Mutsumi Yoshida

Towards Designer Microparticles: Simultaneous Control of Anisotropy, Shape, and Size

Biodegradable, compositionally anisotropic microparticles with two distinct compartments that exhibit controlled shapes and sizes are fabricated. These multifunctional particles are prepared by electrohydrodynamic co-jetting of poly(lactide-co-glycolide) polymer solutions. By varying different solution and process parameters, namely, concentration and flow rate, a variety of non-equilibrium bicompartmental shapes, such as discoid and rod-shaped microparticles are produced in high yields. Optimization of jetting parameters, combined with filtration, results in near-perfect, bicompartmental spherical particles in the size range of 3-5 microm. Simultaneous control over anisotropy, size, shape, and surface structure provides an opportunity to create truly multifunctional microparticles for a variety of biological applications, such as drug delivery, diagnostic assays, and theranostics.

From Advanced Biomedical Coatings to Multi‐Functionalized Biomaterials

In this review, current activities in the development of active biomaterials related to bio‐interfacial design as well as novel concepts for smart polymer systems are highlighted. For instance, advanced biomedical coatings, including vapor‐based polymer coatings for the controlled attachment of biomolecules, and temperature‐responsive coatings for tissue engineering are outlined. Moreover, recent trends with the fabrication of “smart,” switchable surfaces, such as conformationally switching monolayers, will be reviewed. Specific focus will further be given to multi‐functionalized biomaterials including shape‐memory polymers that undergo specific changes in shape in response to stimuli like heat or light. This novel class of intelligent materials highlights a recent trend in biomaterial development targeting is the extension of this concept to multi‐functionalization. In particular, the combination of biodegradability and shape‐memory effects opens a range of opportunities for biomedical applications including intelligent sutures or stents.

Structurally Controlled Bio‐hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured bio-hybrid materials. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.

Purification and Characterization of a Novel Physiological Substrate for Calcineurin in Mammalian Cells

Although the calcium/calmodulin-regulated protein phosphatase calcineurin has been shown to play a role in a number of intracellular processes, relatively few of the downstream phosphoproteins that are dephosphorylated by this enzyme in cells have been described. Calcineurin was previously shown to play a role in amylase secretion by rat pancreatic acinar cells and to specifically dephosphorylate a 24-kDa cytosolic protein. The present study describes the purification and characterization of this novel phosphoprotein, termed CRHSP-24 (calcium-regulatedheat-stable protein with a molecular mass of 24 kDa). Microgram quantities of CRHSP-24 were purified from a large-scale rat pancreas preparation in a procedure involving heat and acid precipitation, anion-exchange chromatography, preparative electrophoresis, electroelution, and two-dimensional electrophoresis. Internal amino acid sequence was obtained from two peptides following trypsin digestion and high pressure liquid chromatography. Both sequences matched with 100% identity nucleotide sequences of expressed sequence tags from human placenta and rat PC-12 cells. Two CRHSP-24 transcripts of 0.7 and 2.9 kilobases were detected in multiple rat tissues by Northern analysis, whereas a single 24-kDa protein was observed by Western blotting. The CRHSP-24 protein is 147 amino acids in length, is composed of nearly 14% proline, and is phosphorylated entirely on serine residues. Western analysis and 32P metabolic labeling of acini revealed CRHSP-24 to be maximally phosphorylated in control cells and to undergo a rapid sustained dephosphorylation on at least 3 serine residues in response to calcium-mobilizing stimuli. Dephosphorylation of CRHSP-24 was completely inhibited by pretreatment of acini with cyclosporin A or FK506. Furthermore, the inhibitory effects of FK506 were blocked by excess rapamycin. The ubiquitous expression of CRHSP-24 in rat tissues suggests that this novel calcineurin substrate plays a common role in calcium-mediated signal transduction.

Molecular aspects of microparticle phagocytosis by dendritic cells

The ability of immature dendritic cells (iDCs) derived from human peripheral blood mononuclear cells to phagocytose poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) as compared to polystyrene MPs and the molecular aspects of this phagocytosis were investigated. Treating iDCs with PLGA or polystyrene fluorospheres of approximately 3 μm in diameter resulted in the internalization of the particles as evidenced by confocal laser scanning micrographs. This uptake of fluorospheres by DCs was decreased by pretreatment of cells with cytochalasin D or by incubation with the fluorospheres at 4°C, and was sensitive to EDTA and trypsin pretreatments in a dose-dependent manner. In agreement with our previous studies, treatment of iDCs with PLGA MPs, but not with polystyrene MPs, led to DC maturation, as measured by increase in release of the autocrine maturation cytokine, tumor necrosis factor-α, which was dependent on ratio of PLGA MPs to DCs. Taken together, this work begins to address the role of phagocytosis on PLGA MP-induced DC maturation and the molecular mechanisms involved.

