Efficacy of SPG-ODN 1826 Nanovehicles in Inducing M1 Phenotype through TLR-9 Activation in Murine Alveolar J774A.1 Cells: Plausible Nano-Immunotherapy for Lung Carcinoma.

Alveolar macrophages are the first line of defense against intruding pathogens and play a critical role in cancer immunology. The Toll-like receptor (TLR) family mediates an important role in recognizing and mounting an immune response against intruding microbes. TLR-9 is a member of the intracellular TLR family, which recognizes unmethylated CG motifs from the prokaryotic genome. Upon its activation, TLR-9 triggers downstream of the MyD-88-dependent transcriptional activation of NF-κB, and subsequently results in abundant inflammatory cytokines expression that induces a profound inflammatory milieu. The present exploratory investigation aimed at elucidating the potency of schizophyllan for entrapping ODN 1826 (SPG-ODN 1826)-mediated stimulation of TLR-9 in provoking an inflammatory-type response in murine alveolar macrophages. Schizophyllan (SPG), a representative of the ß-glucan family, was used in the present study as a nanovehicle for endosomal trafficking of CpG ODN 1826. TEM analysis of SPG-ODN 1826 nanovehicles revealed that the prepared nanovehicles are spherical and have an average size of about 100 nm. Interestingly, SPG-ODN 1826 nanovehicles were competent in delivering their therapeutic payload within endosomes of murine alveolar macrophage (J774A.1) cells. Exposure of these nanovehicles within LPS stimulated J774A.1, resulted in a significant provocation of reactive oxygen species (ROS) (p < 0.01) in comparison to CpG ODN 1826 alone. Moreover, the formulated nanovehicles succeeded in generating a profound Th1-based cytokine profile constituted by enhanced expression of IFN-γ (p < 0.001) and IL-1ß (p < 0.001) inflammatory cytokines. These findings clearly indicated the immunostimulatory potential of SPG-ODN 1826 nanovehicles for inducing the Th1-type phenotype, which would certainly assist in skewing M2 phenotype into the much-desired M1 type during lung cancer.

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine.

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by ß-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

Adsorption of enhanced oil recovery polymer, schizophyllan, over carbonate minerals.

Schizophyllan is a natural polysaccharide that has shown great potential as enhanced oil recovery (EOR) polymer for high-temperature, high-salinity reservoirs. Nevertheless, the adsorption behavior of schizophyllan over carbonate minerals remains ambiguous element towards its EOR applications. Here, we investigate the adsorption of schizophyllan on different carbonate minerals. The effect of mineral type, salinity, and background ions on adsorption is analyzed. Our results indicate the adsorption capacity is higher on calcite and dolomite compared to silica and kaolin and the adsorption capacity decreases with salinity. Moreover, the adsorption kinetics follows pseudo-second order mechanism regardless of the mineral type. Adsorption over calcite is diminished in presence of water structure making ions and enhanced in presence of structure breaking ion and in presence of urea. Gel permeation chromatography results reveal the preferential adsorption of longer chains. The adsorption over carbonate minerals proceed via complex formation between polymer molecule and mineral surface.

Inflammatory responses of macrophage-like RAW264.7 cells in a 3D hydrogel matrix to ultrasonicated schizophyllan.

The water-soluble ß-glucan schizophyllan has diverse immunomodulatory activities. However, its biological activities have only been explored using cells grown in two-dimensional (2D) culture condition, which does not replicate the three-dimensional (3D) microenvironment of actual tissue, resulting in mismatches between in vitro and in vivo findings. In this study, we investigated the immunomodulatory effects of ultrasonicated schizophyllan (uSPG) on RAW264.7 cells encapsulated in 3D poly(ethylene glycol) hydrogel. Cells grown in 3D were less sensitive to uSPG than those grown in 2D, although uSPG upregulated M1 macrophage phenotype markers in both conditions. This might be due to the low availability of uSPG recognition receptors of cells grown in 3D. Conversely, uSPG promoted gene expressions of M2 macrophage phenotype markers in 3D, suggesting uSPG-induced immune-regulation of macrophages in real tissues. These findings imply that the immunomodulatory effects of uSPG on macrophages should be carefully interpreted in terms of the microenvironment.

