loaded performance liposomes of antigenic (Figure proteins8). In the was case further of cells investigated treated with using unmodified DiI-labeled andliposomes, FITC-OVA-loaded red and green liposomes fluorescence (Figure was8). observed In the case as dots of cells within treated cells. with Considering unmodified liposomes liposomes, are redmainly and internalized green fluorescence to cells via was endocytosis, observed bothas dots liposomes within and cells. FITC Considering-OVA molecules liposomes might are be mainly internalized to cells via endocytosis, both liposomes and FITC-OVA molecules might be trapped in endosome/lysosome. For cells treated with MGlu67-Dex-C10-modified liposomes, DiI trappedfluoresce innce endosome/lysosome. was observed as dots For but cells increased, treated with reflecting MGlu67-Dex-C increase10 of-modified cellular liposomes, association DiI of fluorescenceliposomes (Figure was observed 7). According as dots but to the increased, result reflectingof co-staining increase using of cellular LysoTracker, association DiI-fluorescence of liposomes (Figurederived7 ). from According liposomes to the were result totally of co-staining co-localized using with LysoTracker, LytoTracker DiI-fluorescence fluorescence, indicating derived from that liposomes were exist totallyin endosome/lysosomes co-localized with LytoTracker (Figures S1 fluorescence,3 and S14). indicatingIn Figures that 8 and liposomes S13, g existreen influorescence endosome/lysosomes dots were overlapped (Figures with S13 andred and/or S14). Inblue Figure fluorescence8 and Figure but green S13, fluorescence green fluorescence was also dots were overlapped with red and/or blue fluorescence but green fluorescence was also observed observed from different site from red and/or blue fluorescence. This indicates that MGlu67-Dex-C10- frommodified different liposomes site from induced red and/or destabilization blue fluorescence. of endosomal This indicates membrane that responding MGlu67-Dex-C to acidic10-modified pH in liposomes induced destabilization of endosomal membrane responding to acidic pH in endosomes and endosomes and released FITC-OVA molecules from endosomes. CHex42-Dex-C10-modified releasedliposomes FITC-OVA exhibited further molecules strong from intracellular endosomes. delivery CHex42-Dex-C performance10-modified and strong liposomes green fluorescence exhibited furtherwas observed strong from intracellular whole cells delivery reflecting performance their hydrophobic and strong nature green and strong fluorescence interaction was with observed lipid frommembranes whole (Fig cellsures reflecting 4 and their5). Because hydrophobic OVA contents nature andper strongliposomes interaction were almost with lipididentical membranes in each (Figuresliposome,4 andthe 5difference). Because of OVAFITC contentsfluorescence per liposomesdirectly reflect were the almost difference identical of FITC in each-OVA liposome, amounts theinside difference of cells of(Fig FITCure S1 fluorescence5). To investigate directly the reflect effect the of difference CHex group of FITC-OVA contents on amounts cytoplasmic inside delivery of cells (Figure S15). To investigate the effect of CHex group contents on cytoplasmic delivery performance, performance, CHex28-Dex-C10-, CHex42-Dex-C10- and CHex72-Dex-C10-modified liposomes were CHex28-Dex-Capplied to DC2.410-, cells CHex42-Dex-C and observed10- and by CLSM CHex72-Dex-C on same10 microscopy-modified liposomes setting (Fig wereure applied S16). Reflecting to DC2.4 cells and observed by CLSM on same microscopy setting (Figure S16). Reflecting the results of cellular the results of cellular association, CHex42-Dex-C10-modified liposomes delivered FITC-OVA association, CHex42-Dex-C -modified liposomes delivered FITC-OVA molecules into cytosol more molecules into cytosol more10 efficiently than other CHex-Dex-C10-modified liposomes. This was also efficientlyconfirmed thanfrom other the decrease CHex-Dex-C of colocalization10-modified liposomes.of FITC fluorescence This was alsowith confirmed DiI fluorescence from the (Fig decreaseure S17 of). colocalizationThese results indicate of FITC fluorescencethat both carboxylated with DiI fluorescence unit contents (Figure and their S17). spacer These resultsstructures indicate are important that both carboxylatedto obtain liposomes unit contents with high and their intracellular spacer structures delivery performance are important to to dendritic obtain liposomes cells. Cytoplasmic with high intracellulardelivery of antigenic delivery performanceproteins induces to dendritic MHC class cells. I Cytoplasmic-restricted antigen delivery presentation. of antigenic Actually, proteins induces MGlu- MHC class I-restricted antigen presentation. Actually, MGlu-Dex-C -modified liposomes induced Dex-C10-modified liposomes induced MHC class I-restricted antigen10 presentation and antigen- MHC class I-restricted antigen presentation and antigen-specific cellular immune responses [15]. specific cellular immune responses [15]. Therefore, CHex-Dex-C10-modified liposomes are expected to induce MHC class I-restricted antigen presentation and stronger cellular immunity than MGlu- Dex-C10-modified liposomes.