Purification and identification of a 28-kDa calcium-regulated heat-stable protein. A novel secretagogue-regulated phosphoprotein in exocrine pancreas.

This study reports the purification and identification of a novel 28 kDa phosphoprotein from rat pancreatic acini, previously described as being highly regulated by calcium mobilizing secretagogues, which we have designated calcium-regulated heat-stable protein 28 (CRHSP-28). Internal amino acid sequences of purified CRHSP-28 were obtained following trypsin digestion and found to match with >95% identity the predicted amino acid sequence of a novel cDNA recently identified as being highly expressed in human breast carcinomas. Verification that this cDNA codes for human CRHSP-28 was demonstrated by the ability of antiserum raised against purified rat CRHSP-28 to recognize the recombinant human protein when expressed in bacteria. Furthermore, this antibody was found to specifically react with CRHSP-28 in rat acini following one- and two-dimensional electrophoresis and underwent a marked acidic shift in mobility after cholecystokinin stimulation, a phenomenon indicative of an increase in its phosphorylation. CRHSP-28 is predicted to be extremely hydrophilic, is phosphorylated entirely on serine residues, and bears little homology to any known proteins. Finally, the distribution of the CRHSP-28 protein in various rat tissues revealed that although it was present at low levels in almost all tissues, it was most highly expressed in pancreas, followed by the gastric, intestinal, and colonic mucosa. In view of its relative abundance throughout the digestive system and its apparent regulation by calcium-mobilizing agents, this protein may provide valuable insight into the mechanism(s) of calcium signaling in these tissues.

Immunolocalization of CRHSP28 in exocrine digestive glands and gastrointestinal tissues of the rat

The 28-kDa (on SDS-PAGE) Ca2+-regulated heat stable protein (CRHSP28) was recently purified as novel phosphoprotein in exocrine pancreas, since it undergoes an immediate increase in serine phosphorylation when acini are stimulated with Ca2+-mobilizing agonists. Examination of CRHSP28 protein expression in rat revealed that most was highly expressed in pancreas and other morphologically related exocrine tissues, including the parotid, lacrimal, and submandibular glands. Immunofluorescence staining in pancreas indicated that CRHSP28 was specifically concentrated in zymogen granule-rich areas in the apical cytoplasm of acinar cells. Lack of colocalization with pancreatic lipase in dual immunofluorescence studies confirmed localization of CRHSP28 to the area immediately surrounding the granules. Western analysis of pancreatic zymogen granule membrane proteins indicated CRHSP28 was not associated with the granules following their purification. A similar pattern of apical cytoplasmic secretory granule staining was noted in lacrimal and submandibular glands. CRHSP28 protein was also expressed at relatively high levels in mucosal epithelial cells of the stomach and small intestine. CRHSP28 was found in the supranuclear apical cytoplasm of cells lining the small intestinal crypts, including Paneth cells, and was abundant in the cytoplasm of goblet cells. In the stomach, strong CRHSP28 staining was seen in mucus-secreting cells in the upper portion of the gastric glands and in the apical, granule-rich cytoplasm of chief cells located in the lower portions of the glands. Dual labeling with anti-H+-K+-ATPase demonstrated a comparatively lower expression of CRHSP28 in parietal cells. Collectively, the high relative expression of CRHSP28 in various secretory cell types within the digestive system, together with its intracellular localization surrounding the acinar cell secretory granules, strongly supports a role for CRHSP28 in Ca2+-mediated exocrine secretion.

Biocompatible Polymers: Structurally Controlled Bio-hybrid Materials Based on Unidirectional Association of Anisotropic Microparticles with Human Endothelial Cells (Adv. Mater. 48/2009).

Biocompatible anisotropic polymer particles with bipolar affinity towards human endothelial cells are a novel type of building blocks for microstructured biohybrid materials, report Joerg Lahann and co-workers on p. 4920. Functional polarity due to two biologically distinct hemispheres has been achieved by synthesis of anisotropic particles via electro-hydrodynamic co-jetting of two different polymer solutions and subsequent selective surface modification.