Highly Fluorescent Gold Cluster Assembly.

The photoluminescence (PL) of metal nanoclusters (NCs), originating from their molecule-like electronic structure, is one of the most intriguing properties of NCs. Although various strategies such as tailoring the size, structure, and chemical environment of NCs have shown to improve the PL, their quantum yields (QYs) are still lagging far behind those of conventional luminescent materials, including quantum dots and organic fluorophores. Herein, we report the synthesis of highly luminescent gold cluster assembly (GCA) from Zn2+-ion-mediated assembly of Au4(SRCOO-)4 clusters using mercaptocarboxylic acid as a protective ligand and reductant as well as a growth suppressor. The synergetic combination of unique aurophilic interactions among Au4 clusters and the rigidified chemical environment induced by metal ion chelation through carboxylate groups is responsible for the ultrabright greenish-blue fluorescence with a QY up to 90%. Furthermore, the unique flexibility of dis/reassembly and the aggregation-dependent strong fluorescence of GCA offer a great potential for applications in biodegradable and trackable drug delivery systems.

Atom Transfer Radical Polymerization of Multishelled Cationic Corona for the Systemic Delivery of siRNA.

We propose an effective siRNA delivery system by preparing poly(DAMA-HEMA)-multilayered gold nanoparticles using multiple surface-initiated atom transfer radical polymerization processes. The polymeric multilayer structure is characterized by transmission electron microscopy, matrix-associated laser desorption/ionization time-of-flight mass spectrometry, UV-vis spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, and ζ-potential. The amount of siRNA electrostatically incorporated into the nanoparticle can be tuned by the number of polymeric shells, which in turn influences the cellular uptake and gene silencing effect. In a bioreductive environment, the interlayer disulfide bond breaks to release the siRNA from the degraded polymeric shells. Intravenously injected c-Myc siRNA-incorporated particles accumulate in the tumor site of a murine lung carcinoma model and significantly suppress the tumor growth. Therefore, the combination of a size-tunable AuNP core and an ATRP-functionalized shell offers control and versatility in the effective delivery of siRNA.

Electrospun Nanofibrous Sheets for Selective Cell Capturing in Continuous Flow in Microchannels.

Electrospun nanofibrous meshes were surface-modified for selective capturing of specific cells from a continuous flow in PDMS microchannels. We electrospun nanofibrous mats composed of poly(ε-carprolactone) (PCL) and amine-functionalized block copolymers composed of PCL and poly(ethylenimine) (PEI). A mixture of biotinylated PEG and blunt PEG was chemically tethered to the nanofibrous mats via the surface-exposed amines on the mat. The degree of biotinylation was fluorescently and quantitatively assayed for confirming the surface-biotinylation levels for avidin-specific binding. The incorporation level of avidin gradually increased when the blend ratio of biotinylated PEG on the mat increased, confirming the manipulated surfaces with various degree of biotinylation. Biotinylated cells were incubated with avidin-coated biotinylated mats and the specific binding of biotinylated cells was monitored in a microfluidic channel with a continuous flow of culture medium, which suggests efficient and selective capturing of the biotinylated cells on the nanofibrous mat.

Selective Cell-Cell Adhesion Regulation via Cyclic Mechanical Deformation Induced by Ultrafast Nanovibrations.

The adoption of dynamic mechanomodulation to regulate cellular behavior is an alternative to the use of chemical drugs, allowing spatiotemporal control. However, cell-selective targeting of mechanical stimuli is challenging due to the lack of strategies with which to convert macroscopic mechanical movements to different cellular responses. Here, we designed a nanoscale vibrating surface that controls cell behavior via selective repetitive cell deformation based on a poroelastic cell model. The vibrating indentations induce repetitive water redistribution in the cells with water redistribution rates faster than the vibrating rate; however, in the opposite case, cells perceive the vibrations as a one-time stimulus. The selective regulation of cell-cell adhesion through adjusting the frequency of nanovibration was demonstrated by suppression of cadherin expression in smooth muscle cells (fast water redistribution rate) with no change in vascular endothelial cells (slow water redistribution rate). This technique may provide a new strategy for cell-type-specific mechanical stimulation.

Preparation of Stretchable Nanofibrous Sheets with Sacrificial Coaxial Electrospinning for Treatment of Traumatic Muscle Injury.

