Synthesis and Immunestimulating Activity of Lactobacilli-Originated

Article pubs.acs.org/Langmuir

Synthesis and Immunestimulating Activity of Lactobacilli-Originated Polysaccharide−Polymeric Microparticle Conjugates Koji Nagahama,*,† Takayuki Kumano,† Tsubasa Nakata,† Hirokazu Tsuji,‡ Kaoru Moriyama,‡ Kan Shida,‡ Koji Nomoto,‡ Katsuyoshi Chiba,‡ Kazuya Koumoto,*,† and Jun Matsui*,† †

Department of Nanobiochemistry, Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-Minamimachi, Kobe 650-0047, Japan ‡ Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan S Supporting Information *

ABSTRACT: The design and synthesis of biomaterials capable of activating the immune system are of interest in immunology-related fields because of their ability to tune up the immune defenses of the host. Lactobacilli are a major constituent of normal human indigenous flora, and some specific strains are known to activate the immune system of the host as probiotics. In this study, we first fabricated novel biohybrid materials in which lactobacilli (L. casei strain Shirota, LcS)-originated polysaccharide−peptidoglycan complexes (PSPGs) are conjugated with polymeric microparticles (MPs). PSPGs conjugated onto polymeric MPs surfaces bound its specific antibody, suggesting that PS-PGs kept their original molecular recognition ability. The PS-PGs-based hybrid MPs with an appropriate density of conjugated PS-PGs effectively induced high levels of IL-12 production from macrophages without cytotoxicity. These results suggest that LcS-originated PS-PGs could be available bio-originated materials for developing novel biomaterials capable of activating the immune system in a safe manner. activate the immune system of the host as probiotics.16−18 In general, these strains stimulate macrophages to secrete interleukin-12 (IL-12), which plays a key role in activating the innate immunity of the host, such as by augmenting the functions of macrophages and NK cells.19,20 Recently, it was reported that these strains exhibit antitumor and anti-infectious activities without any side effects through the induction of a moderate number of proinflammatory cytokines including IL12 from macrophages.21−23 Among them, L. casei strain Shirota (LcS) effectively induces IL-12 secretion in a safe manner.24 Recently, polysaccharide−peptidoglycan complexes (PS-PGs), which are the main component of its cell wall, has been recognized as a source material to activate the immune system, although the accurate mechanism is not clear. For this reason, we hypothesized that PS-PGs might be a novel bio-originated material available for developing biohybrid materials capable of activating the immune system in a safe manner. However, there have been no reports to assess the potential of PS-PGs yet. In this study, we examined whether LcS-originated PS-PGs can be conjugated with synthetic materials and whether the resultant PS-PGs-based biohybrid materials stimulate macrophages to secrete IL-12 without cytotoxicity.

1. INTRODUCTION The design and synthesis of biomaterials capable of activating the immune system are of interest in immunology-related fields because of their ability to tune up the immune defenses of the host.1−4 A biohybrid approach in which bio-originated immunostimulatory agents are conjugated with synthetic materials has been applied for the development of biomaterials inducing immune activation. The emergence of nanobiotechnology has provided researchers with a robust platform for developing biohybrid materials. Several micro/nanoparticles (MPs/NPs) including liposomes, polymeric MPs/NPs, and gold NPs are promising synthetic platforms, and they have been utilized to display bio-originated immunostimulatory agents. However, one of the most utilized bio-originated immunostimulatory agents is cytosine-guanosine oligodeoxynucleotides (CpG ODNs), which are widely present in the genomic DNA of bacteria and viruses, and CpG ODNs is normally conjugated/encapsulated with/in various kinds of MPs/ NPs.5−12 Although CpG ODN effectively activates Th1-like innate and adaptive immune responses through the induction of proinflammatory cytokines from macrophages, their specific cytotoxicity to T-cells and neurons has been reported.13−15 Hence, the quest for novel bio-originated immunostimulatory agents capable of both conjugating with synthetic micro/ nanoplatforms and activating the immune system in a safe manner should be a significant research field. Lactobacilli are a major constituent of normal human indigenous flora, and some specific strains are known to © 2015 American Chemical Society

Received: October 22, 2014 Revised: December 9, 2014 Published: January 9, 2015 1489

