Scutellaria Root extract-induced hepatocytotoxicity can be controlled by regulating its baicalin content.

Scuellaria Root (SR, root of Scutellaria baicalensis), which has potent anti-inflammatory effects, is a component of useful Kampo formulae. Albeit a low frequency, SR induces serious interstitial pneumonia and liver dysfunction. In this study, to control the adverse effects of SR, we investigated the causal constituent responsible for its hepatocytotoxicity and aimed to develop a method to control it. As a result, we revealed that the hepatocytotoxicity of SR was correlated with its baicalin content, a major constituent in SR. It was confirmed by preparing a baicalin-free SR extract, which exhibited reduced hepatocytotoxicity. The addition of baicalin to the baicalin-free SR extract restored the hepatocytotoxicity, indicating that the hepatocytotoxicity of SR is dependent on its baicalin content. Thus, SR extract-induced hepatocytotoxicity can be controlled by regulating its baicalin content.

Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Development of rice bran-derived nanoparticles with excellent anti-cancer activity and their application for peritoneal dissemination.

BACKGROUND: Rice bran a by-product of the rice milling process is currently underutilized. Recent studies have shown that plant-derived nanoparticles (pdNPs) can be mass-produced at a low cost and exhibit biological and therapeutic activities. Rice bran contains various anti-cancer compounds, including γ-oryzanol and γ-tocotrienol, and rice bran-derived nanoparticles (rbNPs) can be employed as novel therapeutic agents for cancer treatment. RESULTS: Koshihikari rice bran was suspended in water, and the suspension was centrifuged and filtered through a 0.45-µm-pore size syringe filter. The filtrate was ultracentrifuged, and the precipitates were suspended to obtain rbNPs. The rbNPs were negatively charged exosome-like nanoparticles with an average diameter of approximately 130 nm. The rbNPs exhibited cytotoxic activities against cancer cells but not against normal cells. The cytotoxic activity of rbNPs to murine colon adenocarcinoma colon26 cells was significantly greater than DOXIL® or other pdNPs. The rbNPs induced cell cycle arrest and apoptosis, and reduced the expression of proliferative proteins, including ß-catenin and cyclin D1. Intraperitoneal injections of rbNPs into mice bearing peritoneal dissemination of colon26 cells significantly suppressed tumor growth with no significant adverse effects. CONCLUSION: These results indicated that rbNPs are promising nanoparticles, hold significant potential for anti-cancer applications, and are expected to play a vital role in cancer treatment.

Hoechst-Modification on Oligodeoxynucleotides for Efficient Transport to the Cell Nucleus and Gene Regulation.

Various artificial oligodeoxynucleotides (ODNs) that contribute to gene regulation have been developed and their diversity and multifunctionality have been demonstrated. However, few artificial ODNs are actively transported to the cell nucleus, despite the fact that gene regulation also takes place in both the cell nucleus and the cytoplasm. In this study, to prepare ODNs with the ability to accumulate in the cell nucleus, we introduced Hoechst molecules into ODNs that act as carriers of functional molecules to the cell nucleus (Hoe-ODNs). We synthesized Hoe-ODNs and confirmed that they bound strongly to DNA duplexes. When single-stranded Hoe-ODNs or double-stranded ODNs consisting of Hoe-ODNs and its complementary strand were administered into living cells, both ODNs were efficiently accumulated in the cell nucleus. In addition, antisense ODNs, which were tethered with Hoechst unit, were delivered into the cell nucleus and efficiently suppressed the expression of their target RNA. Thus, we constructed a delivery system that enables the transport of ODNs into cell nucleus.

Efficient delivery of mesenchymal stem/stromal cells to injured liver by surface PEGylation.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) have been used in clinical trials for various diseases. These have certain notable functions such as homing to inflammation sites, tissue repair, and immune regulation. In many pre-clinical studies, MSCs administered into peripheral veins demonstrated effective therapeutic outcomes. However, most of the intravenously administered MSCs were entrapped in the lung, and homing to target sites was less than 1%. This occurred mainly because of the adhesion of MSCs to vascular endothelial cells in the lung. To prevent this adhesion, we modified the surface of MSCs with polyethylene glycol (PEG; a biocompatible polymer) using the avidin-biotin complex (ABC) method. METHODS: The surface of MSCs was modified with PEG using the ABC method. Then, the cell adhesion to mouse aortic endothelial cells and the tissue distribution of PEG-modified MSCs were evaluated. Moreover, the homing to the injured liver and therapeutic effect of PEG-modified MSCs were evaluated using carbon tetrachloride-induced acute liver failure model mice. RESULTS: The PEG modification significantly suppressed the adhesion of MSCs to cultured mouse aortic endothelial cells as well as the entrapment of MSCs in the lungs after intravenous injection in mice. PEG-modified MSCs efficiently homed to the injured liver of carbon tetrachloride-induced acute liver failure model mice. More importantly, the cells significantly suppressed serum transaminase levels and leukocyte infiltration into the injured liver. CONCLUSION: These results indicate that PEG modification to the surface of MSCs can suppress the lung entrapment of intravenously administered MSCs and improve their homing to the injured liver.

