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.

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.

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.

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.

Modified keratin sponge: binding of bone morphogenetic protein-2 and osteoblast differentiation.

A keratin sponge was chemically modified to obtain carboxyl and amino sponges by the alkylation of a large amount of active SH group on keratin proteins with iodoacetic acid and 2-bromoethylamine, respectively. The carboxyl sponge having a large amount of carboxyl group was a scaffold that could bind significant amounts of basic bioactive proteins, such as lysozyme and bone morphogenetic protein (BMP)-2, and drugs. Lysozyme (maximum 3.7 mg), a model of basic cytokines such as BMP-2, was absorbed by the carboxyl sponge (4.8 mg), but not by the amino sponge. The lysozyme was rapidly released from the carboxyl sponge in a buffer containing at a concentration higher than 0.5 M, but at 0.15 M, near the physiological ionic strength, after initial burst (only 11%), no significant release was observed (15%, 48 h). BMP-2 also bound the carboxyl sponge. The preosteoblast cells grown inside the BMP-2-loaded sponge differentiated, whereas those grown outside the sponge did not, suggesting that no significant amount of BMP-2 leaked into the surrounding media. The effects of BMP-2 were confined inside modified keratin sponge. Therefore, using in vivo, we expect that only internal osteogenesis will be induced and that no external heterotopis ossification will be induced.

Rapid fabrication of keratin-hydroxyapatite hybrid sponges toward osteoblast cultivation and differentiation.

Wool keratin sponges were reported to be useful scaffolds for long-term and high-density cell cultivation (J. Biotechnol. 93 (2002) 165). The hybrid of the keratin sponges with calcium phosphate materials gave the additional function. Two rapid fabrication methods for calcium phosphate hybrid biomaterials were described. Firstly, the CaP-precipitated sponges were obtained by only the immersion of the carboxyl-sponges, chemically introduced with high amount of carboxyl groups on the sponges, in calcium and phosphate ions containing buffers such as PBS(+) for only 1-3 days. Neither sponge, introduced with amino or amido groups or non-treated, gave significant calcium phosphate precipitation. The carboxyl-sponges were mimics of matrix gamma-carboxyglutamic acid protein, which is responsible for osteoblast calcification. Secondly, the hydroxyapatite particle suspension was added onto carboxyl-sponges to fabricate trapped sponge. The trapped hydroxyapatite particles might interact with keratin protein of the sponge walls. Preliminary experiments measuring the expression of alkaline phosphatase, early osteoblast differentiation marker, suggested that both hybrid sponges, CaP-precipitated and trapped sponges, alter the differentiation pattern of preosteoblasts, MC3T3-E1.

Drug release from hydrogel containing albumin as crosslinker.

Albumin is the major plasma protein and acts as a physiological carrier for various compounds including drugs. To take advantage of the drug-binding ability of albumin for a drug delivery system, we have prepared hydrogels consisting of acrylamide (AAm) and bovine serum albumin (BSA) by introducing three to four vinyl groups into one BSA molecule and subsequently copolymerizing it with AAm. The resultant hydrogel was solubilized by trypsin treatment, since BSA served as a crosslinker in the hydrogel. The BSA-crosslinked hydrogel (BSA-AAm hydrogel) was loaded with salicylic acid or sodium benzoate and their release was investigated. The BSA-AAm hydrogel released much more salicylic acid than sodium benzoate. In addition, the amount of released salicylic acid increased with the BSA content of the hydrogel, despite a decrease in the swelling ratio of the hydrogel. On the other hand, the amount of released sodium benzoate increased with the swelling ratio. When a hydrogel crosslinked with N,N'-methylenebis (acrylamide) was used as a control, both drugs showed release tendencies similar to that of sodium benzoate from the BSA-AAm hydrogel. Furthermore, the salicylic acid release was sustained longer on the BSA-AAm hydrogel than the sodium benzoate release. Taken together, it is thought that albumin in the BSA-AAm hydrogel preferentially adsorbs salicylic acid and contributes to the high drug loading and the sustained release of salicylic acid.

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.

Novel approach to fabricate keratin sponge scaffolds with controlled pore size and porosity.

A compression-molding/particulate-leaching (CM/PL) method was developed to fabricate S-sulfo keratin sponges with the controlled pore size and porosity. The S-sulfo keratin was extracted from wool and was then spray-dried to give S-sulfo keratin powder. The S-sulfo keratin powder mixed with urea in advance was compression-molded together with the sieved NaCl particulates above the melting temperature of urea. The following removal of the salts and urea in water created the sponges composed of interconnected pores and the continuous S-sulfo keratin matrix. The S-sulfo keratin sponges were strong enough to handle and water-insoluble. By contrast, the sponges prepared without urea were very fragile and readily collapsed, because most of S-sulfo keratin matrix remained powdery. The pore size was in good accordance with the size of the salts, indicating that the pores were formed by leaching-out the salts. The S-sulfo keratin sponges with the regulated sizes of pores (<100, 100-300 and 300-500 microm) were fabricated, all of which had more than 90% of the porosity. Thus, CM/PL method is able to give the S-sulfo keratin sponge with the desired pore size and porosity, which might be a good scaffold for the cells in tissue engineering.