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dispersed in 90 μL of 0.5% BSA containing EDTA/PBS (2 mM), and 9 μL of FITC-labeled antimouse IgM antibody solution was added and incubated on ice for 2 h. The reaction solution was centrifuged (3000 rpm, 10 min, 4 °C), and the supernatant was removed. The precipitate was washed three times with 100 μL of pure water. Finally, the precipitate was dispersed in 500 μL of 0.5% BSA containing EDTA/ PBS (2 mM), and flow cytometric analysis (BD FACSAria III) was performed. Moreover, fluorescence microscopic observation of the samples was performed. 2.6. Cell Culture. J774.1 mouse macrophage-like cells were grown in RPMI-1640 medium (Nissui Pharm) supplemented with 10% heatinactivated FBS, 0.15% NaHCO3, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.05 mM 2-mercaptoethanol at 37 °C in humidified air containing 5% CO2. 2.7. Cell Stimulation. J774.1 cells were seeded in a 96-well plate (1.0 × 105 cells/well) with 100 μL of culture medium and incubated for 2 h at 37 °C. Then, certain concentrations of MP-PSPG, LPS, or heat-killed LcS suspensions (100 μL) were added and incubated for 24 h at 37 °C. Supernatants were collected and filtered (pore size 450 nm). Filtrates were used for the determination of IL-12 concentrations. 2.8. Detection of IL-12 Concentrations. Concentrations of IL12 in culture supernatants were determined by sandwich ELISA. Rat antimouse IL-12 monoclonal antibody (clone C15.6) was used as the capture antibody. Biotinylated rat antimouse IL-12 (clone C17.8) monoclonal antibody was used as the detection antibody. These antibodies and standard recombinant mouse IL-12 were purchased from BD Pharmingen. The concentrations were calculated as the mean ± S.D. of three independent measurements. 2.9. Cell Viability Test. J774.1 cells were seeded in a 96-well plate (1.0 × 104 cells/well) with 100 μL of culture medium and incubated for 2 h at 37 °C. Then, certain concentrations of MP-PSPG, LPS, or heat-killed LcS suspensions (100 μL) were added and incubated for 24 h at 37 °C. The cell viability was measured with an LDH assay kit (Takara Bio) according to the manufacturer’s instructions. The viability was calculated as the mean ± S.D. of three independent measurements. Moreover, J774.1 cells treated with MP-PSPG and heat-killed LcS suspensions for 24 h were stained with Calcein-AM and propidium iodide (PI), and then the cells were observed by fluorescence microscope (Biorevo BZ-9000, Keyence).

