Simultaneous analysis of multiple oligonucleotides by temperature-responsive chromatography using a poly(N-isopropylacrylamide)-based stationary phase.

Oligonucleotide therapeutics have contributed remarkably to healthcare, being well suited for the treatment of intractable diseases that are difficult to approach using conventional drug modalities. However, as common techniques of oligonucleotide analysis rely on reversed-phase or ion-exchange liquid chromatography and thus employ toxic organic solvents and/or ion-pairing reagents, better alternatives are highly sought after. Poly(N-isopropylacrylamide) (PNIPAAm) is widely used in temperature-responsive chromatography (TRC), which relies on column temperature variation to control the physical properties of the stationary phase and, unlike conventional reversed-phase liquid chromatography, avoids the use of toxic organic solvents and complicated gradient methods. Herein, PNIPAAm copolymer hydrogel-modified silica beads were used for the simultaneous analysis of multiple synthetic oligonucleotides by TRC to recognize differences in the length of single nucleotides, single bases, and the number of phosphorothioated sites. Temperature-responsive elution was observed in all cases. Each separation of all combinations of multiple oligonucleotides was better at higher temperatures above the lower critical solution temperature and was performed without the use of organic solvents and gradient methods. In the case of multiply phosphorothioated oligonucleotides, good separation was achieved using an aqueous solvent and isocratic elution in the absence of ion-pairing reagents. Thus, the developed procedure was concluded to be well suited for oligonucleotide analysis. Graphical abstract.

Thermoresponsive Cationic Block Copolymer Brushes for Temperature-Modulated Stem Cell Separation.

Recently, cell separation methods have become important for preparing cells for transplantation therapy. In this study, a thermoresponsive cationic block copolymer brush is developed as an effective cell separation tool. This brush is prepared on glass surfaces using two steps of activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP). The cationic segment is prepared in the first step of the ARGET-ATRP of N,N-dimethylaminopropylacrylamide (DMAPAAm). In the second step, the thermoresponsive segment is prepared, attached to the bottom cationic segment, through ARGET-ATRP with N-isopropylacrylamide (NIPAAm). The cell adhesion behavior of the prepared thermoresponsive cationic copolymer, PDMAPAAm-b-PNIPAAm, brush is observed using umbilical cord-derived mesenchymal stem cells (UCMSC), fibroblasts, and macrophages. At 37 °C, all three types of cells adhere to the thermoresponsive cationic copolymer brush. Then, by reducing the temperature to 20 °C, the adhered UCMSC are detached from the copolymer brush, whereas the fibroblasts and macrophages remain adhered to the copolymer brush. Using this copolymer brush, UCMSC can be purified from the cell mixture simply by changing the temperature. Therefore, the prepared thermoresponsive cationic copolymer brush is useful as a cell separation tool for the purification of mesenchymal stem cells.

Adsorption-Desorption Control of Fibronectin in Real Time at the Liquid/Polymer Interface on a Quartz Crystal Microbalance by Thermoresponsivity.

The cell manipulation technique using thermoresponsive polymers is currently attracting much attention for applications in the medical field. To achieve arbitrary and accurate cell control, it is necessary to intensely research fibronectin behavior. A smart surface, which has thermoresponsive wettability and which can adsorb or desorb fibronectin repeatedly without the presence of cells, was fabricated by an electrospinning method. The fabricated coating changed its structure as the temperature was changed, and this transformation could substitute for the pulling force generated by the cytoskeletal contraction of cells. Moreover, a coated quartz crystal microbalance was able to detect the fibronectin behavior as frequency shifts, which could be used in the estimation of the mass shift with the aid of suitable equations. This coating and measurement system can contribute greatly not only to the development in the medical field centered on biomaterial manipulation technologies, but also to the improvement of medical instruments.

Crosslinked Poly(N-Isopropylacrylamide)-Based Microfibers as Cell Manipulation Materials with Prompt Cell Detachment.