Membranes 2017, 7, 41 9 of 15

Therefore, CHex-Dex-C10-modified liposomes are expected to induce MHC class I-restricted antigen presentation and stronger cellular immunity than MGlu-Dex-C -modified liposomes. Membranes 2017, 7, 41 10 9 of 14

Figure 8. Confocal laser scanning microscopic (CLSM) images of DC2.4 cells treated with DiI DiI-labeled-labeled and FITC-OVA-loaded EYPC liposomes modified with or without 30 wt % MGlu67-Dex-C10 or 10 and FITC-OVA-loaded EYPC liposomes modified with or without 30 wt % MGlu67-Dex-C10 or 10 wt % wt % CHex42-Dex-C10 for 4 ◦ h at 37 °C in serum-free medium. Scale bar represents 10 μm. DiI- CHex42-Dex-C10 for 4 h at 37 C in serum-free medium. Scale bar represents 10 µm. DiI-fluorescence fluorescence intensity for liposome modified with CHex42-Dex-C10 was decreased to distinguish the intensity for liposome modified with CHex42-Dex-C10 was decreased to distinguish the distribution of −4 distributionliposomes. Lipid of liposomes. concentration Lipid wasconcentration 5.0 × 10−4 wasM. 5.0 × 10 M.

22.4..4. Activation of Dendritic Cells by Dextran Derivatives For induction of cellular immunity, not only cytoplasmic delivery of antigen but also activation of dendritic cells are necessary. According to our previous results, the introduction of MGlu groups to dextran, curdlan or mannanmannan promotedpromoted theirtheir cytokinecytokine productionproduction [[1616].]. Therefore, the activation properties of dendritic cells by CHex-DexCHex-Dex were evaluated.evaluated. Here, CHex-DexCHex-Dex without anchor groups was used because anchor groups areare buried in thethe lipidlipid membranemembrane ofof polymer-modifiedpolymer-modified liposomes and CHex-DexCHex-Dex on the liposome surface might be be recognizedrecognized by by dendritic dendritic cells. cells. DC2.4 cells were cultured in the presence of dextran derivatives for 24 h and the production of Th1 cytokines (TNF (TNF--α and IL IL-12)-12) was measured by ELISA (Figure(Figure9 9).). AsAs presentedpresented inin FigureFigure9 9,, TNF- TNF-α and IL IL-12-12 production from the the DC2.4 DC2.4 cells cells was was observed observed by treatment by treatment of MGlu65-Dex, of MGlu65 which-Dex, whichis consistent is consistent with our with previous our previousresults [16 results]. Surprisingly, [16]. Surprisingly, CHex40-Dex CHex40 induced-Dex eighteeninduced timeseighteen higher times TNF- higherα production TNF-α production and over and600 timesover 600 higher times IL-12 higher production IL-12 production from dendritic from dendritic cells than cells those than of those MGlu65-Dex. of MGlu65 These-Dex. resultsThese resultsindicate indicate that the that introduction the introduction of hydrophobic of hydrophobic CHex groups CHex is groups quite effective is quite foreffective activation for activation of dendritic of dendriticcells. Although cells. Although further introduction further introduction of CHex groups of CHex to dextran groups decreased to dextran the decreased cytokine the production, cytokine whichproduction, is the oppositewhich is resultthe opposite for the caseresult of for MGlu-Dex the case [of16 ],MGlu CHex-Dex-Dex [16 with], CHex high- CHexDex with group high contents CHex groupstill showed contents over still 100 showed times over higher 100 IL-12times higher production IL-12 thanproduction MGlu65-Dex. than MGlu65 Activation-Dex. Activation of APCs byof APCs by polysaccharide derivatives having carboxyl groups or sulfate groups has been reported [30,31]. But these activation properties are 2–3 times higher than parent . According to our previous report, MGlu65-Dex induced 2 times higher IL-12 production than parent dextran probably because of high density of carboxyl groups on polysaccharide chain [16]. In this study, CHex40-Dex induced over 600 times higher IL-12 production than MGlu65-Dex (Figure 9B). These

