The majority of nanoparticles designed for cellular delivery of drugs and imaging agents enter the cell via endocytotic pathways leading to their entrapment in endosomes that present a robust barrier to further trafficking of the nanoparticles within the cells. A few materials, such as the cell penetrating peptides (CPPs), are known to enter cells not only via endocytosis, but also via translocation through the cell membrane into the cytoplasm, successfully bypassing the endosomes. We report here that random copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate and poly(ethylene glycol) methacrylate, p(DMAPS-ran-PEGMA), are internalized in cells primarily via translocation through the cell membrane rather than endocytosis. The properties of the polymers and their modes of uptake were investigated systematically by dynamic light scattering, confocal fluorescence microscopy, and flow cytometry. Using specific inhibitors of the cellular uptake machinery in a human cervical carcinoma cell line (HeLa), we show that these nontoxic synthetic polyzwitterions exist in cell media as self-assembled nanospheres that unravel as they adsorb on the plasma membrane and translocate through it. Conjugates of p(DMAPS-ran-PEGMA) with rhodamine B were delivered selectively to the mitochondria, whereas doxorubicin (Dox)-p(DMAPS-ran-PEGMA) conjugates were accumulated in both the nucleus and the mitochondria, effectively inducing apoptosis in HeLa cells. These findings suggest that the noncytotoxic and readily synthesized p(DMAPS-ran-PEGMA) can find applications as bioimaging tools and drug nanocarriers.

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Cycloamylose (CA) exhibits differences in geometry and greater colloidal stability compared with amylose. Here we report the synthesis of a cationic CA derivative and its application for gene delivery. Cationic CA (catCA) and cationic amylose (catAmy) were synthesized by introducing spermine groups. The interactions between catCA or catAmy with plasmid DNA encoding firefly luciferase was examined by gel electrophoresis, dynamic light scattering, and transmission electron microscopy. Activity as a gene delivery system was evaluated by flow cytometry and luciferase assays. CatCA formed a condensed pDNA complex ( approximately 250 nm in size). The catCA complex showed enhanced cellular uptake and greater transfection efficiency than the catAmy complex. Hemolysis by membrane destabilization and the effects of hydroxychloroquine on transfection ability suggest that the formation of a supramolecular complex with CA is important for high transfection activity. These results suggested that CA can be used as new polysaccharide-based biomaterials.

Ectopic bone formation after joint replacement or brain injury in humans is a serious complication that causes immobility of joints and severe pain. However, mechanisms underlying such ectopic bone formation are not fully understood. Bone morphogenetic protein (BMPs) are defined as inducers of ectopic bone formation, and they are regulated by several types of inhibitors. ANA is an antiproliferative molecule that belongs to Tob/BTG family, but its activity in bone metabolism has not been known. Here, we examined the role of ANA on ectopic bone formation activity of BMP. In ANA-deficient and wild-type mice, BMP2 was implanted to induce ectopic bone formation in muscle. ANA deficiency increased mass of newly formed bone in vivo compared with wild-type based on 3D-CT analyses. ANA mRNA was expressed in bone in vivo as well as in osteoblastic cells in vitro. Such ANA mRNA levels were increased by BMP2 treatment in MC3T3-E1 osteoblastic cells. Overexpression of ANA suppressed BMP-induced expression of luciferase reporter gene linked to BMP response elements in these cells. Conversely, ANA mRNA knockdown by small interference RNA enhanced the BMP-dependent BMP response element reporter expression. It also enhanced BMP-induced osteoblastic differentiation in muscle-derived C2C12 cells. Immunoprecipitation assay indicated that ANA interacts with Smad8. Thus, ANA is a suppressor of ectopic bone formation induced by BMP, and this inhibitory ANA activity is a part of the negative feedback regulation of BMP function.Ectopic or heterotopic bone formation is one of the major complications associated with joint replacement surgery and brain injury (1, 2). Such pathological ectopic bone formation and heterotopic ossification cause ankylosis of joints or persistent pain and significantly deteriorated quality of life. However, the molecules involved in these ectopic bone formation events have not been fully understood.Bone morphogenetic proteins (BMPs) 3 were first identified as an inducer of ectopic bone formation (3, 4). Although BMP2 is required for fracture healing, skeletal development of the limbs was normal in the absence of BMP2 (5). In contrast, BMP is absolutely required for ectopic bone formation in muscle (3), and no other growth factors could replace such potent ectopic bone formation activity of this molecule. Thus, BMP action on ectopic bone formation is one of the unique features of this molecule.BMP activity is under the control of several groups of modulators against its signaling pathway. One of these groups of modulators that control BMP2 signaling in osteoblasts includes the Tob/BTG antiproliferative protein family, comprising Btg1, Btg2, Tob, Tob2, Pc3, and ANA. These molecules share a homologous region (BTG/Tob homology domain) in their N-terminal ends (6) and suppress cell proliferation when they are overexpressed in NIH3T3 cells (7-13).Among the members of Tob/BTG family, Tob was shown to be a BMP antagonist previously (14, 15). However, effects of other members of Tob/BTG family on BMP activity have not y...

This study describes chitosan-phosphorylcholine (CH-PC) films able to support the formation of cell aggregates (spheroids), which are important for tissue engineering and pharmacological studies. The surface topography, charge, thickness, and rheology of CH-PC thin films were characterized by AFM, zeta-potential measurements, SPR spectroscopy, and QCM-D measurements. The CH-PC films are highly hydrated gels, independently of the level of PC incorporation (15-40 mol-% PC/glucosamine units). QCM-D studies established that the amount of fibrinogen adsorbed on CH-PC films decreased with increasing PC content. CH-PC surfaces underwent a transition from moderately cell-adhesive (CH-PC15) to non-adhesive (CH-PC40). Optical micrographs of HUVEC and MCF-7 cell lines cultured on CH-PC surfaces showed that they form spheroids on CH-PC25 and CH-PC40 films.