Bioconjugate Chem. 2007, 18, 1280−1286
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A Polysaccharide Carrier to Effectively Deliver Native Phosphodiester CpG DNA to Antigen-Presenting Cells Naohiko Shimada,† Cevayir Coban,§,∇ Yoichi Takeda,† Masami Mizu,† Jusaku Minari,† Takahisa Anada,† Yuichi Torii,⊥ Seiji Shinkai,‡,# Shizuo Akira,§,⊥,∇ Ken J. Ishii,§,⊥ and Kazuo Sakurai†,* Department of Chemical Processes & Environments, The University of Kitakyushu, 1-1, Hibikino, Wakamatu-ku, Kitakyushu, Fukuoka 808-0135, Japan, Department of Chemistry & Biochemistry, Graduate School of Engineering, and Center for Future Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), and Department of Host Defense, and The 21st Century COE, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. Received January 18, 2007; Revised Manuscript Received March 22, 2007
Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.
INTRODUCTION Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) can activate the vertebrate immune system (1-3). The initial step of this event is cellular uptake of CpG DNAs by antigen-presenting cells (APCs), particularly dendritic cells, macrophages, and B-cells. Subsequently, CpG DNAs are transported through the endocytosis pathway, and during the transport they are recognized by a pattern recognition receptor called toll-like receptor 9 (TLR-9) in endosome vesicles (4). The recognition by TLR-9 triggers the secretion of cytokines, including IL-6, TNF-R, IFN-γ, and IL-12. These cytokines activate natural killer (NK) cells and dendric cells (DC) (5-8) and finally control adaptive immune responses toward Th1- and IgG2a-secreting B cell inductions. Therefore, the interaction between CpG DNAs and TLR-9 effectively bridges the innate and adaptive immune systems. Recently, clinical trials have demonstrated that CpG DNAs can be useful as adjuvants for vaccines against infectious pathogens, cancer antigens, and allergens (9, 10). However, there is a serious drawback in CpG DNA therapy. Generally, once naked DNAs are exposed to biological liquids, they are quickly * Corresponding author. Tel: +81-93-695-3294, Fax: +81-93-6953368, e-mail:
[email protected]. † The University of Kitakyushu. ‡ Department of Chemistry & Biochemistry, Kyushu University. # Center for Future Chemistry, Kyushu University. § Japan Science and Technology Agency. ⊥ Department of Host Defense, Osaka University. ∇ The 21st Century COE, Combined Program on Microbiology and Immunology, Osaka University.
eliminated by nuclease-mediated degradation and nonspecific binding with plasma proteins. To translate CpG DNAs to practical therapy, it is essentially important to deliver CpG DNAs to their target cells while avoiding these unfavorable interactions. Phosphorothioate (PS) DNA, an analogue of the natural type phosphodiester (PO) DNA, has relatively strong antinuclease capability, so that PS DNAs are used in clinical trials. Some recent studies pointed out the cytotoxicity of PS and unexpected biological responses, for example, inducing a nonspecific response from a complement system (11, 12). These side effects can be ascribed to nonspecific binding to serum proteins and antigen nature of PS. It would be better if we can use PO by effectively protecting it from nuclease-mediated degradation and nonspecific binding. Recent studies categorized CpG DNAs into at least two types of structurally and functionally different classes (10, 13-15). One is denoted as K-type, which contains TCGT or TCGA motifs and activates plasmacytoid DCs to produce IL-6 and IL12, but very little IFN-R or IFN-γ. K-type also stimulates B cells to proliferate and recreate IL-6 and IgM. The PS analogues of K-type show almost the same activity as native phosphodiesters. The other is called D-type. The structural distinction of D-type is an unmethylated CpG dinucleotide flanked by a palindromic sequence and G quartet attached to the 3′ end to increase the cellular uptake (16). D-type activates plasmacytoid DCs to secrete a large amount of IFN-R, causing stimulation of NK-cells to produce IFN-γ and differentiate monocytes into myeloid DCs (14). D-type does not stimulate B-cells and other subsets of DCs. Ballas et al. recently reported that D-type for immunotherapy of cancer was more effective than K-type in the therapy of murine melanoma (17). Verthelyi et al. also
10.1021/bc0700178 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/27/2007
Polysaccharide Carrier to Deliver Native CpG DNA
Figure 1. (A) Repeating unit of SPG, and (B) the structural model of the complex consisting of one polynucleotide and two SPG chains (21). The chemical modifications were carried out by the selective cleavage of the 1,2-diol group of the glycosyl side chain with periodate, leading to formyl group formation, and then subsequently introducing a chemical group (27). As presented in B, the complex forms a triple helix. The black lines and gray lines indicate SPG and polynucleotide, respectively (left). Two main-chain glucoses of SPG interact with one base of nucleotide. Capital letters of G and B indicate the glucoses of SPG and the base of polynucleotides (right).