Stimuli-responsive smart materials are a key to the realization of next-generation medical technologies. Among them, the temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAAm) is attracting particular attention because it is easy to use in physiological conditions. PNIPAAm-grafted surfaces can undergo temperature-modulated cell adhesion and detachment without proteolytic enzymes, and can be used as cell-separating materials through selective cell adhesion/detachment. However, cell detachment at reduced temperatures is problematic because it takes several hours. A novel thermoresponsive crosslinked microfiber system that can greatly reduce the cell detachment time is introduced in this study. The crosslinked fibers provide temperature-dependent volume change, and enable cell detachment within 10 min of reducing the temperature, which is one-sixth of the time required in previous studies. The prompt cell detachment is thought to arise from a completely new mechanism derived from fiber swelling. This system will make a significant contribution as a novel cell manipulating system for next-generation medical technology.

Effect of Polymer Phase Transition Behavior on Temperature-Responsive Polymer-Modified Liposomes for siRNA Transfection.

Small interfering RNAs (siRNAs) have been attracting significant attention owing to their gene silencing properties, which can be utilized to treat intractable diseases. In this study, two temperature-responsive liposomal siRNA carriers were prepared by modifying liposomes with different polymers-poly(N-isopropylacrylamide-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAAm-co-DMAPAAm)) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) P(NIPAAm-co-DMAAm). The phase transition of P(NIPAAm-co-DMAPAAm) was sharper than that of P(NIPAAm-co-DMAAm), which is attributed to the lower co-monomer content. The temperature dependent fixed aqueous layer thickness (FALT) of the prepared liposomes indicated that modifying liposomes with P(NIPAAm-co-DMAPAAm) led to a significant change in the thickness of the fixed aqueous monolayer between 37 °C and 42 °C; while P(NIPAAm-co-DMAAm) modification led to FALT changes over a broader temperature range. The temperature-responsive liposomes exhibited cellular uptake at 42 °C, but were not taken up by cells at 37 °C. This is likely because the thermoresponsive hydrophilic/hydrophobic changes at the liposome surface induced temperature-responsive cellular uptake. Additionally, siRNA transfection of cells for the prevention of luciferase and vascular endothelial growth factor (VEGF) expression was modulated by external temperature changes. P(NIPAAm-co-DMAPAAm) modified liposomes in particular exhibited effective siRNA transfection properties with low cytotoxicity compared with P(NIPAAm-co-DMAAm) modified analogues. These results indicated that the prepared temperature-responsive liposomes could be used as effective siRNA carriers whose transfection properties can be modulated by temperature.

LAT1-Targeting Thermoresponsive Fluorescent Polymer Probes for Cancer Cell Imaging.

L-type amino acid transporter 1 (LAT1) is more highly expressed in cancer cells compared with normal cells. LAT1 targeting probes would therefore be a promising tool for cancer cell imaging. In this study, LAT1-targeting thermoresponsive fluorescent polymer probes based on poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(NIPAAm-co-DMAAm)) were synthesized and their affinity for LAT1 was evaluated. The synthesized polymer probes interacted with LAT1 on HeLa cells, and inhibition of l-[³H]-leucine, one of the substrates for LAT1 uptake, was investigated. l-Tyrosine-conjugated P(NIPAAm-co-DMAAm) inhibited the uptake of l-[³H]-leucine, while P(NIPAAm-co-DMAAm) and l-phenylalanine-conjugated P(NIPAAm-co-DMAAm) did not. This result indicated that l-tyrosine-conjugated polymer has a high affinity for LAT1. The fluorescent polymer probes were prepared by modification of a terminal polymer group with fluorescein-5-maleimide (FL). Above the polymer transition temperature, cellular uptake of the polymer probes was observed because the polymers became hydrophobic, which enhanced the interaction with the cell membrane. Furthermore, quantitative analysis of the fluorescent probe using flow cytometry indicated that l-tyrosine-conjugated P(NIPAAm-co-DMAAm)-FL shows higher fluorescence intensity earlier than P(NIPAAm-co-DMAAm)-FL. The result suggested that cellular uptake was promoted by the LAT1 affinity site. The developed LAT1-targeting thermoresponsive fluorescent polymer probes are expected to be useful for cancer cell imaging.

Mesenchylmal Stem Cell Culture on Poly(N-isopropylacrylamide) Hydrogel with Repeated Thermo-Stimulation.