Traumatic muscle injury with massive loss of muscle volume requires intramuscular implantation of proper scaffolds for fast and successful recovery. Although many artificial scaffolds effectively accelerate formation and maturation of myotubes, limited studies are showing the therapeutic effect of artificial scaffolds in animal models with massive muscle injury. In this study, improved myotube differentiation is approved on stepwise stretched gelatin nanofibers and applied to damaged muscle recovery in an animal model. The gelatin nanofibers are fabricated by a two-step process composed of co-axial electrospinning of poly(ɛ-caprolactone) and gelatin and subsequent removal of the outer shells. When stepwise stretching is applied to the myoblasts on gelatin nanofibers for five days, enhanced myotube formation and polarized elongation are observed. Animal models with volumetric loss at quadriceps femoris muscles (>50%) are transplanted with the myotubes cultivated on thin and flexible gelatin nanofiber. Treated animals more efficiently recover exercising functions of the leg when myotubes and the gelatin nanofiber are co-implanted at the injury sites. This result suggests that mechanically stimulated myotubes on gelatin nanofiber is therapeutically feasible for the robust recovery of volumetric muscle loss.

Gold nanospheres and nanorods for anti-cancer therapy: comparative studies of fabrication, surface-decoration, and anti-cancer treatments.

Various gold nanoparticles have been explored as cancer therapeutics because they can be widely engineered for use as efficient drug carriers and diagnostic agents, and in photo-irradiation therapy. In the current review, we focused on shape-dependent biomedical applications of gold nanoparticles including gold nanospheres and nanorods. Fabrication and functionalization strategies of two different gold nanoparticles for anti-cancer therapy are introduced and the distinguishing performance depending on the shape is discussed to suggest the best carrier shape for specific applications. Moreover, recent advances in anti-cancer immunotherapy using gold nano-carriers are discussed. Thus, this comparative review can be helpful in deciding on suitable shapes and surface-modification strategies for preparing various gold nanoparticle-based therapeutics in anti-cancer therapy.

Biomaterials and controlled release strategy for epithelial wound healing.

The skin and cornea are tissues that provide protective functions. Trauma and other environmental threats often cause injuries, infections and damage to these tissues, where the degree of injury is directly correlated to the recovery time. For example, a superficial skin or corneal wound may recover within days; however, more severe injuries can last up to several months and may leave scarring. Thus, therapeutic strategies have been introduced to enhance the wound healing efficiency and quality. Although the skin and cornea share similar anatomic structures and wound healing process, therapeutic agents and formulations for skin and cornea wound healing differ in accordance with the tissue and wound type. In this review, we describe the anatomy and epithelial wound healing processes of the skin and cornea, and summarize the therapeutic molecules that are beneficial to the respective regeneration process. In addition, biomaterial scaffolds that inherently possess bioactive properties or modified with therapeutic molecules for topical controlled release and enhanced wound healing efficiency are also discussed.

Electrospining of Nanofibrous Meshes Composed of Hypromellose and Poly(vinyl alcohol) for One-Day Release of Cationic Peptide.

Various biocompatible polymers have been developed using electrospun nanofibers for local drug delivery matrices, but many of them are non-FDA-approved polymers or chemicals. Material safety should be considered in biomedical devices, but the effectiveness of electrospun nanofibers is limited with only the approved chemicals. Therefore, we considered the material in FDA-approved polymers and solvents and developed nanofibers using the general additives in the pharmaceutical industry, such as hypromellose, poly(vinyl alcohol) (PVA), and Gellan. The nanofiber, mainly composed of hypromellose and PVA, was applied to a local peptide drug delivery system. Electrostatically complexed Gellan and peptide were loaded in the nanofiber by co-electrospinning. The morphology of the nanofibers with different PVA blending ratios was visualized by scanning electron microscope. The nanofiber composed of only hydrophilic polymers quickly wetted in water and became a transparent gel-like lump. A drug release test of peptide-loaded nanofiber was performed resulted in 37% of initial burst release suppression with the gellan and peptide loaded nanofiber compared with solely peptide loaded hypromellose nanofiber. In addition, higher PVA blending to hypromellose was slightly effective for sustained release of peptide compared with pure peptide-loaded hypromellose nanofiber. Therefore, we suggest the potential application of hypromellose/PVA nanofiber-loaded Gellan/peptide complex to a mucosal layer drug delivery patch.

Coaxial Hydro-Nanofibrils for Self-Assembly of Cell Sheets Producing Skin Bilayers.