Drug Metabolism and Pharmacokinetics of Antisense Oligonucleotide Therapeutics: Typical Profiles, Evaluation Approaches, and Points to Consider Compared with Small Molecule Drugs.

Oligonucleotide therapeutics are attracting attention as a new treatment modality for a range of diseases that have been difficult to target using conventional approaches. Technical advances in chemical modification and drug delivery systems have led to the generation of compounds with excellent profiles as pharmaceuticals, and 16 oligonucleotide therapeutics have been marketed to date. There is a growing need to develop optimal and efficient approaches to evaluate drug metabolism and pharmacokinetics (DMPK) and drug-drug interactions (DDIs) of oligonucleotide therapeutics. The DMPK/DDI profiles of small molecule drugs are highly diverse depending on their structural and physicochemical characteristics, whereas oligonucleotide therapeutics share similar DMPK profiles within each chemistry type. Most importantly, the mechanisms and molecules involved in the distribution and metabolism of oligonucleotides differ from those of small molecules. In addition, there are considerations regarding experimental approaches in the evaluation of oligonucleotides, such as bioanalytical challenges, the use of radiolabeled tracers, materials for in vitro metabolism/DDI studies, and methods to study biodistribution. In this review, we attempt to summarize the DMPK characteristics of antisense oligonucleotide (ASO) therapeutics and discuss some of the issues regarding how to optimize the evaluation and prediction of the DMPK and DDI of ASOs.

Intradermal delivery of Cryj1 loaded in CpG DNA hydrogel for inhibiting allergic reactions in mice.

Immunotherapy for allergic rhinitis alleviates symptoms associated with antigen exposure by administering pathogenic antigens. However, many current immunotherapies fail to induce sufficient immune responses, resulting in frequent and prolonged hospital visits. Consequently, the development of more effective immunotherapies is necessary. In this study, we focused on the skin, which is rich in immune cells, as an administration site for inducing antigen-specific immune responses. To efficiently and sustainably deliver the cedar pollen antigen Cryj1 to immune cells, we attempted to load Cryj1 in an immunostimulatory CpG DNA hydrogel, prepared using self-gelatinizable nucleic acid technology. In this technology, the hydrogel became gelatinized by self-assembly of multiple predesigned DNA units containing potent CpG motifs. Cryj1 loaded in the CpG DNA hydrogel showed sustained release, was taken up by mouse macrophage-like RAW264.7 and mouse dendritic DC2.4 cells, and induced efficient production of interleukin-12 after intradermal injection into mice. Intradermal injection of Cryj1 loaded CpG DNA hydrogel into mice increased the production of Cryj1-specific IgG while suppressing the production of immunoglobulin E (IgE) antibodies. Furthermore, when Cryj1 was resensitized to mice, a stronger induction of IgG production and suppression of IgE production was observed. These results suggest that intradermal administration of Cryj1 loaded CpG DNA hydrogel is a novel immunotherapy for allergic symptoms caused by cedar pollen and can be used as a replacement for current immunotherapies.

Quantitative Model Analysis and Simulation of Pharmacokinetics and Metastasis-Associated Lung Adenocarcinoma 1 RNA Knockdown Effect After Systemic Administration of Cholesterol-Conjugated DNA/RNA Heteroduplex Oligonucleotide Crossing Blood-Brain Barrier of Mice.

