LidNA, a miRNA inhibitor constructed with unmodified DNA, requires an xxxA insertion sequence in miRNA binding site for its potent inhibitory activity.

The involvement of miRNAs in the pathogenesis of various diseases, including cancer, poses the need for developing miRNA inhibitors. Previously, using unmodified DNA, we designed LidNA, which inhibited miRNA function more potently than 2'-O-methylated RNA and locked nucleic acid. LidNA consists of a complementary sequence to miRNA flanked by two structured DNAs. Alterations in the connected sequences between the complementary region and structured region modestly affect miRNA inhibition activity. Surprisingly, variations in the mismatched insertion sequence in the center of the complementary sequence significantly affect activity. The central insertion sequence xxxA is required for the potent miRNA inhibitory effects of LidNA. This suggests that both the structure and insertion sequence of LidNA and other miRNA inhibitors should be considered for maximal miRNA inhibitory activity.

Improved cancer inhibition by miR-143 with a longer passenger strand than natural miR-143.

We improved miR-143, which inhibits the growth of cancer cells, by the replacement of the passenger strand. As a result, new miR-143 variants were developed with a single mismatch at the 4th position from the 3'-terminal of the guide strand and an RNA passenger strand with a G-rich flanking DNA region. A reporter gene assay showed that the 80% inhibitory concentration of the new miR-143, long miR-143, was 69 pM, which was three times lower than that of natural miR-143. Long miR-143 inhibited the growth of two cancer cell lines, HeLa-S3 and MIAPaCa-2, more effectively than natural miR-143. This method could be applied to other miRNA families and should be useful for the development of miRNA drugs.

Chitin degradation enzyme-responsive system for controlled release of fibroblast growth factor-2.

Chitin is widely found in fungal cell walls and arthropod exoskeletons, and is used as a biomedical material. However, chitin is not water-soluble, restricting its use for controlled release materials. We found that water-soluble chitosan can be acetylated to produce a chitin equivalent, or chitin gel. Chitin gel, produced by mixing chitosan solution with acetic anhydride, can be degraded by lysozyme and fetal bovine serum, so could provide an ideal means for controlled release in biological systems. We tested a combination of chitin gel with a chitin binding domain (CBD) fusion protein as a controlled release system regulated by chitin degradation. A fusion protein consisting of fibroblast growth factor 2 (FGF-2) fused to CBD bound the chitin gel, and was released time-dependently rather than as an initial burst during lysozyme degradation, suggesting that this system could provide a means for controlled drug release in biological systems. Contrastingly, the trinitrophenyl residue (TNP-X) covalently bound to chitin gel, and was released by lysozyme degradation with an initial burst. If release of CBD-FGF-2 were simply dependent on lysozyme degradation of the chitin gel, the release behavior of CBD-FGF-2 would be similar to that of TNP-X, with an initial burst. Therefore, we propose that CBD-FGF-2 repeats the cycle of binding, release, and re-binding to the chitin gel during degradation. This system allows for a time-dependent, controlled release of CBD-FGF-2 without an initial burst.

Fluorescein isothiocyanate and blue light irradiation alter cell-adhesiveness of cross-linked albumin films for cell patterning.

Cross-linked albumin films, which are non-cell adhesive, were altered to be cell-adhesive by the combination of varied concentrations of fluorescein isothiocyanate and blue light irradiation. In this system, cell patterning was developed with two different cell lines by sequential seeding.Abbreviations: BSA: bovine serum albumin; EGDE: ethylene glycol diglycidyl ether; PBS: Dulbecco's phosphate buffered saline.

Changes in Cell Adhesiveness and Physicochemical Properties of Cross-Linked Albumin Films after Ultraviolet Irradiation.

We discovered the unique cell adhesive properties of ultraviolet (UV)-irradiated albumin films. Albumin films prepared using a cross-linking reagent with epoxy groups maintained native albumin properties, such as resistance to cell adhesion. Interestingly, the cell adhesive properties of films varied depending upon the UV irradiation time; specifically, cell adhesiveness increased until 2 h of UV irradiation, when the cell number attached to the film was similar to that of culture dishes, and then cell adhesiveness decreased until 20 h of UV irradiation, after which the surface returned to the initial non-adhesive state. To elucidate the molecular mechanisms underlying this phenomenon, we examined the effect of UV irradiation on albumin film properties. The following changes occurred in response to UV irradiation: decreased α-helical structure, cleavage of albumin peptide bonds, and increased hydrophilicity and oxygen content of the albumin film surface. In addition, we found a positive correlation between the degree of cell adhesion and the amount of fibronectin adsorbed on the film. Taken together, UV-induced changes in films highly affect the amount of cell adhesion proteins adsorbed on the films depending upon the irradiation time, which determines cell adhesion behavior.