A comparative study on schizophyllan and chitin nanoparticles for ellagic acid delivery in treating breast cancer.

In this study, following encapsulation of ellagic acid (EA), an anti-cancer agent, loaded in schizophyllan (EA/SPG-NP) and chitin (EA/Ch-NP) nanoparticles, its release in 95% ethanol, and different mediums of digestive systems with pH ranging 1.5 to 7.4, were examined before investigating for treatment of breast cancer MCF-7cells. Following synthesis, the EA was characterized by FT-IR, SEM, XRD, DLS and zeta potential analysis. Loading capacity of schizophyllan and chitin were 30.08 and 79.52%, respectively, while SEM images indicated respective size distributions of 217.8 and 39.82 nm, with the corresponding zeta potentials being +27 and -9.14 mV. As EA was loaded in nanoparticles, antioxidant activity, examined by DPPH method, of the free EA was found to be higher than both EA/SPG-NP and EA/Ch-NP, but lower than the latter at 7.4 pH. Interestingly, scavenging activities for EA and EA/SPG-NP reduced for higher pH. The MTT cytotoxicity indicated that EA/SPG-NP and EA/Ch-NP inhibited effectively cell growth of breast cancer cell lines at IC50 of 60 and 115 µg/ml, respectively.

Complex consisting of antisense DNA and ß-glucan promotes internalization into cell through Dectin-1 and hybridizes with target mRNA in cytosol.

Antisense oligonucleotides (AS-ODNs) hybridize with specific mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs with attached dA40 (AS-ODNs/SPG), and this complex can be incorporated into cells, such as macrophages and dendritic cells, expressing the ß-glucan receptor Dectin-1. We have achieved efficient gene silencing in animal models, but the uptake mechanism and intracellular distribution are unclear. In this study, we prepared the complex consisting of SPG and AS-ODNs (AS014) for Y-box binding protein-1 (YB-1). After treatment with endocytosis inhibitor Pitstop 2 and small interfering RNA targeting Dectin-1, we found that AS014/SPG complexes are incorporated into cells by Dectin-1-mediated endocytosis and inhibit cell growth in a Dectin-1 expression level-dependent manner. After treatment with AS014/SPG complexes, we separated the cell lysate into endosomal and cytoplasmic components by ultracentrifugation and directly determined the distribution of AS014 by reverse transcription PCR using AS014 ODNs as a template or a reverse transcription primer. In the cytoplasm, AS014 clearly hybridized with YB-1 mRNAs. This is the first demonstration of the distinct distribution of the complex in cells. These results could facilitate the clinical application of the complex.

Designing an immunocyte-targeting delivery system by use of beta-glucan.

A ß-1,3-d-glucan called Schizophyllan (SPG) can form a novel complex with homo oligodeoxynucleotides (ODNs) via the combination of hydrogen bonding and hydrophobic interactions. Dectin-1 is a major receptor involved in the recognition of ß-1,3-d-glucans and expressed on antigen presenting cells (APCs) including macrophages, dendritic cells, monocytes, neutrophils, and a subset of T cells. Therefore, the SPG/ODN complex can be used as APCs cell-specific delivery of functional ODNs including unmethylated CpG sequences (CpG-ODNs). In fact, CpG-ODN/SPG complex induced high antibody titers when it was administered to cynomolgus monkeys as adjutant of influenza vaccine. These results indicate that SPG can be an excellent immunocyte-targeting drug delivery system.

Complex Consisting of ß-Glucan and Antigenic Peptides with Cleavage Site for Glutathione and Aminopeptidases Induces Potent Cytotoxic T Lymphocytes.