The cholesterol-conjugated heteroduplex oligonucleotide (Chol-HDO) is a double-stranded complex; it comprises an antisense oligonucleotide (ASO) and its complementary strand with a cholesterol ligand. Chol-HDO is a powerful tool for achieving target RNA knockdown in the brains of mice after systemic injection. Here, a quantitative model analysis was conducted to characterize the relationship between the pharmacokinetics (PK) and pharmacodynamics (PD), non-coding RNA metastasis-associated lung adenocarcinoma 1 (Malat1) RNA, of Chol-HDO, in a time-dependent manner. The established PK model could describe regional differences in the observed brain concentration-time profiles. Incorporating the PD model enabled the unique knockdown profiles in the brain to be explained in terms of the time delay after single dosing and enhancement following repeated dosing. Moreover, sensitivity analysis of PK exposure/persistency, target RNA turnover, and knockdown potency identified key factors for the efficient and sustained target RNA knockdown in the brain. The simulation of an adequate dosing regimen quantitatively supported the benefit of Chol-HDO in terms of achieving a suitable dosing interval. This was achieved via sufficient and sustained brain exposure and subsequent strong and sustained target RNA knockdown in the brain, even after systemic injection. The present study provides new insights into drug discoveries and development strategies for HDO in patients with neurogenic disorders. SIGNIFICANCE STATEMENT: The quantitative model analysis presented here characterized the PK/PD relationship of Chol-HDO, enabled its simulation under various conditions or assumptions, and identified key factors for efficient and sustained RNA knockdown, such as PK exposure and persistency. Chol-HDO appears to be an efficient drug delivery system for the systemic administration of desired drugs to brain targets.

Delivery of Corn-Derived Nanoparticles with Anticancer Activity to Tumor Tissues by Modification with Polyethylene Glycol for Cancer Therapy.

PURPOSE: We recently reported that intratumoral injection of corn-derived nanoparticles (cNPs) affords anticancer activity in tumor-bearing mice. To increase their applicability in cancer therapy, we examined the tissue distribution of cNPs after intravenous injection in mice, modified their surface with polyethylene glycol (PEG) to improve tumor delivery, and examined tissue distribution and anticancer activity of PEG-cNPs in tumor-bearing mice. METHODS: N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) was added to cNPs by sonication to obtain PEG-cNPs, and the ratio of DSPE-PEG to cNPs was optimized by evaluating the modification efficiency. cNPs and PEG-cNPs were labeled with fluorescent dyes DiO or DiR, and their tissue distribution was subsequently examined after intravenous administration to mice. Finally, we determined the anticancer activity and toxicity of PEG-cNPs. RESULTS: No detectable fluorescence intensity was observed in mouse serum after intravenous DiR-cNP injection. DSPE-PEG was successfully modified into cNPs, and a PEG:cNPs ratio of 50 was determined as optimal for preparing PEG-cNPs, based on their size and zeta potential. DiO-PEG-cNPs exhibited significantly higher serum concentrations and lower liver accumulation than DiO-cNPs. Moreover, DiR-PEG-cNPs accumulated in tumor tissues of colon26 tumor-bearing mice. Repeated intravenous PEG-cNP injections significantly retarded tumor growth, with no significant hepatotoxicity or nephrotoxicity. CONCLUSION: Overall, these results indicate that controlling the tissue distribution of cNPs via PEG modification on their surface can be a valuable strategy for developing intravenously injectable cNPs for cancer therapy.

Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device.

Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.

Targeted Delivery of Immunostimulatory CpG Oligodeoxynucleotides to Antigen-Presenting Cells in Draining Lymph Nodes by Stearic Acid Modification and Nanostructurization.

Polypod-like structured nucleic acids (polypodnas), which are nanostructured DNAs, are useful for delivering cytosine-phosphate guanine oligodeoxynucleotides (CpG ODNs) to antigen-presenting cells (APCs) expressing Toll-like receptor 9 (TLR9) for immune stimulation. Lipid modification is another approach to deliver ODNs to lymph nodes, where TLR9-positive APCs are abundant, by binding to serum albumin. The combination of these two methods can be useful for delivering CpG ODNs to lymph nodes in vivo. In the present study, CpG1668, a phosphodiester-type CpG ODN, was modified with stearic acid (SA) to obtain SA-CpG1668. Tripodna, a polypodna with three pods, was selected as the nanostructured DNA. Tripodnas loaded with CpG1668 or SA-CpG1668 were obtained in high yields. SA-CpG1668/tripodna bound more efficiently to plasma proteins than CpG1668/tripodna and was more efficiently taken up by macrophage-like RAW264.7 cells than CpG1668/tripodna, whereas the levels of tumor necrosis factor-α released from the cells were comparable between the two. After subcutaneous injection into mice, SA-CpG1668/tripodna induced significantly higher interleukin (IL)-12 p40 production in the draining lymph nodes than SA-CpG1668 or CpG1668/tripodna, with reduced IL-6 levels in plasma. These results indicate that the combination of SA modification and nanostructurization is a useful approach for the targeted delivery of CpG ODNs to lymph nodes.