Membranes 2017, 7, 41 10 of 15 polysaccharide derivatives having carboxyl groups or sulfate groups has been reported [30,31]. But these activation properties are 2–3 times higher than parent polysaccharides. According to our previous report, MGlu65-Dex induced 2 times higher IL-12 production than parent dextran probably because of high density of carboxyl groups on polysaccharide chain [16]. In this study, Membranes 2017, 7, 41 10 of 14 CHex40-Dex induced over 600 times higher IL-12 production than MGlu65-Dex (Figure9B). These resultsresults suggestsuggest thatthat hydrophobicityhydrophobicity of spacerspacer groupsgroups areare quitequite importantimportant forfor activationactivation ofof dendriticdendritic cellscells ratherrather thanthan thethe densitydensity of carboxyl groups. To thethe bestbest ofof ourour knowledge,knowledge, this is the firstfirst report toto revealreveal thethe effecteffect ofof spacerspacer groupsgroups ofof carboxylatedcarboxylated polysaccharidepolysaccharide derivativesderivatives onon theirtheir adjuvantadjuvant function.function. Moreover, IL-12IL-12 production from dendriticdendritic cells strongly activates cellular immunity [3 [322]].. Therefore,Therefore, CHex-Dex-modifiedCHex-Dex-modified liposomes are expected to not onlyonly deliverdeliver antigenantigen intointo cytosolcytosol butbut alsoalso activate antigen antigen-specific-specific cellular cellular immunity. immunity. We We are are currently currently attempting attempting the theevaluation evaluation of the of theimmuni immunity-inducingty-inducing properties properties in in vivo vivo and and antitumor antitumor effects effects of of CHex CHex-Dex-modified-Dex-modified liposomes to to confirmconfirm theirtheir potencypotency forfor cancercancer immunotherapy.immunotherapy.

Figure 9. TNF-α (A) and IL-12 (B) production from DC2.4 cells treated with liposomes modified with Figure 9. TNF-α (A) and IL-12 (B) production from DC2.4 cells treated with liposomes modified with dextran derivatives (1 mg/mL) for 24 h. ** p < 0.05. * p < 0.01. dextran derivatives (1 mg/mL) for 24 h. ** p < 0.05. * p < 0.01.

