Effect of Pore Size of Honeycomb-Patterned Polymer Film on Spontaneous Formation of 2D Micronetworks by Coculture of Human Umbilical Vein Endothelial Cells and Mesenchymal Stem Cells.

The geometrical control of micronetwork structures ( µ NSs) formed by endothelial cells is an important topic in tissue engineering, cell-based assays, and fundamental biological studies. In this study, µ NSs are formed using human umbilical vein endothelial cells (HUVECs) by the coculture of HUVECs and human mesenchymal stem cells (MSCs) confined in a honeycomb-patterned poly-l-lactic acid film (honeycomb film (HCF)), which is a novel cell culture scaffold. The HCF is produced using the breath figure method, which uses condensed water droplets as pore templates. The confinement of the HUVECs and MSCs in the HCF along with the application of centrifugal force results in µ NS formation when the pore size is more than 20  µ m. Furthermore, µ NS development is geometrically restricted by the hexagonally packed and connected pores in the horizontal direction of the HCF. Network density is also controlled by changing the seeding density of the HUVECs and MSCs. The threshold pore size indicates that µ NSs can be formed spontaneously by using an HCF with a perfectly uniform porous structure. This result provides an important design guideline for the structure of porous cell culture scaffolds by applying a blood vessel model in vitro.

Design of core--shell-type nanoparticles carrying stable radicals in the core.

Utilizing the self-assembled core-shell-type polymeric micelle technique, high-performance nanoparticles possessing stable radicals in the core and reactive groups on the periphery were prepared. The anionic ring-opening polymerization of ethylene oxide (EO) was carried out using potassium 3,3-diethoxypropanolate as an initiator, followed by mesylation with methanesulfonyl chloride to obtain acetal-poly(ethylene glycol)-methanesulfonate (acetal-PEG-Ms; 1). Compound 1 was reacted with potassium O-ethyldithiocarbonate, followed by treatment with n-propylamine to obtain heterobifunctional PEG derivatives containing both sulfanyl and acetal terminal groups (acetal-PEG-SH) (2) in a highly selective and quantitative manner. Poly(ethylene glycol)-block-poly(chloromethylstyrene) (acetal-PEG-b-PCMS) (3) was synthesized by the free-radical telomerization of chloromethylstyrene (CMS) using 2 as a telogen. The chloromethyl groups in the PCMS segment of the block copolymer (3) were quantitatively converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of 3 with 4-amino-TEMPO to obtain acetal-PEG-b-PCMS containing TEMPO moieties (4). The obtained 4 formed core-shell-type nanoparticles in aqueous media when subjected to the dialysis method: the cumulant average diameter of the nanoparticles was about 40 nm, and the nanoparticles emitted intense electron paramagnetic resonance (EPR) signals. The TEMPO radicals in the core of the nanoparticles showed reduction resistance even in the presence of 3.5 mM ascorbic acid. This means that these nanoparticles are anticipated as high-performance bionanoparticles that can be used in vivo.

RAFT-generated polyacrylamide-DNA block copolymers for single-nucleotide polymorphism genotyping by affinity capillary electrophoresis.

Capillary electrophoretic separation of a mixture of 5'-fluorescein isothiocyanate-labeled single-stranded DNA (normal ssDNA) and its single-base-substituted one (mutant ssDNA) was achieved by using a RAFT-generated polyacrylamide-oligodeoxyribonucleotide block copolymer (PAAm-b-ODN) as an affinity polymeric probe. PAAm-b-ODN was synthesized through the Michael addition of thiol-terminated PAAm (PAAm-SH) to 5'-maleimide-modified ODN. PAAm-SH was derived from dithiobenzoate-terminated PAAm prepared via RAFT polymerization. The number-averaged molecular weight (M(n)) and the molecular weight distribution were determined by aqueous size exclusion chromatography. After a capillary tube was filled with the running buffer solution of PAAm-b-ODN, a mixture of normal and mutant ssDNA was subjected to electrophoresis and detected by a laser-induced fluorescent detector. Because the base sequence of PAAm-b-ODN was complementary to part of the mutant ssDNA, including a single-base substitution site, the electrophoretic migration of mutant ssDNA was retarded due to the formation of the equilibrium complex with PAAm-b-ODN. Increasing M(n) of the PAAm segment enhanced this retardation. On the other hand, normal ssDNA was unable to form the complex owing to a single-base mismatch, which was proved by melting curve measurements. The Lineweaver-Burk-type analysis of the mobility of mutant ssDNA revealed that the binding constants for the complexes with different PAAm-b-ODN probes were almost identical to each other. The analysis also demonstrated that the ratio of the hydrodynamic radius of the complex to that of the free mutant ssDNA increased with increasing M(n) of the affinity polymeric probe's PAAm segment. This means that the PAAm segment indirectly provides mutant ssDNA with an additional hydrodynamic friction force via the affinity interaction of the ODN segment. Optimization of the salt concentration of the running buffer and the capillary temperature improved the resolution of the separation. This affinity polymeric probe will be useful for developing a simple and highly reliable single-nucleotide polymorphism genotyping method.