The efficient induction of antigen-specific immune responses requires not only promotion of the uptake of antigens and adjuvant molecules into antigen-presenting cells but also control of their intracellular behavior. We previously demonstrated that the ß-glucan schizophyllan (SPG) can form complexes with CpG oligonucleotides with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses. In this study, we prepared various conjugates composed of antigenic peptide (OVA257-264) and dA40 and made complexes with SPG. The conjugates with a disulfide bond between OVA257-264 and dA40 were easily cleaved by glutathione. The resultant peptides with a hydrophobic amino acid at the C-terminal end was recognized by puromycin-insensitive leucine aminopeptidase (PILS-AP), which trims antigenic peptide precursors and prepares peptides of eight or nine amino acids in length, which is the optimal length for binding to major histocompatibility complex (MHC)-I. The conjugate exposed to such enzymes induced a high antigen presentation level. The antigen presentation level was almost the same before and after the complexation with SPG. Immunization with a mixture of dA-OVA257-264/SPG and CpG-dA/SPG induced high antigen-specific cytotoxic T-lymphocyte activity at a much lower peptide dose than in previous studies. These results can be strongly ascribed to not only the cell-specific delivery by SPG but also the control of the intracellular behavior by the introduction of cleavage sites. Therefore, peptide-dA/SPG complexes could be used as potent vaccine antigens for the treatment of cancers and infectious diseases.

Optimal sequence of antisense DNA to silence YB-1 in lung cancer by use of a novel polysaccharide drug delivery system.

Silencing Y-box binding protein 1 (YB-1) can be an excellent target for cancer therapy and many lung cancer cells express the polysaccharide-recognition receptor Dectin-1. We designed a Dectin-1 targeting vehicle delivering YB-1-antisense DNA. First, we selected five optimal antisense DNA sequences to silence YB-1 from among 153 candidates. We chose the sequence closest to the start codon (AS014), and attached dA40 to the 3' end; dA40 promotes complex formation with a ß-(1➝3)-d-glucan called schizophyllan (SPG). The resultant complexes were applied to 12 human-oriented lung cancer cell lines, and cell viability was examined. The cell lines exhibited decreased viability and showed strong affinity to bind SPG, suggesting the AS014/SPG complex entered the cells via the Dectin-1 mediated pathway.

The targeted delivery of the c-Src peptide complexed with schizophyllan to macrophages inhibits polymicrobial sepsis and ulcerative colitis in mice.

Hyper-inflammatory responses triggered by intracellular reactive oxygen species (ROS) can lead to a variety of diseases, including sepsis and colitis. However, the regulators of this process remain poorly defined. In this study, we demonstrate that c-Src is a negative regulator of cellular ROS generation through its binding to p47phox. This molecule also competitively inhibits the NADPH oxidase complex (NOX) assembly. Furthermore, we developed the schizophyllan (SPG)-c-Src SH3 peptide, which is a ß-1,3-glucan conjugated c-Src SH3-derived peptide composed of amino acids 91-108 and 121-140 of c-Src. The SPG-SH3 peptide has a significant therapeutic effect on mouse ROS-mediated inflammatory disease models, cecal-ligation-puncture-induced sepsis, and dextran sodium sulfate-induced colitis. It does so by inhibiting the NOX subunit assembly and proinflammatory mediator production. Therefore, the SPG-SH3 peptide is a potential therapeutic agent for ROS-associated lethal inflammatory diseases. Our findings provide clues for the development of new peptide-base drugs that will target p47phox.

Immunoregulatory and antitumor activity of schizophyllan under ultrasonic treatment.

Aim of this study was to investigate the effect of ultrasonic treatment on the biological activities of schizophyllan (SPG) from Schizophyllum commune. The immunoregulatory and antitumor activity in vitro and in vivo of SPG and ultrasonic-treated SPG (USPG) were evaluated by splenic lymphocytes, macrophages RAW264.7 and human breast carcinoma T-47D cells. Compared with SPG, USPG fractions had small molecular weight and narrow distribution. Meantime, more enhancement of NO production in macrophages RAW264.7, lymphocytes proliferation rates, IL-2 and TNF-α level from spleen lymphocytes and T-47D cells inhibition rates were observed in USPG fractions groups. This result indicated that the immune-enhancing and antitumor activity of SPG was significantly improved after ultrasonic treatment. USPG60 exhibited the highest biological activity in this study. In conclusion, application of ultrasonic technology on SPG preparation is an efficient approach to get high biological polysaccharide, and USPG60 might be a potential functional component for immunoregulatory and cancer treatment.

Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system.