3. Materials and Methods 3. Materials and Methods

3.1.3.1. Materials EYPCEYPC was kindly donated by NOF Co. (Tokyo, Japan). 1,2 1,2-Cyclohexanedicarboxylic-Cyclohexanedicarboxylic anhydride, OVA,OVA, fluorescein fluorescein isothiocyanate isothiocyanate (FITC), (FITC), pp-xylene-bis-pyridinium-xylene-bis-pyridinium bromide (DPX) (DPX) were were purchased purchased fromfrom Sigma Sigma (St. (St. Louis, Louis, MO MO,, USAUSA).). Dextran having molecular weight of 70,000, 70,000, 1-aminodecane, 1-aminodecane, pyraninepyranine and Triton X X-100-100 were were obtained obtained from from Tokyo Tokyo Chemical Chemical Industries Industries Ltd. Ltd. (Tokyo, (Tokyo, Japan). Japan). 4- 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium morpholinium chloride chloride (DMT (DMT-MM)-MM) was was from from Wako PurePure ChemicalChemical IndustriesIndustries Ltd.Ltd. (Osaka, Japan). 1,10′--Dioctadecyl-3,3,3Dioctadecyl-3,3,30′,3′0--tetramethylindocarbocyaninetetramethylindocarbocyanine perchlorateperchlorate (DiI) was was from from Life Life Technologies. Technologies. Dialysis Dialysis tube tube (molecular (molecular weight weight cutoff: cutoff: 10 kDa 10) kDa) was waspurchased purchased from from Sekisui Sekisui Medical Medical Co., Ltd. Co., (Tokyo, Ltd. (Tokyo, Japan). Japan). FITC- FITC-OVAOVA was prepared was prepared by reacting by reacting OVA (10 mg) with FITC (11.8 mg) in 0.5 M NaHCO3 (4 mL, pH 9.0) at 4 °C◦ for three days and subsequent OVA (10 mg) with FITC (11.8 mg) in 0.5 M NaHCO3 (4 mL, pH 9.0) at 4 C for three days and subsequent dialysis [13]. 3-Methyl glutarylated (MGlu65-Dex and MGlu67-Dex-C10) were prepared as dialysis [13]. 3-Methyl glutarylated dextrans (MGlu65-Dex and MGlu67-Dex-C10) were prepared as previouslypreviously reportedreported [[1515].].

3.2.3.2. Synthesis of DextranDextran Derivatives 2-Carboxycyclohexane-1-carboxylated2-Carboxycyclohexane-1-carboxylated dextrandextran (CHex-Dex)(CHex-Dex) waswas preparedprepared byby reactionreaction ofof dextran withwith 1,2-cyclohexanedicarboxylic1,2-cyclohexanedicarboxylic anhydride anhydride.. Experimental Experimental conditions conditions were were shown shown in in Table Table 1. 1 A. given amount of dextran and LiCl were dissolved in N,N-dimethylformamide (DMF) and 1,2- cyclohexanedicarboxylic anhydride (CHex anhydride) was added to the solution. The mixed solution was kept at various temperatures for 24 h with stirring under argon atmosphere. Then, the reaction mixture was evaporated and saturated sodium hydrogen carbonate aqueous solution was added to the reaction mixture for neutralization and dialyzed against water for 5 days. The product was recovered by freeze-drying. 1H NMR for CHex-Dex (400 MHz, D2O + NaOD, Figures S1–S5): δ 1.3– 2.0 (m, −cyclo−CH2), 2.6 (m, cyclo−CH), 3.4–4.1 (br, glucose 2H, 3H, 4H, 5H, 6H), 5.0 (br, glucose 1H).

Membranes 2017, 7, 41 11 of 15

A given amount of dextran and LiCl were dissolved in N,N-dimethylformamide (DMF) and 1,2-cyclohexanedicarboxylic anhydride (CHex anhydride) was added to the solution. The mixed solution was kept at various temperatures for 24 h with stirring under argon atmosphere. Then, the reaction mixture was evaporated and saturated sodium hydrogen carbonate aqueous solution was added to the reaction mixture for neutralization and dialyzed against water for 5 days. The product 1 was recovered by freeze-drying. H NMR for CHex-Dex (400 MHz, D2O + NaOD, Figures S1–S5): δ 1.3–2.0 (m, −cyclo−CH2), 2.6 (m, cyclo−CH), 3.4–4.1 (br, glucose 2H, 3H, 4H, 5H, 6H), 5.0 (br, glucose 1H). As anchor moieties for fixation of CHex-Dex onto liposome membranes, 1-aminodecane was combined with carboxyl groups of CHex-Dex. Experimental conditions were shown in Table2. Each polymer was dissolved in water around pH 7.4, and 1-aminodecane (0.1 equiv. to hydroxyl group of polymer) was reacted to carboxyl groups of the polymer using DMT-MM (0.1 equiv. to hydroxyl group of polymer) at room temperature for 5–36 h with stirring. The obtained polymers were purified by dialysis in water. The compositions for polymers were estimated 1 1 using H NMR. H NMR for CHex-Dex-C10 (400 MHz, D2O + NaOD, Figures S6–S9):δ 0.9 (br, −CO−NH−CH2−(CH2)8−CH3), 1.2–2.0 (m, −cyclo−CH2, −CO−NH−CH2−(CH2)8−CH3), 2.6 (m, cyclo−CH), 3.2 (br, −CO−NH−CH2−(CH2)8−CH3), 3.4–4.1 (br, glucose 2H, 3H, 4H, 5H, 6H), 5.0 (br, glucose 1H).