2. EXPERIMENTAL SECTION 2.1. Materials. Acetic anhydride, sodium cyanoborohydride, dextran (Mw = 40 000), bovine serum albumin (BSA), and all organic solvents were purchased from Wako Pure Chemicals. Amino-modified polystyrene microparticles (MP, diameter ca. 2.5 μm) were purchased from Invitrogen. Lipopolysaccharides (LPS) from E. coli and trypsin were purchased from Sigma. DNase and RNase were purchased from Roche Diagnostics. The mutanolysin was from Streptomyces globisporus ATCC 21553 and was obtained from Sigma-Aldrich Co. Bacteria: Probiotic strain LcS was originally isolated at the Yakult Central Institute, based on acid and bile tolerance and survival in the gastrointestinal passage. The preparation of heat-killed LcS was carried out as previously described.25 In brief, LcS was cultured at 37 °C for 20 h in lactobacilli MRS broth (Difco), washed with sterilized pure water, held at 100 °C for 30 min, and then lyophilized. 2.2. Isolation of PS-PGs. The isolation of PS-PGs was conducted on the basis of a modified published method.25 In brief, heat-killed LcS (500 mg) was exhaustively digested with mutanolysin (9000 U) for 24 h at 37 °C. The digest was then centrifuged (10 000g, 45 min), and the supernatant was dialyzed against 10 mM phosphate buffer containing 0.25 M NaCl. The nondialyzable material was lyophilized and then dissolved in a small volume of an aqueous solution of pure water. The solution was then subjected to gel filtration on a column of Sephacryl S-200 HR (50 mm × 600 mm) with the same eluent. Fractions were collected and assayed for hexose. The hexose-containing fractions were used as PS-PGs (Mw = 30 000). 2.3. Synthesis of PS-PG-Conjugated Polymer MPs. Isolated PS-PGs (18.0 mg) were dissolved in 3.3 mL of phosphate buffer (50 mM, pH 8.5), and 420 μL of acetic anhydride was added and stirred at room temperature for 6 h. The reaction solution was dialyzed (MWCO 3500) against excess amounts of pure water for 2 days to remove unreacted acetic anhydride and then lyophilized to give acetylated PS-PGs as a white powder (11.6 mg). Then, PS-PGconjugated polystyrene MPs (MP-PSPG) were synthesized through a reductive amination reaction between surface amino groups of MPs and the reducing ends of acetylated PS-PGs. In brief, acetylated PSPGs (4.7 mg) were dissolved in 200 μL of phosphate buffer (50 mM, pH 8.5). Thirty microliters of an MP dispersion (8.4 × 1010 particles) and 90 μL of a sodium cyanoborohydride aqueous solution (100 mM) were added to the acetylated PS-PG solution and stirred at 45 °C for 72 h. The reaction solution was centrifuged (5000 rpm, 10 min, 4 °C), and the supernatant was removed. The precipitate was washed seven times each with 300 μL of pure water. Finally, the precipitate was lyophilized to give MP-PSPG. Dextran-conjugated polystyrene MPs (MP-Dex) were also synthesized through reductive amination reactions as controls (Figure S1, Supporting Information). 2.4. Characterization. FTIR spectrometry was performed using an FTIR-6300A (JASCO) spectrometer. All spectra were recorded in transmission mode with a wavelength range of 600−4000 cm−1. The amounts of PS-PGs conjugated to polystyrene MPs were estimated by gravimetric measurement using an electronic microbalance (AX26, Mettler Toledo). The quantity of PS-PGs conjugated to polystyrene MPs (μg) is equal to the weight of MPs-PSPG after lyophilization (μg) minus the weight of MPs used for the coupling reaction (μg). These values were utilized to estimate the density of PS-PGs on the surfaces of MPs (the number of PS-PG chains per surface area of MP). The size and zeta potential of MP-PSPG were determined on a Malvern Zetasizer Nano ZS at 25 °C. The lyophilized MP-PSPG was dispersed with pure water to a concentration of 0.1 wt % prior to measurement. The diameters and zeta potential of samples were calculated as the mean ± S.D. of three independent measurements. The surface morphology of MP-PSPG was observed by SEM (JEOL, JSM-7001FA) in the dry state. 2.5. Flow Cytometry Analysis. Nine microliters of an L8 monoclonal antibody solution (0.1 μg/mL) was added to the MPPSPG dispersion (5.0 × 106 particles) in PBS (90 μL) and incubated at 4 °C overnight. The reaction solution was centrifuged (5000 rpm, 10 min, 4 °C), and the supernatant was removed. The precipitate was washed two times with 100 μL of pure water. Then, the precipitate was

3. RESULTS AND DISCUSSION 3.1. Synthesis of MP-PSPG. In this study, we designed PSPGs-based biohybrid materials for the first time in which PSPGs were conjugated with the surface of polymeric MPs. We used polystyrene MPs (size ca. 2.5 μm) having amino and carboxyl groups on the surfaces as a microsized platform to conjugate PS-PGs chains. Cell wall components, including PSPGs, were extracted from heat-killed LcS by high-dose mutanolysin treatment based on a modified published method, as illustrated in Figure 1a.26 The PS-PGs were purified by gel filtration on a column of Sephacryl S-200 HR. We previously identified the structures of PS-PGs of LcS, including the amino acid sequences in the PG region, and the monosaccharide sequences and the reducing end monosaccharide residue in the PS region.27 Prior to the synthesis of MP-PSPG, free amino groups of lysine residues in the PG region were acetylated to avoid a coupling reaction between them and the reducing-end monosaccharide of the PS, N-acetylglucosamine. Then, acetylated PS-PGs were conjugated with the surfaces of polystyrene MPs via a reductive amination reaction to give MP-PSPG (Figure 1b). We synthesized MP-PSPG having different numbers of PS-PG chains conjugated by varying the feed molar ratios of PS-PGs to surface amino groups of MPs during the coupling reaction. Dextran-conjugated polystyrene MPs (MP-Dex) were also synthesized via similar methods as negative controls (Figure S1, Supporting Information). We use 1490

