Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Modification of the antigenicity of cancer cells by conjugates consisting of hyaluronic acid and foreign antigens.

Tumor-specific cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens presented on major histocompatibility complex (MHC) class I molecules. However, it is difficult to induce potent CTLs by vaccination because the antigenicity is not so high, compared with that of foreign antigens derived from viruses and microbes. The affinity of binding to MHC class I molecules is proportional to the antigenicity of the antigen that they are presenting. Here, we prepared several conjugates consisting of hyaluronic acid (HA) as a carrier to cancer cells and ovalbumin (OVA) as a foreign protein and changed the antigens on cancer cells from intrinsic antigens to OVA fragments. The conjugate containing multiple HA and OVA molecules (100k4HA-3OVA) adopted a highly condensed structure and was well recognized by recombinant CD44 molecules in quartz crystal microbalance analysis and incorporated into cancer cells (CT26 cells). A mixture of CT26 cells treated with 100k4HA-3OVA and splenocytes including OVA-specific CTLs induced abundant secretion of IFN-γ into the supernatant. At 48 h after mixing with the CTLs, almost all CT26 cells had died. These results indicate that 100k4HA-3OVA is actively internalized into the cells through interaction between HA and CD44. Subsequently, CT26 cells present not only self-antigens, but also OVA fragments on MHC class I molecules and are recognized by OVA-specific CTLs. We thus succeeded in modifying the antigenicity from self- to non-self-antigens on cancer cells. Therefore, this foreign-antigen delivery using HA to cancer cells, followed by antigen replacement, could be used as a novel strategy for treating cancers.

Tyrosinase-Related Protein2 Peptide with Replacement of N-Terminus Residue by Cysteine Binds to H-2Kb and Induces Antigen-Specific Cytotoxic T Lymphocytes after Conjugation with CpG-DNA.

Recent studies have shown the potent efficacy of peptide-based vaccines for cancer immunotherapy. Immunological performance is optimized through the co-delivery of adjuvant and antigenic peptide molecules to antigen-presenting cells simultaneously. In our previous study, we showed that a conjugate consisting of 40-mer CpG-DNA and an antigenic ovalbumin peptide through disulfide bonding could efficiently induce ovalbumin-specific cytotoxic T lymphocyte (CTL) responses in vivo. In this study, based on the conjugation design, we prepared a conjugate consisting of 30-mer CpG-DNA (CpG30) and a cancer antigenic peptide of Tyrosinase-related protein 2 (TRP2180-188) using a cysteine residue attached at the N-terminus of TRP2180-188. However, the immunization of mice with this conjugate did not induce efficient TRP2180-188-specific immune responses. It was thought that the resultant peptide (10-mer) cleaved from the conjugate might be too long to fit into the H-2Kb molecule because the optimal length for binding to it is 8-9 amino acids. We newly designed a conjugate consisting of CpG30 and the C-TRP2181-188 peptide (9-mer), in which the N-terminal serine residue of TRP2180-188 is replaced by a cysteine. By adjusting the peptide length, we succeeded in inducing strong TRP2180-188 peptide-specific CTL activity upon immunization with the CpG30-C-TRP2181-188 conjugate. Furthermore, various CpG30-C-TRP2181-188 conjugates having other CpG-DNA sequences or cysteine analogues also induced the same level of CTL activity. Therefore, CpG-C-peptide conjugates prepared by replacement of the amino acid residue at the N-terminus with a cysteine residue could be a new and effective platform for peptide vaccines for targeting specific antigens of cancers and infectious diseases.

Interaction between CD44 and highly condensed hyaluronic acid through crosslinking with proteins.

Proteins and nucleic acids derived from bioresources have become a novel medical modality. However, the intrinsic features of proteins, such as their high molecular weight and polarity, impede their passage through the cell membrane. In this study, to deliver proteins to cancer cells, we prepared conjugates consisting of bovine serum albumin (BSA) and hyaluronic acid (HA). These conjugates contained multiple BSA and HA molecules and adopted a highly condensed structure by crosslinking through BSA. When the interaction between the HA-BSA conjugates and recombinant CD44 (rCD44) was examined using a quartz crystal microbalance, the conjugates induced a larger decrease of frequency change than HA. CT26 cells treated with FITC-labeled HA-BSA conjugates showed high fluorescence intensity. The uptake of the conjugates decreased upon adding excess HA. Therefore, the conjugates and nanoparticles with densely packed HA structures could be a potent and effective platform for delivering proteins to cancer tissues.

Oligonucleotide delivery to antigen presenting cells by using schizophyllan.