3.3. Titration To 50 mL of an aqueous solution of each polymer (carboxylate concentration: 1.2 × 10−4 M) was added an appropriate amount of 0.1 M NaOH solution to make pH 11.3. The titration was carried out by the stepwise addition of 0.01 M HCl at 4 ◦C and conductivity and pH of the solution were monitored.

3.4. Pyrene Fluorescence A given amount of pyrene in solution was added to an empty flask, and acetone was removed under vacuum. Polymer (0.25 mg/mL) dissolving in 30 mM sodium acetate and 120 mM NaCl solution of a given pH was added to the flask, yielding 0.6 µM concentration of pyrene. The sample solution was stirred overnight at room temperature, and emission spectra with excitation at 337 nm were recorded. The fluorescence intensity ratio of the first band at 373 nm to the third band at 384 nm (I1/I3) was analyzed as a function of solution pH.

3.5. Preparation of Liposomes To a dry, thin membrane of EYPC (10 mg) was added 1.0 mL of OVA/PBS solution (pH 8.2, 4 mg/mL), and the mixture was vortexed at 4 ◦C. The liposome suspension was further hydrated by freezing and thawing, and was extruded through a polycarbonate membrane with a pore size of 100 nm. The liposome suspension was centrifuged with the speed of 55,000 rpm for 2 h at 4 ◦C twice to remove free OVA from the OVA-loaded liposomes. Polymer-modified liposomes were also prepared according to the above procedure using dry membrane of a lipid mixture with polymers (lipids/polymer = 9/1, w/w for CHex-Dex-C10 and 7/3, w/w for MGlu-Dex-C10).

3.6. Dynamic Light Scattering and Zeta Potential Diameters and zeta potentials of the liposomes (0.1 mM of lipid concentration, pH 7.4) were measured using a Zetasizer Nano ZS ZEN3600 (Malvern Instruments Ltd., Worcestershire, UK). Data was obtained as an average of more than three measurements on different samples. Membranes 2017, 7, 41 12 of 15

3.7. Release of Pyranine from Liposome Pyranine-loaded liposomes were prepared as described above except that mixtures of polymers and EYPC were dispersed in aqueous 35 mM pyranine, 50 mM DPX, and 25 mM phosphate solution (pH 8.2). For the study of the interaction of polymers with lipid membranes, a given amount of the polymer dissolved in the same buffer (final concentration: 1.78 µg/mL) at 25 ◦C was added to a suspension of pyranine-loaded liposomes (lipid concentration: 2.0 × 10−5 M) in PBS of varying pH, and fluorescence intensity (512 nm) of the mixed suspension was followed with excitation at 416 nm using a spectrofluorometer (Jasco FP-6500, Tokyo, Japan). For the study of the release behavior of polymer-modified liposomes, polymer-modified liposomes encapsulating pyranine were added to PBS of varying pHs at 37 ◦C and fluorescence intensity of the suspension was monitored (lipid concentration: 2.0 × 10−5 M). The percent release of pyranine from liposomes was defined as:

Release (%) = (Ft − Fi)/(Ff − Fi) × 100 where Fi and Ft mean the initial and intermediary fluorescence intensities of the liposome suspension, respectively. Ff is the fluorescent intensity of the liposome suspension after the addition of TritonX-100 (final concentration: 0.1%).

3.8. Cell Culture DC2.4 cell, which is an immature murine DC line, was provided from Kenneth L. Rock (University of Massachusetts Medical School, Worcester, MA, USA) and were grown in RPMI-1640 supplemented with 10% FBS (MP Biomedical, Inc., Santa Ana, CA, USA), 2 mM L-glutamine, 100 mM nonessential amino acid, 50 µM 2-mercaptoethanol (2-ME, Gibco) and antibiotics at 37 ◦C[33].