The CD40/CD154 co-stimulatory pathway is crucial in alloimmune response. We developed a novel small interfering RNA (siRNA) delivery system with a poly-dA extension at the 5'-end of the siRNA sense strand that was stably incorporated into 1,3-ß-glucan (schizophyllan, SPG). This was captured and incorporated into dendritic cells (DCs) through its receptor, Dectin-1, specifically silencing CD40 genes (siCD40) to exert immunoregulatory activity. siCD40/SPG-treated CBA mice permanently accepted B10 fully mismatched cardiac allografts. Consistent with graft survival, the infiltration of CD4(+), CD8(+) T cells into the graft was lower, and that the numbers of CD40(low)CD11c(+) DCs cells and CD4(+)Foxp3(+)cells were increased in both the graft and in the recipient spleen. In addition, naive CBA recipients given an adoptive transfer of splenocytes from the primary recipients with siCD40/SPG accepted a heart graft from donor-type B10, but not third-party Balb/c mice. In conclusion, the treatment with siCD40/SPG targeting DCs could generate antigen-specific Tregs, resulting in the permanent acceptance of mouse cardiac allografts. These findings have important implications for clarifying the mechanism underlying the induction of tolerance in DCs, and also highlight the potential of immunomodulation and the feasibility of siRNA-based clinical therapy in the transplantation field.

Molecular function of macrophage migration inhibitory factor and a novel therapy for inflammatory bowel disease.

Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD.

A new therapeutic approach using a schizophyllan-based drug delivery system for inflammatory bowel disease.

Antisense technologies for the targeted inhibition of gene expression could provide an effective strategy for the suppression of inflammation. However, the effective use of antisense oligonucleotides (ODN) has been limited because of several problems. Therefore, a delivery system for antisense ODNs that enhances antisense stability, while maintaining the specificity of antisense for its target RNA or DNA is needed. We have developed a delivery system for antisense ODN using schizophyllan (SPG), a polysaccharide that belongs to the ß-(1-3) glucan family. This system has several advantages enabling the effective suppression of targeted RNA or DNA: the SPG complex is stable in vivo and does not dissolve in the presence of deoxyribonuclease, and the SPG complex is effectively taken up into macrophages by phagocytosis through Dectin-1. Macrophage-migration inhibitory factor (MIF), which is mainly produced by macrophages has been shown to have a pathogenetic role in inflammatory bowel disease (IBD). We developed a technique to create an SPG complex that highly conformed to the antisense MIF. The administration of antisense MIF/SPG complex effectively suppressed MIF production and significantly ameliorated intestinal inflammation. Our result demonstrated a possible new therapeutic approach, i.e., the administration of antisense MIF/SPG complex, for the treatment of IBD.

ß-1,3-Glucan/antisense oligonucleotide complex stabilized with phosphorothioation and its gene suppression.

Most of antisense oligonucleotides (ASOs) subjected to current clinical evaluation belong to phosphorothioate (PS) analogues. Although PS has great advantage in DNase resistance, it can induce nonspecific side-effects. Thus it is important to investigate the influence of ASOs with different PS contents. In this paper, we prepared the complex consisting of schizophyllan (SPG) and ASOs attached a dA40 tail with different PS contents to the 3' end of the ODN, which is introduced to stabilize the complex with SPG. With increase of PS content in the dA40, its complexation ability with SPG was improved and the complex showed high thermal stability. The thermal stability of the fully phosphorothioated ASOs was obtained by only replacing 20% of the oxygen of the phosphodiester moiety. The ability of gene suppression between PS and phosphodiester for antisense sequences was almost the same, indicating that the antisense sequences need not to be PS backbone. These data may provide new insight for the interaction between ß-1,3-glucan and DNA and help to deliver therapeutic ODNs.

Blocking effect of anti-Dectin-1 antibodies on the anti-tumor activity of 1,3-beta-glucan and the binding of Dectin-1 to 1,3-beta-glucan.