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MP-PSPG were carried out. Figure S2 (Supporting Information) shows FTIR spectra of MP, acetylated PS-PG, and MP8PSPG. The peaks at 3020−3140, 2850−2950, 1450−1510, and 660−780 cm−1 in −FTIR spectrum of MP can be assigned to the N−H stretching of surface-modified amino groups, the O−H stretching of surface-modified carboxyl groups, the alkane C−H stretching of polystyrene main chains, and the aromatic out-of-plane C−H bending of polystyrene side groups, respectively. The peaks located at 3100−3800, 2780−3040, 1590−1760, 1490−1580, and 950−1140 cm−1 in the FTIR spectrum of acetylated PS-PG can be assigned to the O−H stretching of polysaccharides, the carboxyl O−H stretching of peptidoglycans, the amide CO stretching of peptidoglycans, the amide NH bending of peptidoglycans, and the acetal of polysaccharides, respectively. After conjugation with PS-PGs (in the FTIR spectrum of MP-8PSPG), the peaks attributed to the O−H stretching of polysaccharides, the carboxyl O−H stretching of peptidoglycans, the amide CO stretching and NH bending of peptidoglycans, and the acetal groups of polysaccharides newly appeared, whereas the N−H stretching of surface-modified amino groups of MP was significantly weakened. These results indicate that MP was conjugated with PS-PG chains using surface amino groups, as expected. We investigated the numbers of PS-PG chains conjugated to polystyrene MPs. In this study, the number of PS-PG chains was estimated by gravimetric measurement because there is no characteristic photochemical property available for detecting PS-PGs. These numbers were used to estimate the density of PS-PG chains on the surfaces of MP (Table 1). The density of PS-PG chains on the MP surfaces increased with an increase in the feeding molar ratio, indicating that the density is controllable. In the case of MP-8PSPG, we calculated that approximately 20% of the surface amino groups of MP were bound to reducing-end monosaccharide residues of PS-PG. To investigate the structures of PS-PG chains grafted onto the MPs surface and the surface properties of MP-PSPG, the hydrodynamic diameter (Dh) and zeta potential of MP-PSPG in pure water were analyzed, respectively. We observed both an increases in Dh and a similarity of the zeta potential to that of free PS-PGs with an increase in the density of PS-PG chains for a series of MP-PSPGs (Table 1). Generally, it is well known that polymer chains grafted onto MP/NP surfaces adopt a “mushroom” (random coil) or “brush” (extended) structure depending on the density of polymer chains.28 At a low polymer density, the polymer chains extend only slightly from the surface but remain mostly coiled. When the polymer density increases to the point where the interchain distance is less than the Flory radius, the polymer chains extend significantly because of steric crowding by neighboring chains.29 For instance, poly(ethylene glycol) chains terminally bound to the surface of gold NPs with a relatively high polymer density adopt extended structures, and the Dh of the NPs is significantly larger than that of NPs with a relatively low density of poly(ethylene glycol) chains that adopt coiled structures.30 Thus, the increase in the Dh of MP-PSPG with an increase in the density of PS-PG chains can be attributed to the extended structures of the PS-PG chains grafted onto the MP surface based on the relatively high density. Moreover, the surface properties of MP-PSPG, especially with a high PS-PG density, were found to be governed by PS-PG chains. We examined the surface morphology of PS-PG-conjugated MPs by their SEM observations. Figure S3 shows SEM images of bare MP, MP2PSPG, and MP-8PSPG in the dry state. PS-PG chains

Figure 1. (a) Schematic illustration of the structures of cell-wall components of LcS. (b) Structures of PS-PG-conjugated polymer microparticles (MP-PSPG).

abbreviations for MP-PSPG (Table 1) and MP-Dex according to the molar ratio of PS-PGs or dextran to the surface amino groups of MPs in the feed (PS-PGs/NH2 of MPs), e.g., MP8PSPG (PS-PGs/NH2 of MPs = 8). 3.2. Characterization of MP-PSPG. To assess the conjugation of PS-PG chains to MPs, FTIR measurements of Table 1. Characterization of MP-PSPGs sample MP1PSPG MP2PSPG MP4PSPG MP6PSPG MP8PSPG MP PSPG

no. of PSPGsa

density of PSPGsb (no. of PS-PGs/μm2)

Dh (nm)

zeta potential (mV)

5.75 × 105

1.90 × 104

3110 ± 530

−32.4 ± 1.3

7.18 × 105

2.38 × 104

3310 ± 650

−34.2 ± 0.9

1.58 × 106

5.23 × 104

3330 ± 660

−38.9 ± 1.2

9.48 × 106

3.14 × 105

3470 ± 690

−39.8 ± 0.9

2.85 × 107

9.44 × 105

3580 ± 730

−39.7 ± 2.8

2580 ± 620 130 ± 80

−56.1 ± 1.0 −47.8 ± 6.8

a

Number of PS-PG chains attached to the surface of an MP. bDensity of PS-PG chains attached on the surface of an MP. 1491