reported that D-type as an adjuvant was superior to K-type in the magnitude of elicited antibody response by coadministered heat-killed Leishmania vaccine (18). Delivery system for D-type is required to protect against nuclease because the essential sequence of D-type cannot be replaced by PS (13). There is a natural polysaccharide called schizophyllan (SPG) (Figure 1A), whose main chain consists of β-(1f3)-D-glucan and one β-(1f6)-D-glycosyl side chain links to the main chain at every three glucose residues (19). We found that SPG forms a novel complex with single-stranded homo polynucleotides (Figure 1B), and the fundamental properties of the complex have been examined by our group (20-23). Recently, we started to apply this complex to DNA delivery. Our communication preceding this paper showed a possibility to apply this complex to a CpG DNA carrier, showing that the complex can protect the bound DNA against unfavorable interactions with proteins and thus increase cytokine secretion (24, 25). The previous paper used a classical K-type PS DNA and delivered it to macrophagelike cell lines (24). The promising results in the excellent antinuclease resistance of the complex compelled us to expand this research. This study utilized CpG DNA, having a PO backbone instead of a cytotoxic PS, spleen primary cells in vitro, and a preliminary in vivo assay.
MATERIALS AND METHODS Materials. Mitsui Sugar Co., Ltd. (Japan) kindly supplied the SPG sample. The weight-average molecular weight (Mw) and the number of repeating units were found to be 1.5 × 105
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and 231, respectively. We used this SPG sample unless mentioned. Additionally, two SPG samples with Mw of 2.5 × 106 and 2.5 × 104 were used to study the Mw dependence of the cytokine secretion. The molecular weight control of SPG and purification were described elsewhere (26). The cellular uptake efficiency of SPG itself was not significant in Vitro; therefore, we modified SPG with a functional group that can induce passive cellular uptake. In this work, we introduced spermine (SP), arginine-glycine-aspartic acid tripeptide (RGD), octaarginine (R8), and cholesterol (Chol) (see Table 1). The experimental details and characterization for the modified SPG are described in our previous papers (24, 27) As a CpG DNA and its negative control, we adopted the two sequences of 5′-TCC ATG ACG TTC CTG ATG-3′ and 5′TCC ATG AGC TTC CTG AGT-3′ (key sequence is underlined), respectively. To bind these sequences with SPG, we had to attach a poly(dA)40 tail at the 3′ ends (23). In the present work, 5′-TCC ATG ACG TTC CTG ATG -(dA)40-3′ and 5′TCC ATG AGC TTC CTG ATG -(dA)40-3′ were used and denoted by CpG DNA and non-CpG DNA, respectively. In addition to this classical sequence, we used typical K- and D-type DNAs, which were 5′-(dA)40-ATC GAC TCT CGA GCG TTC TC-3′ ((dA)40-K3) and 5′-(dA)40-GGT GCA TCG ATG CAG GGG GG ((dA)40-D35). PS analogues for the CpG DNA and non-CpG DNA were used to compare with PO. The sequences of the CpG DNA used in this work are summarized in Table 2. All DNAs were synthesized at Hokkaido System Science (Hokkaido, Japan) and purified with high-pressure liquid chromatography. The fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco/BRL. Dulbecco’s modified Eagle’s medium (DMEM) and RPMI1640 were obtained from Nissui Pharmaceutical Co., Ltd. Complex Preparation. A 2 mg amount of PO CpG DNA or PS CpG DNA was dissolved in 10 mM Tris buffer solution (pH ) 7.8) (1 mL). An appropriate concentration of unmodified or modified SPG/DMSO solution was added to the DNA solution so that the water volume fraction was always adjusted to be 0.9 after mixing. The molar ratio (MSPG/MDNA) was controlled to be 1.5, where MSPG and MDNA are the repeating molar concentration of SPG and DNA, respectively. After the DNA and SPG mixture was kept at 5 °C for 1 night to complete the complexation, DMSO was removed by ultrafiltration. After the filtration, the final concentration of DNA was determined by measuring the ultraviolet absorbance. Mice and Cells. Seven- to eight-week-old female BALB/c or C57/B6 mice were purchased from Kyudo Co., Ltd., and CLEA, respectively. TLR-9 deficient mice were generated as previously described (4). Single spleen cell suspensions were prepared in RMPI1640 medium/10% FBS, 2 mM L-glutamine, 10 mM HEPES, and 0.11 mg/mL sodium pyruvate. Bone marrow dendric cells (BMDCs) were prepared as the established method (28). Flt3 ligand (Flt3L)-induced BMDCs (1 × 105 cells/ well) were generated by culturing bone marrow cells with Flt3 ligand (100 ng/mL; PeproTech) for 8-9 days in DMEM medium containing 10% FBS. Murine macrophage-like cells (J774.A1) were cultured in DMEM containing 10% FBS and 1 wt % penicillin/streptomycin mixture. BALB/c spleen cells were maintained in RPMI1640 supplemented with 10% FBS. All medium contains a 1 wt % penicillin/streptomycin mixture. The cells were cultured at 37 °C in a 5% CO2 incubator. Cells were stimulated in the presence of the indicated stimuli, and supernatants were collected for cytokine ELISA. For the in vivo assay, C57/B6 mice were injected intraperitoneally with 50 µg of CpG DNAs or the complex per mouse. Serum was collected in hours. The amount of IL-12p40 in serum was determined by ELISA.