We prepared thermoresponsive hydrogels by mixing poly(N-isopropylacrylamide) (PNIPAAm) derivatives as the main chain components, octa-arm polyethylene glycol (PEG) as a crosslinker, and the Arg-Gly-Asp-Ser (RGDS) peptides as cell adhesion units. Human bone marrow-derived mesenchymal stem cells (hbmMSCs) were cultured on the hydrogels. The PNIPAAm gel prepared by the post-crosslinking gelation method was revealed to be cytocompatible and showed temperature-dependent changes in mechanical properties. Repeated changes in the swelling ratio of the PNIPAAm gel affected the shape of the hbmMSCs. With respect to both cytocompatibility and reversibility of changes in mechanical properties, the PNIPAAm gel system could be potentially useful for the analysis of cell mechanobiology.

The use of a temperature-responsive column for the direct analysis of drugs in serum by two-dimensional heart-cutting liquid chromatography.

A novel pretreatment method, which was performed using a two-dimensional high-performance liquid chromatography (2D-HPLC) system, was proposed for the direct analysis of drugs in human serum. A temperature-responsive column was used as a pretreatment column. The stationary phase of the temperature-responsive column exhibits temperature-regulated hydrophilic/hydrophobic characteristics. Controlling the ionic strength of the eluent enables human serum albumin (HSA) to pass through the column without retention. When serum samples containing barbiturates or benzodiazepines were injected into the temperature-responsive column using 10 mM of ammonium acetate (pH 6.5) as the mobile phase and in the temperature range of 10-40 °C, HSA was eluted from the column near the dead time, followed by the individual drugs. When the column temperature was changed, the retention times of the drugs were altered owing to surface property changes within the pretreatment column. These closely eluted compounds were subsequently introduced into the analytical column using a column-switching valve, with a minimal gap time to avoid foreign substance contamination. This new 2D-HPLC method afforded high-quality chromatograms of multiple drugs without unwanted peaks from foreign substances. The present technique could be an attractive choice in selecting the analytical method for drug analysis.

Transcutaneous drug delivery by liposomes using fractional laser technology.

OBJECTIVE: Transdermal delivery of hydrophilic peptides remains a challenge due to their poor cellular uptake and transdermal penetration. We hypothesize that combination of a CO2 fractional laser to enhance percutaneous absorption and liposomes as transdermal carriers would improve skin penetration of hydrophilic drugs. STUDY DESIGN: NA. METHODS: Liposomes were prepared using membrane fusion lipid dioleoylphosphatidylethanolamine, and used to deliver 5-carboxyfluorescein (CF) and fluorescein isothiocyanate-conjugated ovalbumin (OVA-FITC) as model hydrophilic peptide drugs. Liposome size was estimated by dynamic light scattering. Liposome uptake into murine macrophage cells and penetration or permeation into Yucatan micropig skin after irradiation by CO2 fractional laser at varying energy levels (laser power and exposure duration) were investigated using Franz cell and fluorescence microscopy. Oxidative damage to the irradiated mouse skin was assessed by electron spin resonance. RESULTS: Size of CF and OVA-FITC encapsulated liposomes was 324 ± 75 nm. Cellular uptake of OVA-FITC delivered by liposomes was 10-fold higher (1,370 relative fluorescence units, RFU) than delivered in solution form (130 RFU). Fractional laser irradiation increased skin permeation rate of CF liposomes (0-10%) and OVA-FITC liposomes (4-40%) in a dose-dependent manner. Although peeling off the stratum corneum facilitated CF liposome penetration at low energy levels (2.69-3.29 J/cm2 ; 10-20 W for 500 µs), drug permeation was similar (7-8%) in peeled or untreated skin at higher laser energy levels (6.06 J/cm2 ; 20 W for 1,500 µs). FITC penetrated deeper in the skin after laser irradiation. However, OH, O2-, and VC reactive oxygen species were generated upon irradiation of the skin with a fractional CO2 laser. CONCLUSIONS: Increasing laser power and irradiation, time increased liposome uptake by cells and penetration of peptide drugs across the skin in a dose-dependent manner. High-energy CO2 fractional laser overcomes the rate-limiting barrier function of the stratum corneum. Further investigations are required to establish the safety and efficacy of fractional laser-irradiation assisted delivery of liposome-encapsulated drugs as a transcutaneous drug delivery system. Lasers Surg. Med. 49:525-532, 2017. © 2016 Wiley Periodicals, Inc.