Schizophyllan (SPG), a member of the ß-glucan family, can form novel complexes with homo-polynucleotides such as poly(dA) through hydrogen bonding between two main chain glucoses and the one nucleotide base. Dectin-1, one of the major receptors for ß-glucans, is known to be expressed on antigen presenting cells (APCs) such as macrophages and dendritic cells. This suggests that the above-mentioned complexes could deliver bound functional oligonucleotides (ODNs) including antisense (AS)-ODNs, small interfering RNA, and CpG-ODNs to the APCs. Analysis using a quartz crystal microbalance revealed that a complex consisting of SPG and dA60 with a phosphorothioate backbone was recognized by recombinant Dectin-1 protein. Treatment with this complex containing an AS-ODN for tumor necrosis factor alpha protected mice against lipopolysaccharide-induced hepatitis at a very low AS-ODN dose. Moreover, immunization with CpG-ODN/SPG complex and antigenic proteins induced potent antigen specific immune responses. The present review also represents peptide delivery by conjugation with dA60 and the preparation of a nanogel using DNA-DNA hybridization. These findings indicate that the delivery of a specific ODN using ß-glucans could be used for treating various diseases caused by APCs and for activating antigen specific immune responses.

Binding assay of human Dectin-1 variants to DNA/ß-glucan complex for active-targeting delivery of antisense DNA.

The lectin Dectin-1 is a good target for ß-glucan-mediated drug delivery. Although many murine studies of Dectin-1 have been performed, its human analog has not been studied well in terms of being a drug delivery target. We thus analyzed human Dectin-1 cDNA obtained from chronic myelogenous leukemia-derived cells, CML-1, and confirmed the findings of previous studies that there are many isoforms of human Dectin-1 due to exon skipping, although murine Dectin-1 has only two forms. When we transfected the Dectin-1 gene into a non-Dectin-1-expressing cell line and examined cellular uptake of the antisense DNA/ß-glucan complex, we confirmed that expression of the target gene was effectively suppressed through ß-glucan/Dectin-1-mediated uptake. The present results suggest that the ß-glucan complex would be an effective tool to deliver antisense oligonucleotide (AS-ODN) to Dectin-1-expressing cells not only for mice but also for humans.

Immune Responses and Antitumor Effect through Delivering to Antigen Presenting Cells by Optimized Conjugates Consisting of CpG-DNA and Antigenic Peptide.

Immunotherapy using antigen-specific cytotoxic T lymphocytes (CTLs) has become one of the most attractive strategies for cancer treatment. For the induction of antigen-specific CTLs in vivo, the co-delivery of CpG-DNAs and antigens to the same antigen-presenting cells (APCs) is a promising strategy. In this study, we prepared conjugates consisting of 40mer of CpG-DNA (CpG40) and antigenic peptide (OVA257-264), which have the following distinctive features: (1) multiple CpG motifs in a molecule; (2) cleavage in the cytosol because of the disulfide bonding via cysteine residue between peptide and CpG-DNA; (3) conjugation designed to induce antigen presentation on MHC class I molecules. Immunization with the conjugate CpG40-C-OVA257-264 at the mouse tail base induced strong CTL activity at a very low peptide dose of 20 ng/head. It was found that the conjugates were internalized into C-type mannose receptor 1 (MRC1)-expressing cells in inguinal lymph nodes, indicating that the CpG portion in the conjugate acts as not only an adjuvant for the activation of TLR9 but also a carrier to APCs expressing MRC1. In a tumor-bearing mice model, mice immunized with CpG40-C-OVA257-264 conjugates exhibited long delays in tumor growth compared with those treated with PBS, OVA257-264 alone, or a mixture of CpG40 and OVA257-264. Therefore, CpG-C-peptide conjugates could be a new and effective platform for peptide vaccine for the treatment of cancers and infectious diseases.

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.

Topical Therapy with Antisense Tumor Necrosis Factor Alpha Using Novel ß-Glucan-Based Drug Delivery System Ameliorates Intestinal Inflammation.

Anti-tumor necrosis factor alpha (TNF-α) antibodies are effective in patients with inflammatory bowel disease (IBD). However, the effect is not optimal because a sufficient concentration of antibodies cannot be maintained at the site of inflammation. Thus, a macromolecular complex was developed with schizophyllan (SPG) and antisense oligonucleotides. In the present study, an SPG-antisense TNF-α complex was prepared, and its therapeutic efficacy was examined using a dextran sodium sulfate (DSS)-induced colitis model. The TNF-α production in CD11b+ macrophages significantly increased in the colon of DSS-treated mice. Dectin-1, a receptor of SPG, binds with SPG and is subsequently taken into the cells via phagocytosis. The expression of dectin-1 by CD11b+ macrophages significantly increased in DSS-treated mice. Flow cytometry revealed that the uptake of SPG-antisense TNF-α in the macrophages was efficient. TNF-α production was suppressed significantly by SPG-antisense TNF-α in vitro, which was administered via enema to evaluate its efficacy. The intrarectal administration of SPG-antisense TNF-α ameliorated the intestinal inflammation. In this study, we showed that the delivery system that conjugates SPG and antisense can have higher therapeutic efficacy. Thus, the new therapeutic approach presented in this study may be used in the management of IBD.