Nanostraw membrane stamping for direct delivery of molecules into adhesive cells.

Delivering ions and molecules into living cells has become an important challenge in medical and biological fields. Conventional molecular delivery, however, has several issues such as physical and chemical damage to biological cells. Here, we present a method of directly delivering molecules into adhesive cells with an Au-based nanostraw membrane stamp that can physically inject a target molecule into the cytoplasm through a nanostraw duct. We successfully delivered calcein target molecules into adhesive cells with high efficiency (85%) and viability (90%). Furthermore, we modeled the molecular flow through Au nanostraws and then demonstrated the control of calcein flow by changing the concentration and geometry of Au nanostraws. Our Au membrane stamping provides a new way of accessing the cytoplasm to modulate cellular functions via injected molecules.

Changes in HepG2 spheroid behavior induced by differences in the gap distance between spheroids in a micropatterned culture system.

Micropatterning is a promising technique for modulating culture environments. In this study, we investigated the effect of spheroid separation distance on their properties in a micropatterned chip of HepG2 spheroids. The basic chip design consisted of 37 collagen spots (300 µm in diameter) in a hexagonal arrangement on a glass substrate; the region without collagen-spots was modified by polyethylene glycol to create the non-adhesive surface. Three similar chips were fabricated with gap distances between collagen-spots of 500, 1000, and 1500 µm. HepG2 cells adhered on the collagen spots and then formed spheroids via cell proliferation. Although the albumin secretion activities of HepG2 spheroids were almost the same in all chips, inhibition of spheroid growth and anaerobic metabolism were intensified when the gap distance was less than 1000 µm. Additionally, such phenomena which are induced by interference effects between spheroids, were more pronounced at the inside region of the chip than at the outside region. However, the interference effect between spheroids was nearly avoided when the gap distance was at least 1500 µm. Furthermore, the concentration of dissolved oxygen between neighboring spheroids decreased as the gap distance decreased, indicating that the spheroids competed for oxygen and became hypoxic in a way that depended on the spheroid separation distance. These results indicate that the spheroid separation distance is an important factor that can modulate the spheroid properties.

Turbidimetric detection of ATP using polymeric micelles and DNA aptamers.

Two types of turbidimetric detection of adenosine 5'-triphosphate (ATP) by the naked eye were achieved through a combination of non-cross-linking aggregation of DNA-linked polymeric micelles and molecular recognition of ATP by a DNA aptamer.

Effect of water-soluble phospholipid polymers conjugated with papain on the enzymatic stability.

To maintain enzymatic activity during long-term storage by conjugation with water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers (PMPC-COOH) having various molecular weights with a carboxylic group on the terminal, such compounds were synthesized as a polymer modifier using a photoinduced living radical polymerization technique. A poly(ethylene oxide) with a carboxyl group (PEO-COOH) was used as the control. The PMPC-COOHs were reacted with the amino groups of the enzyme, papain, via amide bonds. With an increase in the molecular weight in the range between 5 and 20K of the PMPC-COOH, the modification degree and alpha-helix content of the conjugated papain slightly decreased, but the remaining enzymatic activity did not depend on the molecular weight of the PMPC-COOH. However, when a much higher molecular weight PMPC-COOH (40K) was conjugated with a reduction in the modification degree, alpha-helix content was higher compared with the other PMPC-conjugated papain. Modification with PEO-COOH showed little reduction of the alpha-helix content of papain. The time dependence of the remaining enzymatic activity of the polymer-conjugated papains was evaluated during storage at 40 degrees C. The native papain diminished activity within one week. PEO-conjugated papain had decreased activity with time, but after one week it had half its initial level. The same tendency was observed when papain was modified with PMPC-COOHs 5 and 40K, that is, the enzymatic activity did not decrease even when they were stored for 4 weeks. We concluded that the PMPC chain could stabilize the enzyme by control of the molecular weight of the PMPC and modification degree to the enzyme.

Enzymatic synthesis of poly(α-ethyl ß-aspartate) by poly(ethylene glycol) modified poly(aspartate) hydrolase-1.