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Table 1. Sample Codes and the Introduced Chemical Groups
a
Determined by N elemental analysis. The experimental details and characterization for the modified SPG are described in our previous papers (24, 27).
Table 2. Sequences of CpG DNAs sample code
sequencea
PS CpG DNA PS non-CpG DNA PO CpG DNA PO non-CpG DNA (dA)40-K3 (dA)40-D35
5′-TCC ATG ACG TTC CTG ATG -(dA)40-3′ 5′-TCC ATG AGC TTC CTG ATG -(dA)40-3′ 5′-TCC ATG ACG TTC CTG ATG -(dA)40-3′ 5′-TCC ATG AGC TTC CTG ATG -(dA)40-3′ 5′-(dA)40-ATC GAC TCT CGA GCG TTC TC-3′ 5′-(dA)40-GGT GCA TCG ATG CAG GGG GG-3′
a The key sequences are indicated as underlined text. All backbones of (dA)40-K3 and (dA)40-D35 are composed of phosphodiester.
ELISA. Spleen cells were plated in 48-well dishes using 2.5 × 106 cells/mL (1 well/1 mL) and were incubated 2 h at 37 °C. Naked CpG DNA or SPG/CpG DNA complexes were added to the cells. The cells were incubated at 37 °C for 24 h, and the supernatants were collected for ELISA assays (n ) 4). The secretion of IL-6 and total IL-12 was determined using commercially available ELISA kits according to the instructions of the manufacturer (Endogen). For the experiments using BMDCs and in vivo experiments, IL-12 p40 and IFN-R (PBL Biomedical Laboratories) were measured either from the supernatants or the serum by ELISA according to the manufacturer’s instructions (n ) 4). R-848 and LPS were added to the cells at the concentration of 1 µM and 1 µg/mL, respectively. J774.A1 cells were plated in 48-well dishes using 1 × 106 cells/mL (1 well/ 200 µL) and were incubated 5 h at 37 °C, and then samples were added to the cells. The cells were incubated at 37 °C for 24 h, and the supernatants were collected for ELISA assays (n ) 4). For all assays, each experiment was repeated at least twice.
RESULTS AND DISCUSSION Comparison of the Cytokine Secretion between Phosphorothioate and Phosphodiester. Figure 2 shows the cytokine secretions from the spleen cells when PS CpG DNA (left panels) and PS non-CpG DNA (right panels) were administered. When naked CpG DNA was exposed at a concentration of 25 µg/mL, the secretion levels of IL-12 and IL-6 were 2.2 and 2.5 ng/mL, respectively. When the amount of dose was doubled, the secretion became almost 2-fold, 3.7 ( 0.5 ng/mL for IL-12 and 3.7 ( 0.4 ng/mL for IL-6. When CpG DNA was complexed with unmodified SPG, the secretions were increased to ∼15% compared with the naked dose for IL-12 and IL-6. This increment can be ascribed to the fact that the complex can
Figure 2. The cytokine secretions by various SPG/CpG DNA complexes when PS CpG was used. The spleen cells were stimulated with various concentrations of CpG DNA. The amount of secretions was determined with ELISA kits, after incubation of the cells in the presence of CpG DNA or its complex for 24 h. The molar ratio (MSPG/ MDNA) is fixed at 1.5. Data represent the average ( SD (n ) 4).