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.

Adjuvant Activity Enhanced by Cross-Linked CpG-Oligonucleotides in ß-Glucan Nanogel and Its Antitumor Effect.

Cancer vaccine has the ability to directly eradicate tumor cells by creating and activating cytotoxic T lymphocytes (CTLs). To achieve efficient CTL activity and to induce Th1 responses, it is essential to administer an appropriate adjuvant as well as an antigen. CpG-ODN is known as a ligand of Toll-like receptor 9 (TLR9) and strongly induces Th1 responses. In our previous study, we developed a CpG-ODN delivery system by use of the formation of complexes between ODN and a ß-glucan SPG, denoted as CpG/SPG, and demonstrated that CpG/SPG induces high Th1 responses. In this study, we created a nanogel made from CpG/SPG complexes through DNA-DNA hybridization (cross-linked (CL)-CpG). Immunization with CL-CpG induced much stronger antigen-specific Th1 responses in combination with the antigenic protein ovalbumin (OVA) than that with CpG/SPG. Mice preimmunized with CL-CpG and OVA exhibited a long delay in tumor growth and an improved survival rate after tumor inoculation. These immune inductions can be attributed to the improvement of cellular uptake by the combination of increased size and the cluster effect of the ß-glucan recognition site in the nanogel structure. In other words, the particle nature of CL-CpG, instead of the semiflexible rod conformation of CpG/SPG, enhanced the efficacy of a cancer vaccine. The present results indicate that CL-CpG can be used as a potent vaccine adjuvant for the treatment of cancers and infectious diseases.

Exploring the relationship between anti-PEG IgM behaviors and PEGylated nanoparticles and its significance for accelerated blood clearance.

Surface PEGylation on nanoparticles has greatly helped prolong their blood circulation half-lives. However, The injection of PEGylated nanoparticles into mice induced poly(ethylene glycol) (PEG)-specific IgM antibodies (anti-PEG IgMs), significantly changing PEG-liposomes' pharmacokinetics. In this study, we used various PEG-conjugates to conduct a mechanistic study of anti-PEG IgMs' binding behavior. The conventional belief has been that anti-PEG IgMs bind to PEG main chains; however, our findings reveal that anti-PEG IgMs did not bind to PEG main chains, whereas anti-PEG IgMs did bind to PEG-hydrophobic polymer blocks. The insertion of a hydrophilic polymer between each PEG chain and each hydrophobic polymer block suppressed anti-PEG IgMs' binding. We prove here that hydrophobic blocks are essential to anti-PEG IgMs' binding, and also that anti-PEG IgMs do not bind to intact PEGs without hydrophobic moiety. These results support our conclusion that anti-PEG IgMs exhibit specificity to PEG; however, the presence of a hydrophobic block at a proximity position from each PEG chain is essential for the binding. Also in the present study, we elucidate relations between anti-PEG IgMs and PEGylated nanoparticles. In one of our previous studies, anti-PEG IgMs scarcely affected the pharmacokinetics of PEG-b-poly(ß-benzyl l-aspartate) block copolymer (PEG-PBLA) micelles, whereas anti-PEG IgMs significantly decreased PEG-liposomes' blood circulation half-life. Finally, we found that the ratio of anti-PEG IgM molecules to PEG-liposome particles is critical to these pharmacokinetic changes, and that a 10-fold increase in the number of anti-PEG IgM molecules permitted them to capture the PEG-liposome particles, thus leading to the aforementioned changes.

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.

Gene silencing using a conjugate comprising Tat peptide and antisense oligonucleotide with phosphorothioate backbones.

Antisense oligonucleotides (ASOs) have a great therapeutic potential for the modulation of gene expression because of the high specificity. The major obstacles for clinical application are enzymatic degradation and low uptake into cells in vivo. In this study, we prepared the conjugate comprising Tat peptide and ASO with phosphorothioate linkages in a simple manner; azide alkyne Huisgen cycloaddition using a copper catalyst. The obtained conjugate showed a high stability in serum, compared with the conjugate with phosphodiester linkages. The conjugates with antisense for c-myb that is transcriptional factor concerning cell growth inhibited the cell proliferation in a dose dependent manner sequence-specifically. These findings suggest Tat-mediated ASOs delivery is useful for the treatment of various diseases.