Bilayered cell sheets were fabricated with coaxial hydro-nanofibrils for three-dimensional (3D) cultivation in a biomimetic environment. Polycaprolactone (PCL) was electrospun and hydrolyzed to release fragmented nanofibrils (NF) in an alkaline condition. Methacrylated gelatin (GelMA) was adsorbed and phototethered on the surface of the fibrils to prepare coaxial NF composed of hydrophilic shells and hydrophobic cores. GelMA layers on the NF were characterized by X-ray photoemission spectroscopy, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The GelMA showed higher decoration level on NF compared to that on native gelatin. GelMA-decorated NF significantly enhanced cell proliferation rate and phenotypic expression of human dermal fibroblasts when spontaneous formation of cell sheets was observed for 7 days. HaCaT cells were layered on top of the fibroblast sheets and further cultivated in air-water interfaces to prepare bilayered skin sheets. After 21 days of incubation, the top layers of the bilayered sheets showed higher expression of pan-keratin, and the dermal cells showed higher proliferation in the GelMA-decorated NF.

Doxorubicin encapsulated clicked gold nanoparticle clusters exhibiting tumor-specific disassembly for enhanced tumor localization and computerized tomographic imaging.

Gold nanoparticles (AuNPs) and matrix metalloproteinase (MMP)-2 cleavable peptides are clicked into gold nanoparticle clusters (AuNCs) for enhanced drug localization and micro computerized tomography (µCT) theranostic of tumors. AuNPs are co-functionalized with doxorubicin (DOX) and an azide-terminated polymer (DOX/N3@AuNPs), and the DOX/N3@AuNPs are associated into DOX@AuNCs in the presence of an alkyne-terminated MMP-2 cleavable peptide (alkyne-peptide-alkyne; APA) by click chemistry. MMP-2-dependent dissociation shows that DOX@AuNCs are highly sensitive to the MMP-2 and are almost completed digested into single nanoparticles. DOX liberation shows that > 75% of the conjugated DOX is bursted out from the digested DOX@AuNCs while < 20% of DOX is released from the integrate DOX@AuNCs within 3 h in acidic conditions, suggesting that DOX is only liberated from dissociated DOX@AuNCs in acidic conditions. In vivo study shows that DOX@AuNCs accumulate in tumor ~ 150 times higher than DOX/N3@AuNPs do and efficiently suppress tumor growth. Mice administered with AuNCs shows clearer µCT images of tumors. Thus, DOX@AuNCs are expected promising carriers for both anticancer therapy and tumor imaging.

Multilayered electrospun fibrous meshes for restenosis-suppressing metallic stents.

Nanofiber is a flexible and highly porous mesh that is advantageous for coating bare metal stent and local drug delivery. Herein, we developed drug-eluting stent coated with PCL/PU blending coaxial nanofiber for controlling drug release manner and suppressing in-stent restenosis, which is a representative side effect of stenting surgery. The shell of coaxial electrospun nanofibrous are composed of poly (ε-caprolactone) (PCL) and polyurethane (PU) for biodegradability and elasticity to the polymeric coating of stent. Paclitaxel (PTX) is loaded into both the core and shell through electrospinning using coaxial nozzle with different weight ratio. The morphology of nanofiber-coated stent, expansion state, and core/shell structure of nanofiber were visualized by scanning electron microscope and transmission electron microscope. As more amount of PCL/PU was infused from the outer nozzle, PTX release speed from the nanofiber was increased. And PTX suppressed L6 cell proliferation in vitro expecting potential possibility of PTX-loaded coaxial nanofiber as a drug-eluting stent coating material. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 628-635, 2017.

Clustering siRNA conjugates for MMP-responsive therapeutics in chronic wounds of diabetic animals.

The MMP-responsive breakdown of siRNA clusters was translated to site-specific gene transfection and enhanced wound healing in diabetic ulcers. MMP-2 siRNA was chemically tethered to the end of multi-armed PEG via MMP-cleavable linkers (4PEG-siRNA) and subsequently clustered into submicron particles complexed with LPEI. 4PEG-siRNA was more tightly complexed with LPEI and the associated cluster showed higher resistance against RNase attack, in comparison to naked siRNA. Because the size of the clusters increased depending on the increase in charge ratio of LPEI to siRNA, cellular uptake of the 4PEG-siRNA/LPEI cluster was significantly attenuated due to the huge size of the cluster. However, upon MMP treatment, the cluster dissociated into smaller particles and was efficiently endocytosed by cells. An in vivo fluorescence resonance energy transfer (FRET) study also revealed that the clusters were effectively dissociated in MMP-rich environments of dorsal wounds in diabetic animals. In addition, diabetic ulcers treated with the clusters showed a faster wound closure rate and the recovered tissue expressed a larger amount of cytokeratin along with a lower expression level of MMP-2 compared to the other groups.

High-yield clicking and dissociation of doxorubicin nanoclusters exhibiting differential cellular uptakes and imaging.