stabilize the bound DNA in the culturing medium, and thus it increases the population of CpG DNA that can reach the cell surface. The stabilization is effective even for PS, because BSA contained in the culturing medium tends to bind to PS to eliminate the activity (29). We carried out the control experiments using naked non-CpG DNA and complexed non-CpG DNA with SPG. Some results are presented in the right panels, showing essentially no secretions for all of the cytokines. When we used the modified SPG samples, all of them increased the amount of secretions for IL-12 and IL-6 2- to
Polysaccharide Carrier to Deliver Native CpG DNA
Figure 3. The cytokine secretions by various SPG/CpG DNA complexes when PO CpG was used. The spleen cells were stimulated with various concentrations of CpG DNA. The amount of secretions was determined with ELISA kits, after incubation of the cells in the presence of CpG DNA or its complex for 24 h. The molar ratio (MSPG/ MDNA) is fixed at 1.5. Data represent the average ( SD (n ) 4), *p < 0.05 for naked PO CpG DNA dose.
4-fold, confirming the previous results obtained for the murine macrophage-like cells J774.A1 (24). The carriers themselves did not show any increment in the cytokines (data not shown). The increments in Figure 2 are ascribed to the increased cellular uptake mediated by the functional groups. Similar to the previous data, the R8-SPG complex was superior to the others. RGD-SPG, SP-SPG, and Chol-SPG ranked second, and RGD-SPG follows. According to previous work, the cellular uptake for the RGD, SP, and Chol complexes can be ascribed to normal endocytosis (30, 31). On the other hand, the R8 complex is seemingly ingested by an endocytosis pathway different from the other functional groups, although there is still controversy over how R8 and its analogues are ingested by cells (32-34). R8 binds to heparan sulfate proteoglycan which is a sulfated polysaccharide anchored to the cell surface (35). The anchored heparan sulfate may release R8 and its cargo during the endocytosis transport (36). Since R8 has cationic charge, it can be ingested by normal electrostatic interactions similar to the other functional groups. The enhanced secretion for the R8-SPG complex may be caused by a combination of all of the endocytosis pathways. Figure 3 shows the cytokine secretions when PO CpG DNAs were administered; here all the assays were carried out with the same conditions as those in Figure 2, except for using PO instead of PS. In contrast to the PS doses, naked PO CpG DNA did not show significant secretions unless the dose was increased over 100 µg/mL (PS showed appreciable secretion even at 25 µg/mL dose). This difference between PS and PO can be ascribed to two factors: PS is more stable against nuclease degradation than PO, and PS itself can be positively ingested by cells with an unidentified pathway (37). When PO CpG DNA was complexed with unmodified SPG, IL-12 level increased almost 11-fold at 50 µg/mL, 3.6-fold at 100 µg/mL, and 3.1-
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fold at 300 µg/mL, compared with the naked dose, respectively. In the case of PS, the complex with unmodified SPG increased the secretion level by 1.5-fold at most. The complexation increased the secretion level more efficiently in the PO system than in the PS system. This feature can be ascribed to the antinuclease protection effect of the complex (25) because naked PO DNAs should be more easily degraded than PS DNAs. The modified SPG samples showed that the amount of secretions for IL-12 and IL-6 increased 4- to 15-fold, compared with the naked ones, similar to the PS results while the order of the superiority was different. R8-SPG and SP-SPG showed the highest increment, and RGD-SPG and Chol-SPG ranked second. Comparison between Figures 2 and 3 offers valuable insight. When we compared PO and PS at the same dose level, the complexed PO did not show the equivalent secretion with those of the complexed PS. For IL-12, we needed to increase the dose level to about 6 times to obtain the same secretion level (e.g., PS CpG DNA/SPG induced 5.5 ng/mL at 50 µg/ mL dose, while PO CpG DNA/SPG induced 5.8 ng/mL at 300 µg/mL dose. Compare the black bars between Figures 2 and 3). This is because PS is positively introduced into cells but PO is not, and the complexation does not perfectly protect the bound PO from degradation. For IL-6, the secretion due to PO was much lower than that of PS (e.g., PS CpG DNA/R8 induced 17 ng/mL at 50 µg/mL dose, while PO CpG DNA/R8 induced 2.8 ng/mL at 300 µg/mL dose. Compare the gray bars between Figure 2 and 3). This difference in IL-6 secretion is due to the intrinsic difference between PS and PO reported by previous papers (38, 39). A large amount of IL-6 secretion induces unfavorable side effects,(40) as