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Figure 2. (a) Fluorescence microscopy images of MP-xPSPG/L8 antibody/IgM-FITC complexes. Scale bar: 50 μm. (b) Flow cytometric analysis of MP-xPSPG/L8 antibody/IgM-FITC complexes. The percentage of fluorescence-positive MPs is shown in the graph.

conjugated to MP are thought to absorb to its surface in the dry state. Thereby, the roughness of the MP surface obviously increased with an increase in the number of PS-PS chains conjugated. In particular, almost the entire surface of MP8PSPG seems to be covered with PS-PG chains. The SEM data is consistent with their surface characterization as revealed by another analysis described above. 3.3. Structure and Molecular Recognition of PS-PGs on MP Surfaces. The specific molecular recognition properties of PS-PGs grafted onto MP surfaces were investigated by fluorescence microscopy observation and flow cytometric analyses using a PS-PG-specific monoclonal L8 antibody as the first antibody and an FITC-labeled IgM antibody as the second antibody. Figure 2a shows fluorescence microscopy images of a series of MP-xPSPG/L8 antibody/IgM-FITC complexes with different densities of surface PS-PG chains. The number of fluorescence-positive complexes and the fluorescence intensity increased with an increase in PS-PG density. Moreover, the number of aggregated complexes also increased with an increase in PS-PG density because an IgM molecule possesses a binding ability with multiple (five) L8 antibodies. These results indicate that PS-PG chains on MP surfaces retain their original molecular recognition ability. Quantitative flow

cytometric analysis was performed to obtain more detailed information on the density of PS-PG chains grafted (Figure 2b). A series of MP-PSPGs showed significantly greater populations of positive complexes, and the populations increased with an increase in PS-PG density, whereas no populations (approximately 0%) of fluorescence-positive complexes were obtained for bare MP or a series of MP-Dex. These results mean that PS-PG chains grafted onto the MP surface bind to the L8 antibody through their specific interactions. It is noteworthy that MP-8PSPG showed a relatively low proportion (74.6%) of fluorescence-positive complexes as compared to MP-4PSPG (98.9%) and MP6PSPG (96.3%). Heat-killed LcS showed a similar proportion of fluorescence-positive complexes (65.2%) with MP-8PSPG, as shown in Figure S4 (Supporting Information). In fact, our previous study revealed that LcS exhibits a very low sensitivity to N-acetylmuramidase treatment, which digests the PG region in the cell wall, owing to the densely packed PS chains located in the PG region.26 The results in our previous study suggested that the moderate populations of fluorescence-positive complexes for heat-killed LcS were due to the high density and packing structures of PS-PG chains in the cell wall. In other words, such a densely packed PS-PG structure sterically 1492

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Figure 3. IL-12 secretion from J774.1 cells and cytotoxicity caused by MP-PSPG stimulation. (a) Concentrations of IL-12 secreted in culture medium after stimulation by MP-1PSPG (●), MP-2PSPG (■), MP-4PSPG (⧫), and MP-8PSPG (▲). (b) Cell viability after stimulation by MPPSPG (●), MP-2PSPG (■), MP-4PSPG (⧫), and MP-8PSPG (▲). Results are expressed as the mean ± standard deviation of three independent measurements.

Figure 4. IL-12 secretion from J774.1 cells and the cytotoxicity caused by the MP-8PSPG stimulus. (a) Concentrations of IL-12 in the culture medium after stimulation by heat-killed LcS. (b) Cell viability after stimulation by heat-killed LcS. (c) Concentrations of IL-12 secreted in the culture medium after stimulation by MP-8PSPG (●) and the corresponding amounts of soluble PS-PGs (○). (d) Cell viability after stimulation by MP8PSPG (●) and the corresponding amounts of soluble PS-PGs (○). Results are expressed as the mean ± standard deviation of three independent measurements.

hindered the access and binding of N-acetylmuramidase as well as the L8 antibody to their corresponding sites. Thus, we thought that the density and the structure of PS-PG chains on the surface of MP-8PSPG closely mimicked those of the cell wall of LcS. 3.4. IL-12 Production Inducing Activity of MP-PSPG. We examined the IL-12 production-inducing activity of a series of MP-PSPGs using macrophage-like cell line J774.1 by ELISA. Moreover, the cell compatibility of a series of MP-PSPGs was investigated by LDH assay. J774.1 cells used in this study secreted a certain amount of IL-12 by LPS stimuli (Figure S5, Supporting Information), as reported previously. Obvious IL12 secretion was observed for a series of PS-PG-conjugated MPs except for MP-1PSPG (Figure 3a), whereas bare MP and MP-Dex did not induce IL-12 secretion (Figure S6, Supporting Information). The amounts of IL-12 secreted by MP-2PSPG,