Approaching over 10 000-fold sensitivity increase in chiral capillary electrophoresis: Cation-selective exhaustive injection and sweeping cyclodextrin-modified micellar electrokinetic chromatography.

A novel and simple method that combines an online concentration technique with an enantioseparation technique for capillary electrophoresis-namely, cation-selective exhaustive injection and sweeping cyclodextrin-modified micellar electrokinetic chromatography (CSEI-sweeping CD-modified MEKC)-realizes the effective enantioseparation of cationic analytes while keeping a significant increase of detection sensitivity. This technique consists of a slight modification of the basic CSEI-sweeping MEKC. The main idea is to simply add an anionic CD as a chiral selector into the micellar buffer including sodium dodecyl sulfate, but not to change any other buffers in order to preserve the online concentration mechanism. When applied to analysis of the street drug, methamphetamine, the method achieved not only a baseline enantioseparation but also limits of detection (LODs; S/N = 3) of 70-90 pg/mL (ppt) for each isomer. This translates to a more than 10 000-fold improvement compared to the LODs by the usual injection method. The present technique, which was made from a slight modification of CSEI-sweeping MEKC, would give an attractive approach that is applicable to almost any analytes for which CSEI-sweeping MEKC is applicable; all that is required is the selection of an appropriate anionic CD to be added to the micellar buffer.

Temperature-responsive molecular recognition chromatography using phenylalanine and tryptophan derived polymer modified silica beads.

Temperature-responsive polymers incorporating molecular-recognition sites were developed as stationary phases for high-performance liquid chromatography (HPLC). The grafted stationary phases consisted of functional copolymers composed of N-isopropylacrylamide (NIPAAm) and N-acryloyl aromatic amino acid methyl esters, i.e., phenylalanine and tryptophan methyl esters (Phe-OMe and Trp-OMe). Three novel temperature-responsive polymers, P(NIPAAm-co-Phe-OMe5), P(NIPAAm-co-Phe-OMe10), and P(NIPAAm-co-Trp-OMe5), were synthesized. These copolymers exhibited a reversible hydrophilic/hydrophobic phase transition at their lower critical solution temperatures (LCSTs). The polymers were grafted onto aminopropyl silica using an activated ester-amine coupling method, and were packed into a stainless steel column, which was connected to an HPLC system. Temperature-responsive chromatography was conducted using water as the sole mobile phase. More hydrophobic analytes were retained longer, and the retention times of aromatic steroids and aromatic amino acids were dramatically increased. This indicated that π-π interactions occurred between the phenyl or indole moieties of phenylalanine or tryptophan, respectively, and the aromatic compounds. Furthermore, the retention times of compounds with hydrogen bond acceptors were higher with P(NIPAAm-co-Trp-OMe5), which contained indole as a hydrogen bond donor, than with P(NIPAAm-co-Phe-OMe5). This indicated that hydrogen bonding occurred between the stationary phase and the analytes. These results indicate that hydrophobic, π-π, and hydrogen bonding interactions all affected the separation mode of the temperature-responsive chromatography, and led to selective separation with molecular recognition. Both temperature-response and molecular recognition characteristics are present in the proposed separation system that utilizes a temperature-responsive polymer bearing aromatic amino acid derivatives.

Design of Environmentally Responsive Fluorescent Polymer Probes for Cellular Imaging.