Preparation of keratin and chemically modified keratin hydrogels and their evaluation as cell substrate with drug releasing ability.

Keratin was extracted as a reduced form from wool, which was then subjected to acetamidation, carboxymethylation or aminoethylation at abundant free cysteine residues to give acetamidated keratin (AAK), carboxymethylated keratin (CMK) and aminoethylated keratin (AEK). Hydrogels were prepared from intact and three chemically modified keratins simply by concentrating their aqueous solution and subsequent cooling. The lowest concentration to form a hydrogel without fluidity was 110 mg/ml for AAK, 120 mg/ml for AEK, 130 mg/ml for keratin and 180 mg/ml for CMK. Comparing with a hydrogel just prepared (swelling ratio: 600-700), each hydrogel slightly shrank in an acidic solution. While AAK hydrogel little swelled in neutral and basic solutions, other hydrogels became swollen and CMK hydrogel reached to dissolution. Hydrogels of keratin, AAK and AEK were found to support cell proliferation, although cell elongation on AAK and AEK hydrogel was a little suppressed. On the other hand, CMK hydrogel did not seem to be suitable for a cell substrate because of its high swelling in culture medium. Evaluation of the hydrogels as a drug carrier showed that keratin and AAK hydrogels were good sustained drug release carriers, which showed the drug release for more than three days, while the release from AEK and CMK hydrogels completed within one day. Thus, keratin and chemically modified keratin hydrogels, especially keratin and AAK hydrogels, were promising biomaterials as a cell substrate and a sustained drug release carrier.

Preparation of keratin hydrogel/hydroxyapatite composite and its evaluation as a controlled drug release carrier.

Infection after artificial joint replacement is a serious problem, which requires the re-implantation of prosthesis. To aim at developing bone filling materials having both osteoconductivity and ability as a sustained drug release carrier, composites of wool keratin or carboxymethylated (CM) keratin hydrogels with hydroxyapatite were prepared and evaluated as a sustained drug release carrier. CM-keratin was prepared by the reaction of keratin extracted from wool with iodoacetic acid. Hydrogels were obtained by dropping keratin or CM-keratin solution into CaCl2 solution. The composites were obtained by immersing hydrogels in simulated body fluid (SBF). The introduction of carboxymethyl groups to keratin facilitated the deposition of hydroxyapatite on hydrogel. After 7 days of immersion in SBF, a 4-5 times higher amount of hydroxyapatite was accumulated on CM-keratin hydrogel than that on keratin hydrogel. When salicylic acid was loaded on keratin and CM-keratin hydrogels, a good sustained release was observed; that is, 90% of a drug was released up to 14 days after 60 and 45% of the initial burst in 1 day. On the other hand, initial release within 1 day was suppressed by forming a composite with hydroxyapatite and the release was almost ceased at 3 days when 60% of the drug was released. Although further improvement to prolong drug release might be necessary, CaKHA and CaCMKHA are expected to be a promising novel type of bone filling materials which has both osteoconductivity and sustained drug release ability.

Binding affinity of ssDNA is improved by attachment of dsDNA regions.

LidNA, a microRNA inhibitor consisting of a microRNA binding ssDNA region sandwiched between dsDNA regions had higher affinity to target oligonucleotides than that without dsDNA region. This enhancement in affinity was found to be owing to the suppressed mobility of ssDNA region by the presence of dsDNA regions.

Long DNA passenger strand highly improves the activity of RNA/DNA hybrid siRNAs.

Small interfering RNAs (siRNAs) are potent tools in biomedical research, which can reduce the expression level of target proteins through RNAi pathway. They are composed of 19-25 bp double strand RNA (dsRNAs), therefore, stimulate dsRNAs dependent interferon responses in a non-specific manner. This problem has prevented siRNAs from being applied as new therapeautic agents. In the present paper, we tried to circumvent interferon responses using RNA/DNA hetero siRNAs (HsiRNAs) composed of RNA guide and DNA passenger strands. It was previously reported that siRNAs which were partially substituted with DNA had RNAi activity and that DNA substitution often caused the activity loss. In our results, HsiRNAs, in which the passenger strand of siRNAs were exchanged with DNA also showed much lower activity than that of parental siRNAs. Here, we found that attachment of 5' flanking sequence to DNA passenger strand improved the activity of HsiRNAs. Furthermore, the effective HsiRNAs induced much lower interferon responses than parental siRNAs. Thus, HsiRNAs with 5' flanking sequence are expected to be novel siRNA drug candidates.