MP-4PSPG, and MP-8PSPG stimuli increased with an increase in concentration and showed a threshold of between 1.0 × 106 and 1.0 × 107 particles/mL. Moreover, the amounts of IL-12 secreted at 1.0 × 107 particles/mL increased with an increase in the density of surface PS-PG chains. These results indicate that the IL-12 production-inducing activity is owing to the PS-PG chains conjugated onto the MP surface and the activity is determined by the PS-PG density and the packed structures. Importantly, MP-PSPG did not show cytotoxicity to J774.1 cells during IL-12 secretion (Figure 3b). In contrast, heat-killed LcS also induced IL-12 production with a threshold of between 1.0 × 106 and 1.0 × 107 cells/mL (Figure 4a); however, obvious cytotoxicity to J774.1 cells was observed over 1.0 × 107 cells/ mL (Figure 4b). The cytotoxicity of heat-killed LcS was also checked by live/dead assay using calcein-AM and PI, as shown in Figure 5. It should be noticed that the amounts of IL-12 1493

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Figure 5. Live/dead staining of J774.1 cells treated with MP-8PSPG and heat-killed LcS. Scale bar: 100 μm.

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production induced by MP-8PSPG with 1.0 × 107 particles/mL were higher than that by heat-killed LcS of the same concentration (1.0 × 107 cells/mL), as shown in Figure 4a,c. Stimulation by heat-killed LcS with 1.0 × 108 cells/mL showed higher IL-12 production as compared to that by MP-8PSPG of the same concentration (1.0 × 108 particles/mL). Importantly, the MP-8PSPG series did not show cytotoxicity even at 1.0 × 108 particles/mL (Figure 4d), whereas heat-killed LcS at 1.0 × 108 cells/mL showed significant cytotoxicity to J774.1 cells. No cytotoxicity of MP-8PSPG was also checked by live/dead assay. Consequently, MP-PSPG having a high PS-PG density possesses a higher IL-12 production-inducing activity and lower cytotoxicity as compared to those of heat-killed LcS. Interestingly, soluble (free) PS-PGs did not show IL-12inducing ability even at a high dose (Figure 4c), indicating that the structure of PS-PGs formed on microparticle surfaces is essential to the exhibition of their IL-12-inducing activity. Thus, we discovered that PS-PG chains reassembled on microparticles effectively induce IL-12 secretion to the macrophage and the constructed lactobacilli-mimicking microparticles have excellent cytocompatibility. Together with all of these results it is concluded that PS-PGs of LcS is a novel material available for the development of biomaterials having immune-stimulating activity. Although we used polystyrene MPs as a primitive platform in this study, polystyrene is not an optimum one because of its nonbiodegradability. Moreover, we should examine the size effects on their immune-stimulating activity. Therefore, we will use biocompatible MPs of biodegradable polymers and examine the correlation of the size with the immune-stimulating activity in our next paper to assess the potential application of PS-PGs-based biohybrid materials as injectable immunostimulants.

ASSOCIATED CONTENT

S Supporting Information *

Characterization of MP-PSPG and MP-Dex, the immunestimulating property, and the cell viability of LPS and MP-Dex. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Masato Nagaoka (Yakult Central Institute for Microbiological Research) for technical support. REFERENCES

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4. CONCLUSIONS We demonstrated the successful fabrication of LcS-originated PS-PGs-conjugated MPs by using simple surface modification chemistry. The PS-PGs grafted onto MPs surfaces kept their original molecular recognition, and the resultant MP-PSPG with appropriate PS-PGs density induced high levels of IL-12 production from macrophages without cytotoxicity. Accordingly, LcS-originated PS-PGs was concluded to have potential as a novel bio-originated immunostimulant. Moreover, PS-PGsconjugated materials could be utilized for basic research on probiotics as an artificial lactobacillus to clear the mechanism of their immune-stimulation activity. 1494

DOI: 10.1021/la5041757 Langmuir 2015, 31, 1489−1495

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DOI: 10.1021/la5041757 Langmuir 2015, 31, 1489−1495