Gold nanoparticles (AuNPs) and quantum dots (Qdots) were clicked into doxorubicin nanoclusters that showed enzyme-dependent dissociation behaviors for differential cellular uptakes and imaging. The AuNPs were co-functionalized with doxorubicin (DOX) and azide-terminated polymer (DOX/azide@AuNP), while an enzyme-cleavable peptide and alkyne-terminated polymer were sequentially conjugated on Qdot surface (Alkyne-MMP@Qdot). Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and fluorescence imaging detected the azide and alkyne groups on DOX/azide@AuNP and Alkyne-MMP@Qdot, respectively, and the click-reactivity was also confirmed. In the presence of the catalyst, two nanoparticles were clicked to doxorubicin nanoclusters, which increased the volume of the particles ca. 343-fold within 30min. Upon matrix metalloproteinase-2 (MMP-2) digestion, the nanoclusters were clearly dissociated into smaller particles, and the fluorescence of the quenched Qdot was also recovered, which suggests that the nanoclusters respond to MMP-2 concentrations and can thus be employed for cancer imaging. Confocal microscopy and an elemental analysis of the cancer cells revealed that the cellular uptakes of doxorubicin nanoclusters significantly increased at higher MMP-2 concentrations, and doxorubicin could also be cleaved for anti-cancer effects. In vivo and in vitro cytotoxicity assay accordingly showed that the cytotoxicity of doxorubicin nanoclusters against cancer cells increased in MMP-2-rich environments such as tumor site. Thus, these nanoclusters containing DOX/azide@AuNP and Alkyne-MMP@Qdot are expected to be multifunctional carriers for targeted anti-cancer treatments and imaging.

Antibacterial Nanofibrous Mats Composed of Eudragit for pH-Dependent Dissolution.

A pH-responsive nanofibrous mesh was prepared for the controlled release of antibiotics in response to pH changes. Eudragit EPO (EPO) and Eudragit L100 (L100) were injected through inner and outer needle and simultaneously electrospun through coaxial nozzles composed of inner and outer needles. Various amounts of EPO and L100 were coejected with tetracycline through the needle and simultaneously electrospun to the fibrous meshes. The mass erosion rates of the meshes at pH 6.0 gradually decreased as the amounts of EPO increased, whereas those at pH 2.0 showed negligible differences; these differences were confirmed by scanning electron microscopy and monitoring the dry weight changes. At pH 6.0, the fibrous structures of the meshes rapidly disappeared compared to those under acidic conditions because Eudragit L100 is localized to the shell of the nanofiber during the electrospinning process. Both the pH changes and the blend ratio of the two polymers significantly affected the tetracycline release; tetracycline was rapidly released from the meshes at pH 6.0, whereas the release rates were attenuated at pH 2.0. Tetracycline was released faster from the mesh at higher blend ratios of EPO for both pH values. The electrostatic interaction between EPO and L100 is expected to yield different release profiles of tetracycline. Consequently, higher amounts of encapsulated drugs were released from the mesh at neutral pH and successfully inhibited bacterial growth.

Therapeutic applications of electrospun nanofibers for drug delivery systems.

Electrospun nanofiber drug delivery systems have been studied using various techniques. Herein, we describe the fabrication of a drug-incorporating nanofiber. Drugs, such as proteins, peptide, antibodies, and small molecule drugs, can be loaded within or on the surface of nanofibers according to their properties. Hydrophobic drugs are directly dissolved with a polymer in an organic solvent before electrospinning. However, it is preferred to surface-immobilize bioactive molecules on nanofibers by physical absorption or chemical conjugation. Especially, chemically surface-immobilized proteins on a nanofiber mesh stimulate cell differentiation and proliferation. Using a dual electrospinning nozzle to create nanofiber sheet layers, which are stacked on top of one another, the initial burst release is reduced compared with solid nanofibers because of the layers. Furthermore, hybridization of electrospun nanofibers with nanoparticles, microspheres, and hydrogels is indirect drug loading method into the nanofibers. It is also possible to produce multi-drug delivery systems with timed programmed release.

Multifunctional nanorods serving as nanobridges to modulate T cell-mediated immunity.