3.9. Cellular Association of Liposomes Liposomes containing DiI were prepared as described above except that a mixture of polymer and lipid containing DiI (0.1 mol%) was dispersed in PBS. DC2.4 cells (1.5 × 105 cells) cultured for 2 days in 12-well plates were washed with Hank’s balanced salt solution (HBSS), and then incubated in serum-free RPMI medium (0.5 mL). The DiI-labeled liposomes (1 mM lipid concentration, 0.5 mL) were added gently to the cells and incubated for 4 h at 37 ◦C. After the incubation, the cells were washed with HBSS three times. Fluorescence intensity of these cells was determined by a flow cytometric analysis (Cyto FLEX, Beckman Coulter, Inc., Brea, CA, USA). DiI fluorescence of each liposome was measured and the cellular fluorescence shown in Figure7 was corrected using liposomal fluorescence intensity.

3.10. Intracellular Behavior of Liposomes The FITC-OVA-loaded liposomes containing DiI were prepared as described above except that a mixture of polymer and lipid containing DiI (0.1 mol %) was dispersed in PBS containing FITC-OVA (4 mg/mL). DC2.4 cells (1.5 × 105 cells) cultured for 2 days in 35-mm glass-bottom dishes were washed with HBSS, and then incubated in serum-free RPMI medium (1 mL). The FITC-OVA-loaded liposomes (1 mM lipid concentration, 1 mL) were added gently to the cells and incubated for 4 h at 37 ◦C. After the incubation, the cells were washed with HBSS three times. Confocal laser scanning microscopic (CLSM) analysis of these cells was performed using LSM 5 EXCITER (Carl Zeiss Co. Ltd., Oberkochen, Germany).

3.11. Cytokine Production from Cells Treated with Dextran Derivatives The DC2.4 cells (5 × 104 cells) cultured for 2 days in 12-well plates were washed with HBSS, and then incubated in serum-free RPMI medium (0.5 mL). Dextran derivatives (2 mg/mL, 0.5 mL) were added gently to the cells and incubated for 24 h at 37 ◦C. After the incubation, supernatants Membranes 2017, 7, 41 13 of 15 of cultured cells were collected for measurements of TNF-α and IL-12 using an enzyme-linked immunosorbent assay kit (ELISA Development Kit, PeproTech EC Ltd., London, UK) according to the manufacture’s instruction.

3.12. Statistical Analysis Tukey-Kramer method was employed in the statistical evaluation of the results in Figures7 and9.

4. Conclusions In this study, carboxylated dextran derivatives having hydrophobic spacer groups were developed. CHex-Dex formed strong hydrophobic domains with pH decreasing and destabilized liposomal membrane immediately. These polymer-modified liposomes also showed contents release at low pH and efficient intracellular delivery performance compared with conventional dextran derivatives (MGlu-Dex). CHex-Dex also exhibited several hundred times higher IL-12 production from dendritic cells than MGlu-Dex. Therefore, CHex-Dex-modified liposomes are promising as antigen carriers with both intracellular delivery function and strong adjuvant functions to activate cellular immunity.

Supplementary Materials: The following are available online at www.mdpi.com/2077-0375/7/3/41/s1, Figures S1–S9: NMR spectra for dextran derivatives, Figure S10: Time course of pyranine release from liposomes after addition of CHex-Dex, Figure S11: Time course of pyranine release from liposomes modified with dextran derivatives, Figure S12: Effect of polymer/lipid ratio on pH-sensitivity of CHex-Dex-C10-modified liposomes, Figures S13 and S14: Co-staining of end/lysosomes for liposome-treated DC2.4 cells, Figure S15: OVA amounts in liposomes, Figure S16: CLSM images of DC2.4 cells treated with CHex-Dex-C10-modified liposomes, Figure S17: Analysis of colocalization for FITC and DiI. Acknowledgments: This work was supported by Grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan (15H03024). Author Contributions: Eiji Yuba and Kenji Kono conceived and designed the experiments; Shinya Uesugi performed most of experiments; Maiko Miyazaki and Yuna Kado performed CLSM analysis (Figure S16) and ELISA (Figure9), respectively; Atsushi Harada analyzed the data; Eiji Yuba wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

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