in the case of TNF-R. Therefore, the lower induction of IL-6 from PO CpG DNA/SPG complexes can be an advantage as an adjuvant. It is interesting that the complex of unmodified SPG showed a comparable effect with that of Chol-SPG and probably with that of RGD-SPG for IL-6. These results suggest that we can use unmodified SPG for CpG DNA carriers. A neutral or slightly anionic nature is favorable for the delivery in vivo, providing prolonged circulation time (41). This is because cations are generally easily absorbed by anionic proteins or the cellular surface and thus eliminated and/or inactivated in short period. Additionally, polycations can indeed induce side effects in the case of frequent doses, such as activation of complement, erythrocyte aggregation, and hemolysis. This is the main reason that the carriers with neutral and negative charge show good performance in vivo. On the basis of these facts, we decided to use unmodified SPG. An additional advantage to using unmodified SPG is that recent immunology (42, 43) and our data (44) showed that SPG and other β-(1f3)-d-glucans can be recognized by dectin-I and thus ingested by APCs. This means that the unmodified SPG complex has the ability to deliver its cargo to APCs in vivo, where the target TLR-9 is present. Many RGD-attached molecules have been studied as drug carriers to target to tumor cells in vivo (45-47), because the RGD motif can bind to integrin on the surface of the tumor cells (48) and also bind to newly formed endothelial cells (49). CpG DNA is required for uptake by APCs. This is why we did not use RGD-SPG. SPG Molecular Weight Dependence of the Secretion. The Mw of SPG used in the above experiments was 1.5 × 105, which means 230 repeating units in one molecule. The stoichiometric number of the complex shows that two repeating units of SPG and three bases are interacting with each other; that is to say, two main-chain glucoses and one base are interacting (Figure 1B) (21). CpG DNA consists of 58 bases; therefore, one complex contains at least 11 CpG DNA molecules. [In this calculation, we assumed that the hetero sequence of CpG DNA
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Figure 4. SPG molecular weight dependence of the secretion. J774.A1 (1 × 106 cells/mL, 200 µL) was stimulated with 25 µg/mL of PS CpG DNA and its SPG complex for 24 h. Data represent the average ( SD (n ) 4), *p < 0.001.
is complexed with SPG and one SPG chain is involved in one complex. In ref 23, we attached the (dA)40 tail to a hetero sequence at the 3′ or 5′ end, where (dA)40 can form a complex but the hetero sequence itself does not. After complexation with SPG, we compared the hydrolysis rate between the 3′- and 5′(dA)40 tailed hetero sequences using exonuclease I (hydrolyzes DNA from the 3′ end). We found that the hydrolysis rate was dramatically reduced for both 3′- and 5′-(dA)40 tailed hetero sequences and there was no appreciable difference between them. This result implies that the hetero part is protected, suggesting that the complexation of the (dA)40 part triggers additional complexation between the hetero part and SPG. If the hetero part is not involved in the complexation, 17 CpG DNA molecules are present in the complex.] We prepared two additional SPG samples with different Mw: 2.5 × 104 and 2.5 × 106. The same calculation shows that 2 and 200 CpG DNA molecules can be present in one complex, respectively. Figure 4 shows Mw dependence of the cytokine secretion for IL-12. The secretion was increased with increasing Mw. These results imply that the more CpG DNA molecules contained in the complex, the more cytokine secretion can be induced. A recent study reported the dramatic enhancement of cytokine secretion in the case of multiple CpG pairs in one DNA (50). Their results can be rationalized with the “cluster effect”, which is normally termed for the case that branched oligosaccharides are bound much more tightly to receptors than monosaccharides or single chain oligosaccharides. We presume that the multiple CpG DNA pairs in one sequence can play the same role as the multiple oligosaccharides. A more recent study, by Wu et al., reported that the aggregation of CpG DNA due to the polymerization of G-quartet and palindrome enhanced the IL-12 secretion (51). This result can be explained with the cluster effect of CpG pairs. Our results in Figure 4 can be understood in the same framework as these two studies. With increasing Mw, the size of the complex increases. Our dynamic light scattering determined the hydrodynamic diameter of the complexes using SPG with Mw ) 1.5 × 104 to be 100300 nm and with Mw ) 2.5 × 106 to be 1000 nm, respectively (data not shown). Since SPG is internalized by dectin-I-mediated phagocytosis, such a large size of complex may not hinder the cellular uptake.