We report the development of environmentally responsive fluorescent polymers. The reversible temperature-induced phase transition of copolymers composed of N-isopropylacrylamide and a fluorescent monomer based on the fluorescein (FL), coumarin (CO), rhodamine (RH), or dansyl (DA) skeleton was used as a molecular switch to control the fluorescence intensity. The poly(N-isopropylacrylamide) (PNIPAAm) chain showed an expanded coil conformation below the lower critical solution temperature (LCST) due to hydration, but it changed to a globular form above the LCST due to dehydration. Through the combination of a polarity-sensitive fluorophore with PNIPAAm, the synthetic fluorescent polymer displayed a response to external temperature, with the fluorescence strength dramatically changing close to the LCST. Additionally, the P(NIPAAm-co-FL) and P(NIPAAm-co-CO) polymers, containing fluorescein and coumarin groups, respectively, exhibited pH responsiveness. The environmental responsiveness of the reported polymers is derived directly from the PNIPAAm and fluorophore structures, thus allowing for the cellular uptake of the fluorescence copolymer by RAW264.7 cells to be temperature-controlled. Cellular uptake was suppressed below the LCST but enhanced above the LCST. Furthermore, the cellular uptake of both P(NIPAAm-co-CO) and P(NIPAAm-co-RH) conjugated with a fusogenic lipid, namely, l-α-phosphatidylethanolamine, dioleoyl (DOPE), was enhanced. Such lipid-conjugated fluorescence probes are expected to be useful as physiological indicators for intracellular imaging.

Thermoresponsive anionic copolymer brushes containing strong acid moieties for effective separation of basic biomolecules and proteins.

A thermoresponsive copolymer brush possessing the sulfonic acid group, poly(N-isopropylacrylamide (IPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-tert-butylacrylamide (tBAAm)), was grafted onto the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer and copolymer brushes on silica beads were characterized by observing the phase transition profile, CHNS elemental analysis, X-ray photoelectron spectroscopy, and gel permeation chromatography. The phase transition profile indicated that an appropriate AMPS composition for enabling thermally modulated property changes is 5 mol %, while excessive amounts of sulfonic acid groups prevented copolymer phase transition. Chromatographic elutions of catecholamine derivatives and basic proteins were observed, using the prepared copolymer brush-modified beads as chromatographic matrices, and the results suggest that the beads interact with these analytes through relatively strong electrostatic interactions. Thus, poly(IPAAm-co-AMPS-co-tBAAm) brush-modified beads will be useful for effective thermoresponsive chromatography matrices that separate basic biomolecules through strong electrostatic interactions.

Monolithic silica rods grafted with thermoresponsive anionic polymer brushes for high-speed separation of basic biomolecules and peptides.

Thermoresponsive anionic copolymer brushes, poly(N-isopropylacrylamide-co-acrylic acid-co-tert-butylacrylamide) [P(IPAAm-co-AAc-co-tBAAm)], were grafted onto a monolithic silica rod column through surface-initiated atom-transfer radical polymerization (ATRP) to prepare an effective thermoresponsive anionic chromatography matrix. An ATRP initiator was attached to the rod surface. N-Isopropylacrylamide (IPAAm), tert-butyl acrylate (tBA), tert-butylacrylamide (tBAAm), and the ATRP catalyst CuCl/CuCl2/tris[2-(N,N-dimethylamino)ethyl]amine were dissolved in 2-propanol, and the reaction mixture was pumped into the initiator-modified column. After grafting P(IPAAm-co-tBA-co-tBAAm) on the monolithic silica surfaces, deprotection of the tert-butyl group of tBA was performed. Chromatographic analysis showed that the prepared column was able to separate catecholamine derivatives and angiotensin subtypes within a shorter analysis time (5 min) than a silica-bead-packed column modified with the same copolymer brush could. These results indicated that the prepared copolymer-modified monolithic silica rod column may be a promising bioanalytical and bioseparation tool for rapid analysis of basic bioactive compounds and peptides.

Thermoresponsive copolymer brushes possessing quaternary amine groups for strong anion-exchange chromatographic matrices.

A thermoresponsive copolymer incorporating a quaternary amine group, poly(N-isopropylacrylamide-co-3-acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butylacrylamide), was conjugated to the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer- and copolymer brush-modified beads were characterized by CHN elemental analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, and observation of phase transition profiles. Phase transition profiles of the prepared copolymer indicated that 5 mol % APTAC is suitable for enabling thermally modulated property changes in the copolymer. Chromatographic elution behaviors of adenosine nucleotides and proteins were observed using prepared beads as chromatography matrices. Higher retention time of adenosine nucleotides and strong protein adsorption behavior were observed compared with those on beads with tertiary amine groups, because of the strong basic properties. Therefore, copolymer brush modified beads will be useful as thermoresponsive ion-exchange chromatographic matrices.