Intravenous injection of mesenchymal stem cell spheroids improves the pulmonary delivery and prolongs in vivo survival.

BACKGROUND: Because of the excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. METHODS AND RESULTS: The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 µm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. CONCLUSIONS: These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

Development of nanoparticles derived from corn as mass producible bionanoparticles with anticancer activity.

Recent studies showed that plant-derived nanoparticles (NPs) can be easily produced in high yields and have potential applications as therapeutic agents or delivery carriers for bioactive molecules. In this study, we selected corn as it is inexpensive to grow and mass-produced globally. Super sweet corn was homogenized in water to obtain corn juice, which was then centrifuged, filtered through a 0.45-µm-pore size syringe filter, and ultracentrifuged to obtain NPs derived from corn, or corn-derived NPs (cNPs). cNPs obtained were approximately 80 nm in diameter and negatively charged (- 17 mV). cNPs were taken up by various types of cells, including colon26 tumor cells and RAW264.7 macrophage-like cells, with selective reduction of the proliferation of colon26 cells. Moreover, cNPs induced tumor necrosis factor-α release from RAW264.7 cells. cNPs and RAW264.7 in combination significantly suppressed the proliferation of colon26/fluc cells. Daily intratumoral injections of cNPs significantly suppressed the growth of subcutaneous colon26 tumors in mice, with no significant body weight loss. These results indicate excellent anti-tumor activity of cNPs.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

Mesenchymal stem/stromal cells as next-generation drug delivery vehicles for cancer therapeutics.

INTRODUCTION: Drug delivery to solid tumors remains a significant therapeutic challenge. Mesenchymal stem/stromal cells (MSCs) home to tumor tissues and can be employed as tumor targeted drug/gene delivery vehicles. Reportedly, therapeutic gene- or anti-cancer drug-loaded MSCs have shown remarkable anti-tumor effects in preclinical studies, and some clinical trials for assessing therapeutic MSCs in patients with cancer have been registered. AREAS COVERED: In the present review, we first discuss the source and interdonor heterogeneity of MSCs, their tumor-homing mechanism, and the route of MSC administration in MSC-based cancer therapy. We then summarize the therapeutic applications of MSCs as a drug delivery vehicle for therapeutic genes or anti-cancer drugs and the drug delivery mechanism from drug-loaded MSCs to cancer cells. EXPERT OPINION: Although numerous preclinical studies have revealed significant anti-tumor effects, several clinical trials assessing MSC-based cancer gene therapy have failed to demonstrate corroborative results, documenting limited therapeutic effects. Notably, a successful clinical outcome with MSC-based cancer therapy would require the interdonor heterogeneity of administered MSCs to be resolved, along with improved tumor-homing efficiency and optimized drug delivery efficiency from MSCs to cancer cells.

Critical contribution of macrophage scavenger receptor 1 to the uptake of nanostructured DNA by immune cells.

Despite the efficient uptake of polypod-like nanostructured DNA, or polypodna, by macrophage-like RAW264.7 and other immune cells, the detailed mechanism has not been fully elucidated. Our previous study using HEK-Blue hTLR9 cells showed that transfection of macrophage scavenger receptor 1 (MSR1) increased the uptake of tetrapod-like structured DNA. Here, we investigated the involvement of MSR1 in the structure-dependent uptake of polypodna. Transfection of MSR1 to HEK-Blue hTLR9 cells pod number-dependently increased the uptake of polypodna, and its knockout in RAW264.7 cells reduced the uptake and subsequent cytokine release. To examine the binding of DNA with MSR1, biotinylated DNA added to RAW264.7 cells was cross-linked with cell surface proteins. Then, MSR1 cross-linked with polypodna, but not with single-stranded DNA. Similar results were obtained with murine primary immune cells. Taken together, MSR1 discriminates between simple and nanostructured DNAs and plays a dominant role in the efficient uptake of polypodna by immune cells.

Combined use of chemically modified nucleobases and nanostructured DNA for enhanced immunostimulatory activity of CpG oligodeoxynucleotide.