LidNA, a novel miRNA inhibitor constructed with unmodified DNA.

Many miRNA inhibitors have been developed and they are chemically modified oligonucleotides such as 2?-O-methylated RNA and locked nucleic acid (LNA). Unmodified DNA was not yet reported as a miRNA inhibitor because of the low affinity of DNA/miRNA compared to mRNA/miRNA. We designed a structured unmodified DNA that significantly inhibits miRNA function. The clue structure for activity is the miRNA binding site between double stranded regions which is responsible for the miRNA inhibitory activity and tight binding to miRNA. We developed the miRNA inhibitor constructed with unmodified DNA, and named it LidNA, DNA that puts a lid on miRNA function.

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

Enhanced internalization and endosomal escape of dual-functionalized poly(ethyleneimine)s polyplex with diphtheria toxin T and R domains.

A new multifunctional gene delivery system was constructed with diphtheria toxin's functional domains. Used functional domains are T domain for endosomal escape and R domain for efficient internalization into cell. In order to conjugate these domains into PEI polyplex, diphtheria toxin T and R domains-streptavidin fusion protein (DTRS) was prepared. The conjugation of the DTRS with biotinylated PEI polyplex (DTRS-polyplex) lead to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex in CHO-K1 cell. It was demonstrated that DTRS-polyplex had high endosomal escape efficiency and internalization efficiency by several measurements, such as in vitro intracellular trafficking observation and the internalization inhibition with several inhibitors. These results suggest that this multifunctional non-viral vector may contribute to the future cancer gene therapy.

Drug-carrying albumin film for blood-contacting biomaterials.

Surface-induced thrombosis is a major complication in the development of blood-contacting medical devices. Serum albumin has the ability to bind to a wide variety of compounds, including drugs, and neither cells nor proteins adsorb to an albumin-coated surface. These properties of albumin are useful for improving the blood compatibility of biomaterial surfaces. In the present study, we prepared a water-insoluble film by cross-linking pharmaceutical grade recombinant human serum albumin aiming to the clinical applications, and loaded the film with a synthetic antiplatelet drug, cilostazol. The resultant film possessed native albumin characteristics such as drug binding ability and resistance to cell adhesion. Mouse fibroblast L929 cells did not adhere on the albumin film, just as they did not adhere on native albumin-coated surfaces. Furthermore, when the albumin film carrying cilostazol was placed in PBS containing Tween-80, the release of cilostazol was sustained over 144 h. The results indicate that the surface coating with thus prepared albumin film can confer the biomaterials with antithrombogenic surface by virtue of its non-adhesiveness to cells and its release of cilostazol.

Preparation of long sticky ends for universal ligation-independent cloning: sequential T4 DNA polymerase treatments.

Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.

Cell micropatterning on an albumin-based substrate using an inkjet printing technique.

Positioning of cells in a desired pattern on a substrate is an important technique for cell-based technologies, including the fundamental investigation of cell functions, tissue-engineering applications, and the fabrication of cell-based biosensors and cell arrays. Recently, the inkjet printing technique was recognized as a promising approach to the creation of cellular patterns on substrates, and it has been achieved by the printing of living cells or cell adhesive proteins. In this article, we created complex cellular patterns by using an albumin-based substrate and inkjet printing technique. Albumin was cross-linked using ethylene glycol diglycidyl ether. Subsequent casting of the cross-linked albumin solution onto glass plates prevented cells from adhering to their surfaces. Through screening various chemical reagents, we found that these cross-linked albumin surfaces dramatically changed into cell adhesive surfaces after immersion in cationic polymer solutions. Based on this finding, cell adhesive regions were prepared with a desired pattern by printing the polyethyleneimine (PEI) solution onto a cross-linked albumin substrate using a modified commercial inkjet printer. Various cellular patterns including figures, letters, and gradients could be fabricated by seeding mouse L929 fibroblast cells or mouse Neuro-2a neuroblastoma cells onto the printed PEI-patterned substrate. Compared with the printing of fragile living cells or proteins, printing of stable PEI circumvents clogging of printer head nozzles and enables reproducible printing. Therefore, the present method will allow the creation of complex cell patterns.

The conjugation of diphtheria toxin T domain to poly(ethylenimine) based vectors for enhanced endosomal escape during gene transfection.

