Membrane Translocation and Organelle-Selective Delivery Steered by Polymeric Zwitterionic Nanospheres.

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.

Novel artificial nerve transplantation of human iPSC-derived neurite bundles enhanced nerve regeneration after peripheral nerve injury.

BACKGROUND: Severe peripheral nerve damage always requires surgical treatment. Autologous nerve transplantation is a standard treatment, but it is not sufficient due to length limitations and extended surgical time. Even with the available artificial nerves, there is still large room for improvement in their therapeutic effects. Novel treatments for peripheral nerve injury are greatly expected. METHODS: Using a specialized microfluidic device, we generated artificial neurite bundles from human iPSC-derived motor and sensory nerve organoids. We developed a new technology to isolate cell-free neurite bundles from spheroids. Transplantation therapy was carried out for large nerve defects in rat sciatic nerve with novel artificial nerve conduit filled with lineally assembled sets of human neurite bundles. Quantitative comparisons were performed over time to search for the artificial nerve with the therapeutic effect, evaluating the recovery of motor and sensory functions and histological regeneration. In addition, a multidimensional unbiased gene expression profiling was carried out by using next-generation sequencing. RESULT: After transplantation, the neurite bundle-derived artificial nerves exerted significant therapeutic effects, both functionally and histologically. Remarkably, therapeutic efficacy was achieved without immunosuppression, even in xenotransplantation. Transplanted neurite bundles fully dissolved after several weeks, with no tumor formation or cell proliferation, confirming their biosafety. Posttransplant gene expression analysis highlighted the immune system's role in recovery. CONCLUSION: The combination of newly developed microfluidic devices and iPSC technology enables the preparation of artificial nerves from organoid-derived neurite bundles in advance for future treatment of peripheral nerve injury patients. A promising, safe, and effective peripheral nerve treatment is now ready for clinical application.

Tuning the properties and functions of 17ß-estradiol-polysaccharide conjugates in thin films: impact of sample history.

In addition to its role in the regulation of sex-related processes, 17ß-estradiol (E2) participates in the prevention and treatment of cardiovascular diseases via nongenomic pathways mediated by estrogen receptors (ER-α) located in the cell membrane. To achieve specific nongenomic activity of E2, we linked E2 (4.4 mol %) to chitosan-phosphorylcholine (CH-PC) (20 mol % PC). Injections of ER-α solutions (5 to 100 nmol L(-1)) over rehydrated CH-PC-E2 thin films led to permanent adsorption of ER-α to the film surface, as detected by quartz crystal microbalance with dissipation (QCM-D). However, ER-α did not bind onto CH-PC-E2 films formed in situ and never dried. X-ray photoelectron spectroscopy (XPS) analysis of spin-cast CH-PC-E2 films revealed significant E2 enrichment of the topmost section of the film, attributed to the preferential migration of E2 toward the film/air interface upon drying. Mechanical analysis of CH-PC-E2 films in the frequency domain probed by QCM-D indicated that rehydrated films behave as an entangled network with junction points formed by self-assembly of hydrophobic E2 moieties and by ion pairing among PC groups, whereas films formed in situ are entangled polymer solutions with temporary junctions. The structural analysis presented offers useful guidelines for the study of amphiphilic biomacromolecules designed for therapeutic use as thin films.

Functional cycloamylose as a polysaccharide-based biomaterial: application in a gene delivery system.

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.

Protein-conjugated quantum dots effectively delivered into living cells by a cationic nanogel.

Quantum dots (QDs) have attracted attention for their potential as a cell imaging regent. However, the development of effective intracellular delivery system for QDs is needed to apply various cell lines without affecting cellular function. We reported here new QDs delivery system by using cationic nanogel consisting of cholesterol-bearing pullulan modified with an amino group (CHPNH2). The uptake of hybrid nanoparticles into HeLa cells was followed by flow cytometry, and confocal laser scanning fluorescence microscopy. Protein-conjugated QDs were effectively internalized into cells by the nanogel compared with a cationic liposome system. The hybrid nanoparticle was used to stain rabbit mesenchymal stem cells (MSCs) so as to evaluate their effect on cell function. CHPNH2-QD hybrid nanoparticles remained detectable inside MSCs for at least 2 weeks of culture and had little effect on the in vitro chondrogenic ability of MSCs. The hybrid nanoparticles are a promising candidate as a cell tracer in tissue engineering.

Phosphorylcholine-modified chitosan films as effective promoters of cell aggregation: correlation between the films properties and cellular response.

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.

Polysaccharide nanogel gene delivery system with endosome-escaping function: Co-delivery of plasmid DNA and phospholipase A2.

We developed a novel gene delivery system capable of endosome disruption using a polysaccharide-based cationic nanogel composed of a hexadecyl group-bearing cationic cycloamylose nanogel (C16-catCA nanogel) and phospholipaseA(2) (PLA(2)) to hydrolyze membrane phospholipids. C16-catCA nanogel formed nanoparticles with PLA(2) and pDNA by hydrophobic and electrostatic interactions. Both pDNA and PLA(2) were effectively internalized into cells by the C16-catCA nanogel. In addition, the pDNA expression level was enhanced when complexed with specific concentrations of PLA(2). PLA(2) complexed with C16-catCA nanogel also showed a similar hemolytic activity against red blood cells to that observed using native PLA(2). These results suggest that the C16-catCA nanogel/PLA(2) complex possesses membrane disruption ability when delivered into cells and triggers the subsequent release of pDNA from the endosome to the cytoplasm. This is the first report of co-delivery of pDNA and PLA(2) using the same carrier to achieve effective gene delivery.

DNA separation by cholesterol-bearing pullulan nanogels.

We present an application of a novel DNA separation matrix, cholesterol-bearing pullulan (CHP) nanogels, for microchip electrophoresis. The solution of the CHP showed a unique phase transition around 30 mg∕ml and formed gel phase over this critical concentration. This gel phase consists of the weak hydrophobic interactions between the cholesterols could be easily deformed by external forces, and thus, loading process of the CHP nanogels into microchannels became easier. The high concentration of the CHP nanogels provided excellent resolutions especially for small DNA fragments from 100 to 1500 bp. The separation mechanism was discussed based on Ogston and Reptation models which had developed in gels or polymer solutions. The result of a single molecule imaging gave us an insight of the separation mechanism and the nanogel structures as well.

ANA deficiency enhances bone morphogenetic protein-induced ectopic bone formation via transcriptional events.

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-muCT 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.