Oligodeoxynucleotide (ODN) containing a cytosine-phosphate-guanine (CpG) motif, or CpG ODN, is considered suitable for treating immune diseases, including allergies. Although the phosphorothioate modification is used to enhance the stability and immunostimulatory activity of CpG ODNs, it is associated with the risk of adverse effects. Construction of nanostructured DNA assemblies, such as tripod- and hexapod-like structured DNAs, tripodna and hexapodna, respectively, were also found to increase this activity. The chemical modification of nucleobases could be another approach for enhancing CpG ODN activity. Here, we examined whether chemically modified nucleobase substitutions can enhance CpG ODN activity by measuring tumor necrosis factor α (TNF-α) release after addition to murine macrophage-like RAW264.7 cells. First, the guanine at the 18th position of phosphodiester CpG 1668 was substituted with several chemically modified guanines, and then the various guanines were substituted. Among all tested substitutions, 15,18-thdG, in which two guanines outside the CpG motif were substituted with the 2-aminothieno[3,4-d]pyrimidine guanine mimic (thdG), was the most effective. Compared to 32P-CpG 1668, 32P-15,18-thdG was taken up more efficiently by the RAW264.7 cells. Then, 15,18-thdG was incorporated into tripodna and hexapodna. 15,18-thdG/tri- or hexapodna induced higher TNF-α release from the RAW264.7 cells than PO CpG 1668/tri- or hexapodna, respectively. These results indicate that the thdG substitution is a useful effective strategy for enhancing the immunostimulatory activity of CpG DNAs in both single stranded and DNA nanostructure forms.

Polypod-like structured guanine-rich oligonucleotide aptamer as a selective and cytotoxic nanostructured DNA to cancer cells.

Guanine-rich oligonucleotide (GRO) can be developed as an effective anticancer agent owing to its high selectivity, affinity and antiproliferative activity in cancer cells. In this study, to increase the potency of GRO29A, a 29-mer GRO aptamer against nucleolin, an overexpressed protein in cancer cells, GRO29A was incorporated into three or six pods of polypod-like structured DNA (polypodna), tripodna or hexapodna, respectively. The polypod-like structured GROs, tri-G3, consisting of one tripodna and three GRO29A, or hexa-G1, hexa-G3 or hexa-G6, each of which comprises one hexapodna and one, three or six GRO29A, respectively, were designed. Tri-G3, hexa-G1 and hexa-G3 were prepared in high yield, except for hexa-G6. Polypod-like structured GROs had quadruplex structures under physiological salt conditions, and degraded at a slower rate in buffer containing serum. Cellular interaction experiments using fluorescently labelled DNA samples showed that the uptake of hexa-G3 by nucleolin-positive MCF-7 cells was more than 2-fold higher than GRO29A, and the interaction was increasingly dependent on the number of GRO29A in the structures. Hexa-G3 inhibited the proliferation of MCF-7 cells in more than 40%, but not of CHO cells. These results indicate that polypod-like structured GROs are useful DNA aptamers with high selectivity and cytotoxicity against nucleolin-positive cancer cells.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy.

Mesenchymal stem cells (MSCs) have a tumor-homing ability-they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

Enhanced Immunostimulatory Activity of CpG Oligodeoxynucleotide by the Combination of Mannose Modification and Incorporation into Nanostructured DNA.

The immunostimulatory activity of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) could be improved via delivery to immune cells expressing Toll-like receptor 9 (TLR9). Previously, we showed that the polypod-like structured nucleic acid (polypodna), a nanostructured DNA comprised of three or more ODNs, was an efficient system for the delivery of CpG ODNs to immune cells. Because some TLR9-positive immune cells express mannose receptors (MR), the uptake of polypodna by immune cells can be further increased by its modification with mannose. In this study, we selected the phosphodiester CpG ODN, ODN1668, which has a sequence identical to CpG1668, and a hexapodna, a polypodna with six pods, to design a hexapodna that harbored ODN1668 or the mannosylated CpG ODN (Man-ODN1668) synthesized via modification of the 5'-terminal of ODN1668 with a synthesized mannose motif. By mixing ODN1668 or Man-ODN1668 with the hexapodna, ODN1668/hexapodna and Man-ODN1668/hexapodna were successfully formed with high yields. However, Man-ODN1668/hexapodna was found to induce a greater tumor necrosis factor-α release from TLR9- and MR-positive mouse peritoneal macrophages and macrophage-like J774.1 cells than Man-ODN1668 or ODN1668/hexapodna. These results indicate that the combination of mannose modification and incorporation into nanostructured DNA is a useful approach for enhancing the immunostimulatory activity of CpG ODN.