Thermoresponsive bio-affinity interfaces for temperature-modulated selective capture and release of targeted exosomes.

The existing methods for exosome isolation, such as ultracentrifugation, size exclusion, and affinity separation, suffer from some limitations. Herein, we aimed to develop temperature-modulated exosome-capturing materials using thermoresponsive polymers and peptides with affinity for exosomes. Poly(2-hydroxyethyl methacrylate-co-propargyl acrylate)-b-poly(N-isopropylacrylamide) (P(HEMA-co-PgA)-b-PNIPAAm) was grafted on silica beads via a two-step process of activator regenerated by electron transfer atom transfer radical polymerization. Peptides with affinity for exosomes were conjugated to the propargyl group of the bottom P(HEMA-co-PgA) segment of the copolymer via a click reaction. The prepared copolymer-grafted beads were characterized by elemental analysis, X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, gel permeation chromatography, and the turbidity of the polymer solution. Results indicated that the copolymer and peptide were successfully modified on the silica beads. Exosomes from SK-BR-3 â€‹cells, a human breast cancer cell line, were selectively captured on the prepared beads at 37 â€‹°C, as the upper PNIPAAm segment shrank and the affinity between the peptide and exosome was enhanced. Upon lowering the temperature to 4 â€‹°C, the captured exosomes were released from the copolymer brush because of the extension of the PNIPAAm segment that reduced the affinity between peptides and exosomes. These findings demonstrated that the prepared copolymer brush-grafted silica beads can capture and release targeted exosomes via temperature modulation. Taken together, the developed copolymer brush-grafted silica beads would be useful for the separation of exosomes using simple procedures such as temperature modulation.

Thermoresponsive mixed polymer brush to effectively control the adhesion and separation of stem cells by altering temperature.

During the last few decades, thermoresponsive materials for modulating cell adhesion have been investigated for the application of tissue engineering. In this study, we developed thermoresponsive mixed polymer brushes consisting of poly(N-isopropylacrylamide) (PNIPAAm) and poly(N,N-dimethylaminopropylacrylamide) (PDMAPAAm). The mixed polymer brushes were prepared on a glass substrate via the reversible addition-fragmentation chain transfer polymerization of DMAPAAm and subsequent atom transfer radical polymerization of NIPAAm. The mixed polymer brushes grafted to glass exhibited increased cationic properties by increasing the grafted PDMAPAAm length. The shrinking and extension of PNIPAAm exposed and concealed PDMAPAAm, respectively, indicating that the surface cationic properties can be controlled by changing the temperature. At 37 â€‹°C, the prepared mixed polymer brushes enhanced cell adhesion through their electrostatic interactions with cells. They also exhibited various thermoresponsive adhesion and detachment properties using various types of cells, such as mesenchymal stem cells. Temperature-controlled cell adhesion and detachment behavior differed between cell types. Using the prepared mixed polymer brush, we separated MSCs from adipocytes and HeLa cells by simply changing the temperature. Thus, the thermoresponsive mixed polymer brushes may be used to separate mesenchymal stem cells from their differentiated or contaminant cells by altering the temperature.

Real-time in situ X-ray micro-computed tomography study of the effect of impurities on the crystallization of amorphous nifedipine.

Controlling the physical stability of noncrystalline active pharmaceutical ingredients remains a major challenge in the development of amorphous formulations such as amorphous solid-dispersion (ASD) formulations. To establish new evaluation and formulation strategies, the spatial distribution of the crystal phase in bulk amorphous nifedipine (NFD) was investigated as a model. The crystallization of amorphous NFD and the effect of a deliberately added impurity were investigated using powder X-ray diffraction (PXRD), differential scanning calorimetry and real-time in situ X-ray micro-computed tomography (X-ray CT). The stability data of amorphous samples, i.e., NFD and a mixture of NFD with an oxidative degradation product of NFD, impurity A (Imp A), at a weight ratio of 90:10, presented as percent amorphous remaining, suggests that Imp A accelerates the bulk crystal growth of NFD. Real-time in situ X-ray CT results showed surface-enhanced crystal growth and cavity formation in solid NFD samples. Moreover, the crystals were heterogeneous in density. These results suggest that Imp A affects the physical stability of the amorphous NFD. X-ray CT equipped with a heating unit can aid in-situ evaluation and assessment of physicochemical properties and physical stability of amorphous samples and formulations.