Schizophyllan (SPG) is used to treat cervical cancer in combination with irradiation to enhance the immunological surveillance system. Dectin-1 is a cell surface receptor for 1,3-beta-glucan. In this study, we prepared two anti-Dectin-1 monoclonal antibodies, 4B2 and SC30 having a K(D) of 7.04 x 10(-8) M and 1.55 x 10(-7) M, respectively, and evaluated the role of Dectin-1 in SPG-induced anti-tumor activity in mice. Expression of Dectin-1 on peritoneal macrophages and binding of SPG to the cells were decreased by administration of 4B2 and SC30. SPG-mediated anti-tumor activity was inhibited by 4B2 and SC30. 4B2 and SC30 inhibited the binding of SPG to splenocytes from mice. The binding of SPG-biotin to Dectin-1-transfected HEK293 cells was inhibited by 4B2, but not SC30. 4B2 and SC30 differ in their influence on Dectin-1 between primary cells and transduced cells, and Dectin-1 effects 1,3-beta-glucan-mediated anti-tumor activity in mice by binding to SPG.

Cell wall beta-glucan derived from Candida albicans acts as a trigger for autoimmune arthritis in SKG mice.

SKG mice are a recently established experimental model for rheumatoid arthritis (RA). Although they spontaneously develop chronic autoimmune arthritis under conventional conditions, SKG mice failed to develop chronic arthritis in a strictly controlled specific pathogen-free (SPF) environment. Beta-glucan (BG) from Laminaria digitata, laminarin (LAM), induced arthritis under SPF conditions, thus BG would be a pathogenic factor for arthritis in SKG mice. Therefore, we prepared BG from Candida albicans, a pathogenic fungus and investigated whether BG from C. albicans induced arthritis in SKG mice under SPF conditions. SKG mice were injected intraperitoneally with particulate BG (oxidative-Candida albicans (OX-CA)), soluble BG (Candida soluble beta-glucan (CSBG)) from C. albicans and LAM as a positive control. In addition, schizophyllan (SPG) from Schizophyllum commune or Mycobacterium whole cells were injected into SKG mice to induce arthritis. Mice injected with OX-CA, CSBG and SPG had more severe arthritis than with LAM, and whole Mycobacterium cells. IL-6 concentration in sera from SKG mice injected with OX-CA or CSBG was high, whereas not detected in sera from mice treated with LAM. In histological analysis, infiltration of inflammatory cells was observed in SKG mice injected with BG. These results suggest that fungal infection may be a factor to induce and exacerbate autoimmune diseases such as RA.

Ternary complex consisting of DNA, polycation, and a natural polysaccharide of schizophyllan to induce cellular uptake by antigen presenting cells.

A natural polysaccharide called schizophyllan (SPG) can form a complex with polynucleotides, and the complex has been shown to deliver biofunctional short DNAs such as antisense DNAs and CpG-DNAs. Although it is a novel and efficient method, there is a drawback: attachment of homo-polynucleotide tails [for example, poly(dA) or poly(C)] to the end of DNA is necessary to stabilize the complex, because DNA heterosequences cannot bind to SPG. The aim of this paper is to present an alternative method in which SPG/DNA complexes can be made without using the tails. The basic strategy is as follows: since SPG can form hydrophobic domains in aqueous solutions, hydrophobic objects should be encapsulated by this domain. DNA alone is highly hydrophilic; however, once DNA/polycation complexes are made, they should be included by the SPG hydrophobic domain. The aim of this paper is to prove the formation of the polycation/DNA/SPG ternary complex. Gel electrophoresis showed that presence of SPG influenced the migration pattern of polycation+DNA mixtures. With increasing the SPG ratio, the zeta potential (zeta) of the polycation+DNA+SPG mixture decreased drastically to reach almost zeta = 0 and the particle size distributions were altered due to the ternary complex formation. Confocal laser scanning microscopy revealed that the polycation/DNA/SPG ternary complexes showed high uptake efficiency when the complexes were exposed to macrophage-like cells (J774.A1). IL-12 secretion was enhanced when CpG-DNA was added as the ternary complex. These features can be ascribed to the fact that J774.A1 has a SPG recognition site called Dectin-1 on the cellular surface and the ternary complex can be ingested by this pathway.

PEG-appended beta-(1-->3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation.

Schizophyllan is a natural beta-(1-->3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier.