Chlorin e6 (Ce6)-loaded plaque-specific liposome with enhanced photodynamic therapy effect for atherosclerosis treatment.

Recently, photodynamic therapy (PDT) has been considered as a new strategy for atherosclerosis treatment. Targeted delivery of photosensitizer could significantly reduce its toxicity and enhance its phototherapeutic efficiency. CD68 is an antibody that can be conjugated to nano-drug delivery systems to actively target plaque sites, owing to its specific binding to CD68 receptors that are highly expressed on the surfaces of macrophage-derived foam cells. Liposomes are very popular nanocarriers due to their ability to encapsulate a wide range of therapeutic compounds including drugs, microRNAs and photosensitizers, and their ability to be surface-modified with targeting moieties leading to the development of nanocarriers with an improved targeted ability. Hence, we designed a Ce6-loaded liposomes using the film dispersion method, followed by the conjugation of CD68 antibody on the liposomal surface through a covalent crosslinking reaction, forming CD68-modified Ce6-loaded liposomes (CD68-Ce6-mediated liposomes). Flow cytometry results indicated that Ce6-containing liposomes were more effective in promoting intracellular uptake after laser irradiation. Furthermore, CD68-modified liposomes significantly strengthened the cellular recognization and thus internalization. Different cell lines have been incubated with the liposomes, and the results showed that CD68-Ce6-mediated liposomes had no significant cytotoxicity to coronary artery endothelial cells (HCAEC) under selected conditions. Interestingly, they promoted autophagy in foam cells through the increase in LC3-Ⅰ, LC3-Ⅱ expression and the decrease in p62 expression, and restrained the migration of mouse aortic vascular smooth muscle cells (MOVAS) in vitro. Moreover, the enhancement of atherosclerotic plaque stability and the reduction in the cholesterol content by CD68-Ce6-mediated liposomes were dependent on transient reactive oxygen species (ROS) generated under laser irradiation. In summary, we demonstrated that CD68-Ce6-mediated liposomes, as a photosensitizer nano-drug delivery system, have an inhibitory effect on MOVAS migration and a promotion of cholesterol efflux in foam cells, and thereby, represent promising nanocarriers for atherosclerosis photodynamic therapy.

Characterization and Pharmacokinetic Evaluation of Oxaliplatin Long-Circulating Liposomes.

The clinical efficacy of Oxaliplatin (L-OHP) is potentially limited by dose-dependent neurotoxicity and high partitioning to erythrocytes in vivo. Long-circulating liposomes could improve the pharmacokinetic profile of L-OHP and thus enhance its therapeutic efficacy and reduce its toxicity. The purpose of this study was to prepare L-OHP long-circulating liposomes (L-OHP PEG lip) by reverse-phase evaporation method (REV) and investigate their pharmacokinetic behavior based on total platinum in rat plasma using atomic absorption spectrometry (AAS). A simple and a sensitive AAS method was developed and validated to determine the total platinum originated from L-OHP liposomes in plasma. Furthermore, long-circulating liposomes were fully characterized in vitro and showed great stability when stored at 4°C for one month. The results showed that the total platinum in plasma of L-OHP long-circulating liposomes displayed a biexponential pharmacokinetic profile with five folds higher bioavailability and longer distribution half-life compared to L-OHP solution. Thus, long-circulating liposomes prolonged L-OHP circulation time and may present a potential candidate for its tumor delivery. Conclusively, the developed AAS method could serve as a reference to investigate the pharmacokinetic behavior of total platinum in biological matrices for other L-OHP delivery systems.

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds.

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.

Efficient generation of functional cardiomyocytes from human umbilical cord-derived virus-free induced pluripotent stem cells.

We have previously demonstrated that human umbilical cord-derived mesenchymal stem cells (UC-MSCs) can differentiate into cardiomyocyte-like cells. However, no contracting cells were observed during differentiation. In this study, we generated induced pluripotent stem cells (iPSCs) from UC-MSCs using mRNA reprogramming and focused on the differentiation of reprogrammed iPSCs into functional cardiomyocytes. For cardiac differentiation, the spontaneously contracting cell clusters were present on day 8 of differentiation. Immunostaining studies and cardiac-specific gene expression confirmed the cardiomyocyte phenotype of the differentiated cells. Electrophysiology studies indicated that iPSCs derived from UC-MSCs had a capacity for differentiation into nodal-, atrial-, and ventricular-like phenotypes based on action potential characteristics, and the derived cardiomyocytes exhibited responsiveness to ß-adrenergic and muscarinic stimulations. Moreover, the derived cardiomyocytes displayed spontaneous intracellular Ca2+ transients. These results demonstrate that functional cardiomyocytes can be generated from reprogrammed UC-MSCs, and the methodology described here will serve as a useful protocol to obtain functional cardiomyocytes from human mesenchymal stem cells.

Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy.

The safe and effective delivery of drugs is a major obstacle in the treatment of ischemic stroke. Exosomes hold great promise as an endogenous drug delivery nanosystem for the treatment of cerebral ischemia given their unique properties, including low immunogenicity, innate stability, high delivery efficiency, and ability to cross the blood-brain barrier (BBB). However, exosome insufficient targeting capability limits their clinical applications. In this study, the c(RGDyK) peptide has been conjugated to the exosome surface by an easy, rapid, and bio-orthogonal chemistry. In the transient middle cerebral artery occlusion (MCAO) mice model, The engineered c(RGDyK)-conjugated exosomes (cRGD-Exo) target the lesion region of the ischemic brain after intravenous administration. Furthermore, curcumin has been loaded onto the cRGD-Exo, and administration of these exosomes has resulted in a strong suppression of the inflammatory response and cellular apoptosis in the lesion region. The results suggest a targeting delivery vehicle for ischemic brain based on exosomes and provide a strategy for the rapid and large-scale production of functionalized exosomes.

Soft Hydrogels Featuring In-Depth Surface Density Gradients for the Simple Establishment of 3D Tissue Models for Screening Applications.

Three-dimensional (3D) cell culture models are gaining increasing interest for use in drug development pipelines due to their closer resemblance to human tissues. Hydrogels are the first-choice class of materials to recreate in vitro the 3D extra-cellular matrix (ECM) environment, important in studying cell-ECM interactions and 3D cellular organization and leading to physiologically relevant in vitro tissue models. Here we propose a novel hydrogel platform consisting of a 96-well plate containing pre-cast synthetic PEG-based hydrogels for the simple establishment of 3D (co-)culture systems without the need for the standard encapsulation method. The in-depth density gradient at the surface of the hydrogel promotes the infiltration of cells deposited on top of it. The ability to decouple hydrogel production and cell seeding is intended to simplify the use of hydrogel-based platforms and thus increase their accessibility. Using this platform, we established 3D cultures relevant for studying stem cell differentiation, angiogenesis, and neural and cancer models.

Accelerating and increasing nano-scaled pore formation on electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers.

Porous fibers are advantageous for filtration systems, drug delivery systems, and in the field of tissue engineering, in comparison to their non-porous counterparts. In this study, we developed a facile technique including two steps to generate poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) porous fibers with a controllable pore size. An electrospinning technique was employed to obtain five types of PHBV/poly(ethylene oxide) (PEO)-blended fibers (PHBV:PEO = 9:1, 8:2, 7:3, 6:4, 5:5) with PEO as the porogen. PEO was leached out by simulated body fluid (SBF) and water, respectively. The pore morphology and calcium deposition of the resulting fibers were compared to those formed on film through the SEM-EDX analysis. It was revealed that pore size and number increased with increasing PEO percentage in the fiber or film. The pore size on the films (at micrometer scale) was much larger than that of nanofibers, which was in the range of 70-120 nm. The simultaneous removal of PEO and deposition of calcium phosphate through SBF buffer enhanced synergistically both the pore formation and mineral deposition. The different phase separation mechanisms explain the different pore morphologies in the film and the nanofibers. The cellular experimental results show that fibers with nanometer-scale pores and minerals can enhance the proliferation of bone marrow-derived mesenchymal stem cells.

Effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal stem cells in vitro and bone regeneration in vivo.

Among a variety of polymers, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a microbial polyester, with biodegradable, nonantigenic, and biocompatible properties, is attracting more and more attention in tissue engineering. Hydroxyapatite (HA), similar to the mineral component of natural bone, is known to be osteoconductive, nontoxic, and noninflammatory. In this study, aligned and random-oriented PHBV nanofibrous scaffolds loaded with HA nanoparticles were fabricated through electrospinning technique. Mesenchymal stem cells (MSCs) derived from rat bone marrow were used to investigate the effects of HA and orientation of fibers on cell proliferation and differentiation in vitro. Cell proliferation tested with CCK-8 assay indicated that the MSCs attached and proliferated more favorably on random-oriented PHBV nanofibrous meshes without HA. After one, two and four weeks of cell seeding, osteogenic markers including alkaline phosphate (ALP), osteocalcin (OCN), and mineralized matrix deposits were detected, respectively. The results indicated that the introduction of HA could induce MSCs to differentiate into osteoblasts. Moreover, 3D PHBV/HA scaffolds made from aligned and random-oriented nanofibers were implanted into critical-sized rabbit radius defects and exhibited significant effects on the repair of critical bone defects, implying their promising applications in bone tissue engineering.

Dynamics of exosome internalization and trafficking.

Cells release exosomes into extracellular medium. Although the important roles of exosomes in many physiological and pathological processes are being revealed, the mechanism of exosome-cell interaction remains unclear. In this article, employing real-time fluorescence microscopy, the motion of exosomes on the plasma membrane or in the cytoplasm of recipient PC12 cells was observed directly. In addition, several motion modes of exosomes were revealed by single particle tracking (SPT). The changes between motion modes were also detected, presenting the dynamic courses of exosome attachment onto plasma membrane and exosome uptake. Octadecyl rhodamine B chloride (R18) was found to be useful to distinguish endocytosis from fusion during exosome uptake. Colocalization with organelle markers showed exosomes were sorted to acidic vesicles after internalization. The results provide new sight into the exosome-cell interaction mode and the intercellular trafficking of exosomes. This study will help to understand the roles of exosomes at cell level.

The effects of PHBV electrospun fibers with different diameters and orientations on growth behavior of bone-marrow-derived mesenchymal stem cells.

Microenvironments in which cells live play an important role in the attachment, growth and interactions of cells. To mimic the natural structure of extracellular matrices, electrospinning was applied to fabricate biomaterials into ultrafine fibers. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biocompatible and biodegradable polyester, has been shown to be an excellent biomaterial candidate for tissue engineering. In this study, five types of PHBV fibrous scaffolds with different diameters and orientations were obtained by changing solvents, concentration of electrospun solution and collector. Three kinds of scaffolds with good continuity and suitable mechanical properties, selected according to the morphology and mechanical properties of the scaffolds, were used for studying the influence of fiber diameter and orientation on growth behavior of bone-marrow-derived mesenchymal stem cells (MSCs). The results indicated that the random-oriented nanofibrous scaffold is most favorable for cell growth compared to other scaffolds, while the microfibrous scaffold resulted in the lowest viability of MSCs. The orientation of nanofibers showed a distinct effect on cell morphology by guiding cell skeleton extension. Both the random-oriented and aligned PHBV nanofibrous scaffolds showed to be good candidates for applications in tissue engineering.

Efficient creation of cellular micropatterns with long-term stability and their geometric effects on cell behavior.

Cellular micropatterning with bio-adhesive and nonadhesive areas has attracted increasing interest for the precise design of cell-to-surface attachment in cell biology studies, tissue engineering, cell-based biosensors, biological assays, and drug development and screening. In this paper we describe a simple and efficient method to create a two-dimensional stable cellular microenvironment, which is based on (1) forming a protein-resistant oligo(ethylene glycol) methyl ether methacrylate polymer layer on the substrates via surface-initiated atom transfer radical polymerization; (2) placing a defined photomask on the substrate and exposing the substrate to ultraviolet light; and (3) immersing the patterned surface in a fibronectin solution to form cell-adhesive protein patterns in a cell-resistant background. The resulting surfaces are tailored into cell-adhesive and cell-resistant regions. Three different types of cells (NIH-3T3, PC12, bone marrow-derived mesenchymal stem cells) are seeded on such patterned surfaces to form cellular patterns. The geometric effects on cell behavior are investigated. The long-term stability is tested by NIH-3T3 fibroblasts and mesenchymal stem cells and excellent retention of cellular patterns is observed. The strategy illustrated here offers an efficient way to create a stable, patterned cellular microenvironment, and could be employed in tissue engineering to study the effect of micropatterns on the proliferation and differentiation of cells, and in particular mesenchymal stem cells.

Nanomechanical interactions of phenylalanine-glycine nucleoporins studied by single molecule force-volume spectroscopy.

Phenylalanine-glycine (FG)-repeat nucleoporins (Nups) form the major components of the selective gating mechanism in the nuclear pore complex (NPC). Hence, a primary requirement is to understand how they vacillate between preventing the access of passively diffusing molecules and promoting the translocation of receptor-bound cargo into the NPC. To shed light on such behavior, we have studied the nanomechanical properties of a cysteine-modified FG-rich C-terminal domain of hNup153 (i.e., cNup153) and its interactions with importin-beta. This is carried out using single molecule force spectroscopy (SMFS) with the atomic force microscope (AFM). In the absence of importin-beta, cNup153 is highly flexible and can be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, indicating a lack of intra-FG interactions, i.e., natively unfolded. Importin-beta-modified AFM tips reveal complex binding topologies with cNup153, and provide evidence for binding promiscuity in FG-receptor interactions. These differences suggest that cooperativity between FG-domains arises from FG-receptor interactions instead of FG-FG interactions. On a technical note, this work highlights an improved SMFS technique which involves pre-passivating the underlying substrate surface with polyethylene glycol to reduce undesirable AFM tip-surface effects. A high yield of acceptable data is subsequently obtained from the low surface coverage of target molecules by implementing SMFS measurements in force-volume (FV) mode.