Shimada et al.
Figure 5. Difference of the cytokine secretions between K-type and D-type DNA with unmodified SPG. Flt3L-induced BMDCs were stimulated with CpG DNAs (30 µg/mL), R-848 (1 µM), or LPS (1 mg/mL) for 24 h. WT and TLR-9 (-/-) denote BMDCs obtained from wild type and TLR-9 knockout mice, respectively. Data represent the average ( SD (n ) 4), *p < 0.001.
Distinct Cytokine Secretion with K-Type and D-Type CpG DNAs in Vitro. A distinctive difference between K- and D-type in the immunological response for the Flt3L-induced BMDCs can be determined by whether the secretion of IFN-R is induced. The resultant BMDCs contain CD11c+/B220+ and CD11c+/ B220- cells (52). K-type leads both cells to secrete IL-12; on the other hand, D-type leads to IFN-R secretion from CD11c+/ B220+ and to IL-12 secretion from CD11c+/B220-. Therefore, when K-type is administered to the Flt3L-induced BMDCs, only IL-12 should be observed and the secretion level of IL-12 should be higher than that of D-type. On the other hand, both IL-12 and IFN-R should be secreted from the D-type administration. The upper panel of Figure 5 shows the IL-12 secretion comparing (dA)40-K3 and (dA)40-D35, when the wild type and TLR-9-knockout (TLR-9 -/-) BMDCs were used. The IL-12 secretion was dramatically increased when (dA)40-K3 was complexed. The increment due to the complexation was also observed for the case of (dA)40-D35; however, the amount of the secretion was much smaller than those of (dA)40-K3. These features are consistent with the difference between K- and D-types. The control experiment used with TLR-9 -/- BMDCs did not secret IL-12 at all for both (dA)40-K3 and (dA)40-D35 systems, while it showed a large amount of IL-12 for R848 and LPS doses, which are recognized by TLR-7 and TLR-4, respectively (53). This result indicates that the IL-12 productions by treatments of complex of both (dA)40-K3 and (dA)40-D35 are ascribed to the signal induced by TLR-9. The lower panel shows the IFN-R secretions. As in the case of IL-12, the advantage of the complexation is evidently demonstrated. The K-type did not show any secretion; on the other hand, the D-type clearly showed the secretion and the complex increased the secretion. The present IFN-R assay clarifies the advantage of using the SPG complex for the CpG DNA delivery. IL-12 Secretion Induced by the Complex in Vivo. Figure 6 shows IL-12 p40 secretion of unmodified SPG/CpG DNA complex in vivo. IL-12 secretion was hardly observed by injection of unmodified SPG and naked CpG DNAs. On the other hand, injection of the complexes clearly exhibited 2-9 fold increase in IL-12 productions compared with those of naked
Polysaccharide Carrier to Deliver Native CpG DNA
Figure 6. Time course of IL-12 p40 secretion in vivo, when CpG DNAs (50 µg) were intraperitoneally injected to C57/B6 mouse. Results represent the average ( SD of three mice per group. *p < 0.05 when compared with naked DNAs at each time. Naked (dA)40-K3 and its SPG complex are indicated with unfilled and filled squares, respectively. Naked (dA)40-D35 and its complex are indicated with unfilled and filled circles, respectively. PBS alone and SPG alone are indicated with open triangles and filled triangles, respectively.
doses. Maximum secretions appeared at 2-4 h. These enhancements can be ascribed to the stabilization due to the complex. Moreover, the complex with (dA)40-K3 produced a large amount of IL-12 than that with (dA)40-D35. This result agrees with in vitro study (Figure 5).
CONCLUSIONS This paper showed that a natural polysaccharide called schizophyllan (SPG) can be used as a CpG DNA carrier. Kand D-type of phosphodiester CpG DNA were administered to Flt3L-induced BMDCs. K-type induced a large amount of IL12 secretion; on the other hand, D-type led to the secretion of both IL-12 and IFN-R. The complexation increased the secretion in both cases, showing that the difference between K- and D-type was further enhanced. When CpG DNA was complexed with SPG and intraperitoneally injected to mouse, the maximum value of IL-12 for (dA)40-K3 was 9-fold that of the naked dose. On the basis of these promising findings, the present paper proposes a new delivery method of CpG DNA and could lead to development of this therapy.
ACKNOWLEDGMENT This work was finically supported by JST SORST and ERATO programs and Grant-in-Aid for Scientific Research (16350068 and 16655048).
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