Immunization with antigenic peptides complexed with ß-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that ß-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases.

Macrophage-Targeting Gene Delivery Using a Micelle Composed of Mannose-Modified Lipid with Triazole Ring and Dioleoyl Trimethylammonium Propane.

Gene carriers with cell specific ligand molecules are needed for the treatment of several diseases. Mannose is known to be recognized and incorporated into the cells through mannose recognition lectins that are exclusively expressed on macrophages. In this study, we synthesized two types of mannose-modified lipids with different stereoisomer (α-mannose and ß-mannose). To make a complex with plasmid DNA (pDNA), termed "lipoplex," we prepared a two-component micelle made from cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP); and mannose-modified lipid (D/α-Man or D/ß-Man). The prepared D/α-Man lipoplexes were able to bind to one of the α-mannose lectins concanavalin A (ConA) immobilized on gold substrate in the quartz-crystal microbalance sensor cell. D/ß-Man lipoplexes did not show any frequency changes. These results indicate that the mannose residues were exposed on the lipoplexes, leading to not only the binding to ConA but also the prevention of nonspecific interactions with proteins. Both lipoplexes showed high transfection efficiencies to RAW264.7 cells which have several kinds of mannose lectins. This delivery system to macrophages may overcome the problems for gene therapy and may be used for the treatment of immune diseases involved in macrophages.

Determination of polymeric micelles' structural characteristics, and effect of the characteristics on pharmacokinetic behaviors.

We evaluated structural factors characterizing PEG-b-P(Asp-Bzl) micelles including core size, aggregation number (Nagg), and core surface PEG density by means of small-angle X-ray scattering (SAXS), field flow fractionation with multi-angle light scattering (FFF-MALS) analysis, and DLS. Furthermore, we evaluated the stability of PEG-b-P(Asp-Bzl) micelles by means of GPC. This paper reports the correlation between the evaluated micelles' structural factors and the micelles' behaviors including the micelles' in vivo pharmacokinetic behaviors. One micelle PEG(12)-b-P(Asp-Bzl) (PEG=12,000) exhibited a high core surface density (~0.99 chain/nm(2)). In these circumstances, PEG(12)-b-P(Asp-Bzl) micelles exhibited a highly stretched PEG brush form. However, the evaluated core surface PEG densities could not fully explain the micelles' in vivo pharmacokinetic behaviors. In contrast, GPC will become a strong tool for predicting PEG(12)-b-P(Asp-Bzl) micelles' in vivo behaviors, as well as the micelles' in vitro behaviors. The stability results correlated strongly with the area-under-the-curve (AUC) values of PEG-b-P(Asp-Bzl) micelles' in vivo pharmacokinetics. Finally, we evaluated PEG(12)-b-P(Asp-Bzl) micelles' most effective structural factor for determining the micelles' behaviors, and the micelles' outermost shell surface's PEG density (DOS, PEG) correlated with the micelles' behaviors. We revealed that the evaluated DOS, PEG is the most important factor for understanding PEG(12)-b-P(Asp-Bzl) micelles' behaviors.

Hepatocyte-targeting gene delivery using a lipoplex composed of galactose-modified aromatic lipid synthesized with click chemistry.

Highly efficient drug carriers targeting hepatocyte is needed for treatment for liver diseases such as liver cirrhosis and virus infections. Galactose or N-acetylgalactosamine is known to be recognized and incorporated into the cells through asialoglycoprotein receptor (ASGPR) that is exclusively expressed on hepatocyte and hepatoma. In this study, we synthesized a galactose-modified lipid with aromatic ring with click chemistry. To make a complex with DNA, termed 'lipoplex', we prepared a binary micelle composed of cationic lipid; dioleoyltrimethylammoniumpropane (DOTAP) and galactose-modified lipid (D/Gal). We prepared lipoplex from plasmid DNA (pDNA) and D/Gal and examined the cell specificity and transfection efficiency. The lipoplex was able to interact with ASGPR immobilized on gold substrate in the quartz-crystal microbalance (QCM) sensor cell. The lipoplex induced high gene expression to HepG2 cells, a human hepatocellular carcinoma cell line, but not to A549 cells, a human alveolar adenocarcinoma cell line. The treatment with asialofetuin, which is a ligand for ASGPR and would work as a competitive inhibitor, before addition of the lipoplexes decreased the expression to HepG2 cells. These results indicate that D/Gal lipoplex was incorporated into HepG2 cells preferentially through ASGPR and the uptake was caused by galactose specific receptor. This delivery system to hepatocytes may overcome the problems for gene therapy and be used for treatment of hepatitis and hepatic cirrhosis.