Preparation and physicochemical properties of compression-molded keratin films.

The S-sulfo keratin was extracted from wool and was then spray-dried to give S-sulfo keratin powder. Differential scanning calorimetry analysis showed that the glass transition temperature of S-sulfo keratins became lowered with the increase of moisture content, while perfectly dried S-sulfo keratin powder did not give thermal transition in the temperature range 30-130 degrees C. The compression molding of the S-sulfo keratin powder supplemented with one-tenth weight of water afforded a plastic-like transparent proteinous film above the glass transition temperature. The film obtained from the powder without water addition or compression molded below glass transition temperature partly remained powdery. The film compression molded at 120 degrees C gave the maximum ultimate strength and Young's modulus, 27.8 +/- 2.9 and 1218 +/- 80 MPa, respectively. Obtained film was insoluble and slightly swelled in water, but, in the presence of reducing agent, the film significantly swelled at pH 7.0 and even dissolved at pH 9.0, suggesting the relevance of abundant disulfide linkage. The film supported the mammalian cell adhesion and proliferation, demonstrating the biocompatibility of S-sulfo keratin films.

Preparation and characterization of keratin-chitosan composite film.

Keratin-chitosan composite film was prepared by casting the mixed solution of both biopolymers in 75% acetic acid. Although keratin film without any additive is very fragile, 10-30 wt% of chitosan addition gave strong and flexible film (ultimate strength: 27-34 MPa, ultimate elongation: 4-9%). Glycerol (20 wt%) also afforded flexibility to keratin film (ultimate strength: 1 MPa, ultimate elongation: 28%). Further addition of chitosan to glycerol-containing keratin film increased the ultimate strength to 9-14 MPa but gave little effect on ultimate elongation. These data suggest that mechanical properties of keratin film are adjustable by appropriately adding chitosan and glycerol. Waterproof characteristics such as swelling behavior and mechanical properties after swelling were much ameliorated for the composite film compared with keratin and chitosan films, respectively. Furthermore, keratin-chitosan composite film as well as chitosan film decreased bacteria number when the bacteria suspension was treated with a film owing to the irreversible adsorption of bacteria onto the film. The composite film as well as keratin and chitosan films supported fibroblast attachment and proliferation, demonstrating to be a good substrate for mammalian cell culture.

Preparation of collagen/calcium phosphate multilayer sheet using enzymatic mineralization.

The multilayer sheets (2-10 layers), which consisted of alternately cumulated collagen and calcium phosphate layers with the thickness of 6-8microm in each layer, were prepared. The inorganic layer was mineralized by means of an alkaline phosphatase-catalyzed hydrolysis of water-soluble phosphate esters in the presence of calcium ions. The calcium phosphate, which was formed on the collagen, was assayed as a mixture of hydroxyapatite (main) and amorphous calcium phosphate. The multilayer sheets were not only strong mechanically but also semitransparent and flexible in a dry state. Furthermore, the collagen/calcium phosphate multilayer sheets did not swell in water to keep the original morphology. As a scaffold, the sheets having the calcium phosphate layer on the top supported the attachment and growth of L929 fibroblast cells. The enzymatic mineralization and the collagen/calcium phosphate composite sheets were discussed in conjunction with physicochemical and biological properties.

Fabrication of wool keratin sponge scaffolds for long-term cell cultivation.

Wool keratin sponge scaffolds were fabricated by lyophilization of an aqueous wool keratin solution after controlled freezing. Freezing at -20 degrees C for 3 days was needed for the preparation of stable sponges, which did not show significant changes against heat treatment at 60 degrees C for several hours. Scanning electron microscopic observation revealed that the wool keratin sponges had a homogeneously porous microstructure, the pore size was approximately equal to 100 microm. At 1 h from seeding, the adhesion of cells was observed and at 1 day, cells spread on the sponge surface. Rapid cell growth on the sponge (doubling time: 29.0 h) was observed for at least 7 days, as well as on a commercially available plastic culture dish (doubling time: 27.4 h). At long-term (23-43 days) cultivation, cells were constantly counted to be approximately 4.2-7.4 million per sponge (1 cm in diameter). The maximum cell number was 7.4 million, approximately 37 times higher than on the same area dish. Living cells on the sponge were observed at 23-43 days by SEM observation and no abnormal morphology of the cells was observed. These results show that wool keratin sponges are useful scaffolds for long-term and high-density cell cultivation.

Keratin sponge: immobilization of lysozyme.

A porous wool keratin sponge was used for immobilization of lysozyme, a model bioactive substance and was demonstrated to be a unique biomaterial in terms that the activity of lysozyme linked to the sponge through disulfide bond was gradually released, while lysozyme through thioether bond was stably maintained.

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.

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.

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.