The endosomal escape is a well-known serious obstacle for non-viral gene delivery. This is because of an acidic and enzymatic degradation of the contents of the endosome/lysosome. Therefore, the internalized gene needs to be efficient released into the cytosol to obtain the efficiently transfection efficiency. On the other hand, the diphtheria toxin T domain fuses with endosome membrane by pH decrease, then enhances the endosomal escape of the diphtheria toxin C fragment. In this study, we constructed diphtheria toxin T domain-conjugated poly(ethylenimine)s (PEI) polyplex for enhancing the endosomal escape of exogenous gene. The conjugation of diphtheria toxin T domain with PEI/pDNA polyplex leads to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex. The pH-responsive increase in hydrophobicity of the diphtheria toxin T domain might not only trigger the perturbation of the endocytic vesicle membrane but might also increase the membrane permeability.

Facile cell patterning on an albumin-coated surface.

Fabrication of micropatterned surfaces to organize and control cell adhesion and proliferation is an indispensable technique for cell-based technologies. Although several successful strategies for creating cellular micropatterns on substrates have been demonstrated, a complex multistep process and requirements for special and expensive equipment or materials limit their prevalence as a general experimental tool. To circumvent these problems, we describe here a novel facile fabrication method for a micropatterned surface for cell patterning by utilizing the UV-induced conversion of the cell adhesive property of albumin, which is the most abundant protein in blood plasma. An albumin-coated surface was prepared by cross-linking albumin with ethylene glycol diglycidyl ether and subsequent casting of the cross-linked albumin solution on the cell culture dish. While cells did not attach to the albumin surface prepared in this way, UV exposure renders the surface cell-adhesive. Thus, surface micropatterning was achieved simply by exposing the albumin-coated surface to UV light through a mask with the desired pattern. Mouse fibroblast L929 cells were inoculated on the patterned albumin substrates, and cells attached and spread in a highly selective manner according to the UV-irradiated pattern. Although detailed investigation of the molecular-level mechanism concerning the change in cell adhesiveness of the albumin-coated surface is required, the present results would give a novel facile method for the fabrication of cell micropatterned surfaces.

Preparation of water-insoluble albumin film possessing nonadherent surface for cells and ligand binding ability.

Serum albumin is the most abundant protein in blood plasma. Albumin has the ability to bind to a wide variety of compounds including drugs, and cells as well as proteins do not attach to an albumin-coated surface. These properties of albumin are attractive for biomaterials utilized in biomedical fields. In the present study, we aimed to prepare a water-insoluble albumin film possessing suitable flexibility and native albumin characteristics, such as drug binding ability and resistance to cell adhesion. To confer the film with both water insolubility and flexibility without losing albumin characteristics, we searched a suitable condition for the cross-linking of albumin. As a result, we found that a film having aimed properties could be obtained by conducting the cross-linking reaction at room temperature for 24 h using 215 mM ethylene glycol diglycidyl ether. Mouse fibroblast L929 cells did not adhere on thus obtained film in a similar manner to a native albumin-coated surface. In addition, the film could bind 2-(4'-hydroxyphenylazo)-benzoic acid, a representative albumin binding dye, and gradually release it in a pH-dependent manner.

Dual-ligand effect of transferrin and transforming growth factor alpha on polyethyleneimine-mediated gene delivery.

In order to enhance the internalization of exogenous gene and add cell specificity to non-viral vectors, receptor-binding elements have been widely utilized to mimic the virus infection. Herein, for the purpose of intensifying the effects of the ligand on gene delivery, dual receptor-binding elements, transferrin (Tf) and transforming growth factor alpha (TGFalpha), were introduced into the polyethyleneimine polyplex. The transfection and internalization efficiency by dual Tf- and TGFalpha-introduced polyplex (Tf&TGFalpha-polyplex) was examined in A549 and CHO-K1 cells, respectively. In A549, Tf&TGFalpha-polyplex had higher transfection efficiency when compared to that by single Tf- or TGFalpha-introduced polyplex (Tf-polyplex and TGFalpha-polyplex), respectively, while no enhancement was observed in CHO-K1. Moreover, in A549, the internalization efficiency of dual Tf&TGFalpha-polyplex was higher than that of single Tf- and TGFalpha-polyplex. In contrast, in CHO-K1, no difference in internalization efficiency was observed. In the presence of excess free transferrin or TGFalpha, the internalization efficiency of Tf&TGFalpha-polyplex was strongly inhibited only in A549, not in CHO-K1. In summary, the enhancement of internalization efficiency by dual ligands is an important factor for improving transfection efficiency. In addition, the effect of dual ligands depends on cell species; receptor-mediated and efficient internalization may be related to this enhanced transfection efficiency.