Adjustable bioadhesive control of PEGylated hyperbranch brushes on polystyrene microplate interface for the improved sensitivity of human blood typing.

A PEGylated 96-well polystyrene (PS) microplate was first introduced for applications in high-throughput screening for selective blood typing to minimize the risks in blood transfusions. Herein, we present a hemocompatible PS 96-well microplate with adjustable PEGylated hyperbranch brush coverage prepared by ozone pretreated activation and thermally induced surface PEGylation. The grafting properties, hydration capacity, and blood compatibility of the PEGylated hyperbrush immobilized PS surfaces in human blood were illustrated by the combined chemical and physical properties of the surface, and the dependence of the specific absorption of human plasma fibrinogen onto the PEGylated surfaces on the grafting density was analyzed by monoclonal antibodies. The surface coverage of PEGylated brushes plays a major role in the bioadhesive properties of modified PS microplates, which in turn control the level of agglutination sensitivity in blood typing. The bioadhesive resistance toward proteins, platelets, and erythrocytes in human whole blood showed a correlation to the controlled hydration properties of the PEGylated hyperbrush-modified surfaces. Therefore, we suggested that the surface coverage of PEGylated hyperbrushes on PS surfaces can increase the sensitivity of cross-matching blood agglutination by up to 16-fold compared to that of the conventional 96-well virgin PS due to the regulated biorecognition of hematocrit and antibodies of the PEGylated hyperbrush-modified surfaces.

Modulation of osteogenic, adipogenic and myogenic differentiation of mesenchymal stem cells by submicron grooved topography.

Topographic cues have been recognized crucial on the modulation of cell behavior, and subsequent important for the design of implants, cell-based biomedical devices and tissue-engineered products. Grooved topography direct cells to align anisotropically on the substrates, resulting in an obvious morphological difference compared with the flat and the other topographies. This study aimed at investigating the effects of grooved topography on the differentiation of mesenchymal stem cells (MSCs) into osteoblasts, adipocytes and myoblasts. A series of submicron-grooved polystyrene substrates with equal groove-to-ridge ratio but different width and depth (width/depth (nm): 450/100, 450/350, 900/100, and 900/550) were fabricated based on electron beam lithography and soft lithography techniques. Primary rat MSCs (rMSCs) were cultured on these substrates without induction for differentiation for 6 days, and then subjected to induction for osteogenesis, adipogenesis and myogenesis. While the alignment of rMSCs strongly complied with the direction of the grooves and increased with groove depths, cell attachment on day 1 (~1.5 × 10(4)/cm(2)) and cell proliferation after 6 days of culture (~5 × 10(4)/cm(2)) were not significantly affected by substrate types. Osteogenesis, indicated by alkaline phosphatase activities and calcium deposit, was not significantly modulated by the grooved substrates, compared with the flat control, suggesting that cell alignment may not determine osteoinduction of rMSCs. On the other hand, adipogenesis, indicated by lipid production, was significantly enhanced by the grooved substrates compared with the flat surface (P < 0.001). On the other hand, myogenesis, indicated by desmin and MHC staining, was enhanced by the grooves in a time- and groove size-dependent manner compared with the flat control. The results suggested that grooved topography has an in-depth potential for modulating the commitment of the stem cell lineages, which could benefit the development of advanced biomaterials for biomedical applications.

Effect of Sulfonation Group on Polyaniline Copolymer Scaffolds for Tissue Engineering with Laminin Treatment under Electrical Stimulation.

Sulfonated copolyanilines (SPANs), SPAN-40 and SPAN-75, were prepared and applied in this tissue engineering study. SPAN scaffolds (SPANs) and control group polyaniline (PANI) were synthesized by performing oxidative polymerization. To further research the effects of neuron regeneration, PC12 cells were cultured on as-prepared PANI and SPANs with laminin (La) treatment under electrical stimulation. The effects on PC12 cell differentiation were investigated by controlling the amount of sulfonated groups (-SO3H) in the SPAN chain, the electrical stimulation voltage, and the presence or absence of La coating. The adhesion and proliferation of cells increased with the degree of sulfonation; La and electrical stimulation further promoted neuronal cell differentiation as increased neurite length was demonstrated in the micrograph analyses. In summary, the sulfonated copolyaniline coated with La had the best effect on neuronal differentiation under electrical stimulation, suggesting its potential as a substrate for nerve tissue engineering.

Modulated electro-hyperthermia-enhanced liposomal drug uptake by cancer cells.

PURPOSE: Modulated electro-hyperthermia (mEHT) stands to be a significant technological advancement in the hyperthermia field, utilizing autofocusing electromagnetic power on the cell membrane to create massive apoptosis. Since mEHT possesses the unique ability to excite cell membranes, we hypothesized that mEHT could enhance the uptake of liposomal drugs by enhancing phagocytic activity. MATERIALS AND METHODS: Water bath control and mEHT were used to compare the enhancement of liposome-encapsulated doxorubicin (Lipodox®) uptake by cancer cells. Cancer cells were made visible by doxorubicin fluorescence to investigate drug uptake. Viable cell yield was determined via the Trypan Blue exclusion method. Various substrates were used to investigate the mechanism of drug-uptake enhancement. The murine colon carcinoma model, CT26, was used to confirm the tissue infiltration of Lipodox® and its therapeutic effect. RESULTS: mEHT treatment showed a significant enhancement of Lipodox® uptake of doxorubicin fluorescence compared with 37°C or 42°C water bath treatment. Tumor tissue sections also confirmed that mEHT treatment achieved the highest doxorubicin concentration in vivo (1.44±0.32 µg/g in mEHT group and 0.79±0.32 µg/g in 42°C water bath). Wortmannin was used to inhibit the macropinocytosis effect and 70 kDa dextran-FITC served as uptake substance. The uptake of dextran-FITC by cancer cells significantly increased after mEHT treatment whereas such enhancement was significantly inhibited by wortmannin. CONCLUSION: The result showed mEHT-induced particle-uptake through macropinocytosis. mEHT-enhanced uptake of Lipodox® may amplify the therapeutic effect of liposomal drugs. This novel finding warrants further clinical investigation.

Evaluation of different promoters and host strains for the high-level expression of collagen-like polymer in Escherichia coli.

The increased expression of collagen-like polymer, CLP3.1-his which consists of 52 repeating peptide (GAPGAPGSQGAPGLQ), in Escherichia coli was investigated. The effects of three promoters, thermally inducible promoter, T7 promoter and T7lac promoter, and three Escherichia coli host strains, BL21, BL21(DE3) and BL21(DE3)[pLysS] which differ in stringency of suppressing basal transcription, were compared. Based on the CLP3.1-his expression level, solubility of CLP3.1-his in cells and basal transcription that occurred in the absence of induction, two expression systems, BL21(DE3) containing plasmid pJY-2 with T7lac promoter and BL21(DE3)[pLysS] containing plasmid pJY-1 with T7 promoter, were selected. With these two expression systems, CLP3.1-his expression levels greater than 40% (g/g) of total cellular proteins and CLP3.1-his concentrations of 0.1-0.2 gl(-1) can be achieved by using Luria-Bertani medium in shake flask batch cultures. The CLP3.1-his accumulated in the cells is totally soluble and no basal transcription was found before induction. These two high-level expression systems are promising for use in scale-up production.