Electrodeposited nanorods serving as multivalent bridges were fabricated and surface-decorated with ligands for immune cells. Gold and nickel solutions were sequentially electrodeposited on nanoporous anodized disc templates and the template was dissolved to retrieve bisegmented nanorods with different lengths. Gold and nickel segmented nanorods were surface-immobilized with mannose and RGD peptides to prepare immune-cell recruiting nanorods. Surface-functionalization of nanorods were confirmed by fluorescence-labeling of each ligands and confocal microscopy. Dendritic cells and T cells were co-incubated with the surface-functionalized nanorods, and the proximity between the nanorods and the immune cells was visualized by variable pressure scanning electron microscopy and confocal microscopy. The long nanorods were associated with the immune cells, whereas the shorter nanorods were rather endocytosed by cells, suggesting a feasibility of the longer nanorods as bridging for the cells. Cytokine releases from the immune cells were monitored by cultivating lipopolysaccharide-activated dendritic cells with T cells. Interleukine-2 and interferon-γ release profiles showed a strong correlation with the length of the nanorod, where the 4 µm nanorods induced the highest levels of cytokine release compared to 1 or 2 µm nanorods. Thus, we concluded that the proximity of the immune cells increased by bridging the immune cells with the nanobridging system, which subsequently increased cytokine release by facilitating the antigen presentation process.

Electrospun catechol-modified poly(ethyleneglycol) nanofibrous mesh for anti-fouling properties.

Electrospun nanofibrous mesh composed of catechol-conjugated 8-arm PEG (8cPEGa) and thiolated PLGA (PLGA-SH) was prepared with various blending ratios of PLGA-SH and 8cPEGa. Cross-linking between the two polymers via catechol-thiol reactions and catechol-catechol conjugation was performed by brief soaking with sodium periodate solution. The chemical conjugation of PLGA-SH and 8cPEGa in the nanofibrous mesh was confirmed by the spectral differences of the Raman spectra and changes in the thermal properties. The crosslinked meshes showed lower degradation rates and their fibrous morphologies remained intact even after 15 days. When the blend ratio of 8cPEGa was increased from 0 to 50%, the crosslinked meshes showed a dramatic decrease in the water-contact angles due to the surface-exposed PEG chains tethered on the mesh. The crosslinked meshes had superior anti-fouling effect on protein and mammalian cell binding in proportion to the amount of 8cPEGa in the mesh compared to non-crosslinked meshes.

Dexamethasone-incorporated nanofibrous meshes for antiproliferation of smooth muscle cells: thermally induced drug-loading strategy.

Pluronic-immobilized nanofibrous meshes were tailored for thermally induced incorporation of dexamethasone. A diblock copolymer composed of poly(e-caprolactone)-poly (ethyleneglycol) (NH(2)) (PCL-PEG (NH(2))) was electrospun to a nanofibrous mesh, and Pluronic was subsequently surface-immobilized on the mesh in aqueous phase. Surface-wettability analysis and (1)H NMR spectroscopy confirmed surface-decoration of nanofibrous meshes with Pluronic moieties depending on the blend ratios of PCL-PEG(NH(2)). Fluorescently-labeled micelles were incorporated in the nanofibrous meshes by temperature modulation and showed attenuated release profiles of the micelles were for 1 month. The suppression degree of drug-loaded micelle releases was proportional to the blend ratio of PCL-PEG(NH(2)). Dexamethasone was formulated into micellar nanoaggregates, and the dexamethasone micelles-loaded nanofibrous meshes were used for antiproliferation studies of smooth muscle cells. Flow cytometric analysis of the arrested cells at a G(0)-G(1) phase revealed that the dexamethasone micelles-loaded nanofibrous meshes effectively controlled proliferation of the smooth muscle cells when cells were cultivated with the nanofibrous meshes. The antiproliferation effects of the nanofibrous meshes were closely correlated to the release profiles of the micelles from the nanofibrous meshes with different blend ratios. Thus, dexamethasone-incorporated nanofibrous meshes can be potentially used for treatments of restenosis after percutaneous transluminal coronary angioplasty.

[Effects of a problem-based learning program on health education for elders].

PURPOSE: The purposes of this study was to analyze the effects of a health education program using problem-based learning on health related knowledge, behavior, and quality of life in elderly people. METHODS: The participants included 44 elders, of whom 23 took the health education program and 21 did not. All participants were over 60 yr of age and were selected from residents of nursing homes or participants in activities of social welfare facilities in Jeju Province. Elders in both groups completed pre- and post-tests. Elders in the education group participated in 5 weekly sessions, 100-120 min/session of problem-based learning on health education. Data were analyzed using SPSS WIN 12.0. RESULTS: Scores for health knowledge, health behavior, and quality of life for the education group were significantly higher than those of the control group. CONCLUSION: A problem-based learning health education program can be recommended as a method to promote the health of the elders. Indirectly, the results seem to indicate that proper assessment and support should be provided simultaneously in the management of elders' health. Finally, future study is needed to examine whether problem-based learning is more helpful compared to traditional education.