We recently discovered that poly(aspartate) (PAA) hydrolase-1 from Pedobacter sp. KP-2 has a unique property of specifically cleaving the amide bond between ß-aspartate units in thermally synthesized PAA (tPAA). In the present study, the enzymatic synthesis of poly(α-ethyl ß-aspartate) (ß-PAA) was performed by taking advantage of the substrate specificity of PAA hydrolase-1. No polymerization of diethyl L-aspartate by native PAA hydrolase-1 occurred because of the low dispersibility of the enzyme in organic solvent. Poly(ethylene glycol) (PEG) modification of the enzyme improved its dispersibility and enabled it to polymerize the monomer substrate. MALDI-TOF MS analysis showed that the synthesized polymer was observed in the range of m/z = 750-2 500. This analysis also revealed that the polymer was composed of ethyl aspartate units, containing either an ethyl ester or a free carboxyl end group at its carboxyl terminus. (1) H NMR analysis demonstrated that the synthesized polymer consisted of only ß-amide linkages. Thus, the present results indicate that PAA hydrolase-1 modified with PEG is useful for the synthesis of ß-PAA due to its unique substrate specificity and good dispersibility in organic solvent.

Completely dispersible PEGylated gold nanoparticles under physiological conditions: modification of gold nanoparticles with precisely controlled PEG-b-polyamine.

A novel water-soluble, biocompatible polymer, poly(ethylene glycol)-block-poly((2-N,N-dimethylamino)ethyl methacrylate) (PEG-b-PAMA), possessing controlled molecular weight with a narrow molecular weight distribution, was synthesized by the atom-transfer radical polymerization (ATRP) method. PEG-b-PAMA having a short PAMA chain length was successfully synthesized under suitable polymerization conditions. Gold nanoparticles (GNPs) were modified using PEG-b-PAMA prepared under a variety of PEGylation conditions. Under alkaline conditions (pH >10) and an [N]/[GNP] ratio of more than 3300, the PEGylated GNPs (PEG-GNPs) showed complete dispersion stability, avoiding coagulation. The amino groups of the PAMA segment of the block copolymers were completely deprotonated above pH 10. This means that PEG-b-PAMA interacted with the GNP surface via multipoint coordination of the tertiary amino groups of PAMA, not electrostatically. The effect of the number of amino groups in the PAMA segment on GNP surface modifications was investigated by zeta potential and dynamic light scattering (DLS) measurements. When the PEG-GNPs were prepared in excess polymer solution, almost the same diameter was observed regardless of the PAMA chain length. After the PEG-GNPs were purified by centrifugation, the zeta potentials of all PEG-GNPs were shielded to almost 0 mV, indicating the effective modifications of the GNP surface by PEG-b-PAMA regardless of the chain length. However, the particle size and particle size distribution of the purified PEG-GNPs were strongly affected by the PAMA chain length. PEG-GNPs with longer PAMA segments underwent coagulation after purification, whereas PEG-GNPs with shorter PAMA segments increased their dispersion stability. The experimental results of the thermal gravimetric analysis confirmed that the PEG density on the GNP surface increased as the AMA units decreased to 3. Thus, the dispersion stability depended significantly on the PEG density on the GNP surface. GNPs modified with PEG-b-PAMA having short AMA units showed excellent dispersion stability under a variety of pH conditions. The excellent dispersion stability of the obtained PEG-GNP was also confirmed both in bovine serum albumin (BSA) solution and 95% human serum.

Design of poly(ethylene glycol)/streptavidin coimmobilized upconversion nanophosphors and their application to fluorescence biolabeling.

Infrared-to-visible upconversion phosphors (i.e., rare earth ion-doped Y2O3 nanoparticles (UNPs)) were synthesized by the homogeneous precipitation method. Because the charge on the erbium (Er) ion-doped Y2O3 (Y2O3:Er) NP (UNP1) surface is positive under neutral conditions, the UNP1 surface was electrostatically PEGylated using negatively charged poly(ethylene glycol)- b-poly(acrylic acid) (PEG- b-PAAc). The adsorption of PEG- b-PAAc was confirmed by Fourier transform infrared (FT-IR) measurements and thermal gravimetric analysis (TGA). The surface charge of the PEGylated UNP1s (PEG-UNP1s) was effectively shielded by the PEGylation. The dispersion stability of the UNP1s was also significantly improved by the PEGylation. The PEG-UNP1s were dispersed over 1 week under physiological conditions as a result of the steric repulsion between the PEG chains on the UNP1 surface. The upconversion emission spectrum of PEG-UNP1s was observed under physiological conditions and was confirmed by near-infrared excited fluorescence microscope observation. Streptavidin (SA)-installed ytterbium (Yb) and Er ion-codoped Y2O3 (Y2O3:Yb,Er) NPs (UNP2s) were prepared by the coimmobilization of PEG- b-PAAc and streptavidin. The PEG/SA coimmobilized UNP2s (PEG/SA-UNP2s) specifically recognized biotinylated antibodies and emitted strong upconversion luminescence upon near-infrared excitation. The obtained PEG/streptavidin coimmobilized UNPs are promising as high-performance near-infrared biolabeling materials.