Umbilical cord-derived mesenchymal stem cell sheets transplanted subcutaneously enhance cell retention and survival more than dissociated stem cell injections.

BACKGROUND: Human umbilical cord-derived mesenchymal stem cell (hUC-MSC) sheets have recently attracted attention as an alternative approach to injected cell suspensions for stem cell therapy. However, cell engraftment and cytokine expression levels between hUC-MSC sheets and their cell suspensions in vivo have not yet been compared. This study compares hUC-MSC in vivo engraftment efficacy and cytokine expression for both hUC-MSC sheets and cell suspensions. METHODS: hUC-MSC sheets were prepared using temperature-responsive cell culture; two types of hUC-MSC suspensions were prepared, either by enzymatic treatment (trypsin) or by enzyme-free temperature reduction using temperature-responsive cell cultureware. hUC-MSC sheets and suspensions were transplanted subcutaneously into ICR mice through subcutaneous surgical placement and intravenous injection, respectively. hUC-MSC sheet engraftment after subcutaneous surgical transplantation was investigated by in vivo imaging while intravenously injected cell suspensions were analyzing using in vitro organ imaging. Cytokine levels in both transplant site tissues and blood were quantified by enzyme-linked immunosorbent assay. RESULTS: After subcutaneous transplant, hUC-MSC sheets exhibited longer engraftment duration than hUC-MSC suspensions. This was attributed to extracellular matrix (ECM) and cell-cell junctions retained in sheets but enzymatically altered in suspensions. hUC-MSC suspensions harvested using enzyme-free temperature reduction exhibited relatively long engraftment duration after intravenous injection compared to suspensions prepared using trypsin, as enzyme-free harvest preserved cellular ECM. High HGF and TGF-ß1 levels were observed in sheet-transplanted sites compared to hUC-MSC suspension sites. However, no differences in human cytokine levels in murine blood were detected, indicating that hUC-MSC sheets might exert local paracrine rather than endocrine effects. CONCLUSIONS: hUC-MSC sheet transplantation could be a more effective cell therapeutic approach due to enhanced engraftment and secretion of therapeutic cytokines over injected hUC-MSC suspensions.

Induction of different reactive oxygen species in the skin during various laser therapies and their inhibition by fullerene.

BACKGROUND AND OBJECTIVES: The production of reactive oxygen species (ROS) is one of the mechanisms of laser irradiation in the skin, and there are beneficial and detrimental aspects to this reaction. Detrimental side effects after laser treatments, such as redness and pigmentation, can be reduced by using anti-oxidants. MATERIALS AND METHODS: Electron spin resonance (ESR) analysis using a free radical trapping agent revealed that different free radicals, including hydroxyl ((·) OH) and superoxide anion (O 2-) radicals, were generated in the skin of hairless mice by irradiation with intense pulsed light (IPL), plasma, and radio frequency lasers. RESULTS: Generation of O 2- and (·) OH radicals was significantly inhibited in a dose-dependent fashion by fullerene and fullerene did not have any pro-oxidant effects as no radical adduct signal was detected. Although ROS can increase expression of COX-2 mRNA, an inflammatory marker, laser-induced COX-2 expression was significantly suppressed by the antioxidant activity of fullerene. In addition, imaging analysis of human skin has shown that erythema-associated redness caused by laser-induced inflammation is inhibited by fullerene gel. CONCLUSION: These data suggest that laser-induced inflammation is suppressed by the ROS-scavenging activity of fullerene and that application of fullerene is effective against oxidative skin damage caused by laser irradiation. Thus, fullerene has potential as an after-care therapy following laser irradiation of the skin.