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Hyaluronic acid (HA) was chemically immobilized on the surface of electrospun nanofibrils to form a cell/NF complex. Poly(caprolactone) (PCL) was electrospun into nanofibrous mats that were subsequently aminolyzed into nanofibrils. The aminolyzed nanofibrils were surface-decorated with methacrylated HA via Michael type addtion and by photo-cross-linking. Fourier transform infrared spectroscopy revealed the presence of HA on the surface of the nanofibrils. The thermogravimetric and colorimetric analyses indicate that the degree of HA immobilization could be varied by varying the photo-cross-linking duration. Thus, on increasing the photo-cross-linking duration, the swelling ratios increased gradually, and the surface charge of the decorated nanofibrils decreased. NIH3T3 cells and surface-decorated nanofibrils spontaneously assembled into the cell/NF complex. A higher degree of surface-immobilized HA enhanced cell viability and proliferation compared to nanofibrils without surface-immobilized HA. Thus, we envision that HA-immobilized nanofibrils can be employed as a tissue-engineering matrix to control cell proliferation and differentiation.

Monitoring tumor progression is important for elucidating appropriate therapeutic strategies in response to anticancer therapeutics. To fluorescently monitor the

Gold nanoparticles (AuNPs) were surface-engineered with a cationic corona to enhance the incorporation of photosensitizers for photodynamic therapy (PDT). The cationic corona composed of poly(2-(dimethylamino)ethyl methacrylate) was atom transfer radical-polymerized on the surface of the AuNPs. The cationic corona of the engineered surface was characterized by dynamic light scattering, electron microscopy, Raman spectroscopy, and mass spectroscopy. Chlorin-e6 (Ce6) incorporated onto the surface-engineered AuNPs exhibited higher cell incorporation efficiency than bare AuNPs. Ce6-incorporated AuNPs were confirmed to release singlet oxygen upon NIR irradiation. Compared to Ce6, Ce6-incorporated AuNPs exhibited higher cellular uptake and cytotoxicity against cancer cells in an irradiation time-dependent manner. Near-infrared-irradiated animals administered Ce6-incorporated AuNPs exhibited higher levels of tumor suppression without noticeable body weight loss. This result was attributed to the higher localization of Ce6 at the tumor sites to induce cancer cell apoptosis. Thus, we envision that engineered AuNPs with cationic corona can be tailored to effectively deliver photosensitizers to tumor sites for photodynamic therapy.

Gold nanoparticles (AuNPs) are the predominant and representative metal nano-carriers used for the tumor-targeted delivery of therapeutics because they possess advantages such as biocompatibility, high drug loading efficiency, and enhanced accumulation at tumor sites via the size-dependent enhanced permeability and retention (EPR) effect. In this study, we designed an AuNP functionalized with block polymers comprising polyethylenimine and azide group-functionalized poly(ethyl glycol) for the electrostatic incorporation of cytosine–guanine oligonucleotide (CpG ODN) on the surface. The ODN-incorporated AuNPs were cross-linked to gold nanoparticle clusters (AuNCs) via click chemistry using a matrix metalloproteinase (MMP)-2 cleavable peptide linker modified with alkyne groups at both ends. In the presence of Cu(I), azide groups and alkyne groups spontaneously cyclize to form a triazole ring with high fidelity and efficiency, and therefore allow single AuNPs to stack to larger AuNCs for increased EPR effect-mediated tumor targeting. 1H-NMR and Fourier transform infrared spectroscopy revealed the successful synthesis of an azide–PEG-grafted branched polyethylenimine, and the size and morphology of AuNPs fabricated by the synthesized polymer were confirmed to be 4.02 ± 0.45 nm by field emission-transmission electron microscopy. Raman spectroscopy characterization demonstrated the introduction of azide groups on the surface of the synthesized AuNPs. Zeta-potential and gel-retardation analysis of CpG-loaded AuNPs indicated complete CpG sequestration by AuNPs when the CpG : AuNP weight ratio was higher than 1 : 2.5. The clustering process of the CpG-loaded AuNPs was monitored and was demonstrated to be dependent on the alkyne linker-to-AuNP ratio. Thus, the clicked AuNC can be tailored as a gene carrier where a high accumulation of therapeutics is required.

Cell sheet engineering has attracted great attention because thin layers of tissue can be easily transplanted to defect sites. Wound-dressing materials are required to support fast re-epithelization, both with keratinocytes and fibroblasts, to enhance the prognosis and therapeutic outcomes. We prepared self-assembled cell sheets composed of adipocyte-derived stem cells (ADSCs) and surface-engineered nanofibrils (NFs). NFs were surface-engineered with multilayers of gelatin so that the cell sheets could spontaneously assemble within 3 days in cell culture plates. Dorsal wounds transplanted with the cell sheets exhibited higher wound-healing rates when a high concentration of gelatin was immobilized on the surfaces of the NFs. Histochemical staining revealed that those with gelatin-immobilized NFs showed a higher expression of cytokeratin and collagen in the re-epithelized epidermis. Keratinocytic differentiation of the epidermis was molecularly evidenced by the higher expression of keratinocyte-specific genes.

The therapeutic efficacy of nanoparticles depends on their ability to release encapsulated photosensitizers. Here, surface-engineered metallic gold nanoparticles (AuNP) were irradiated with dual near-infrared (NIR) light to enhance the release of photosensitizer. Dopamine hydrochloride was surface-polymerized to polydopamine (PDA) layers on AuNP, and chlorin-e6 (Ce6) was chemically tethered to primary amines of PDA. The resulting Ce6-conjugated AuNP were characterized by Raman and X-ray photoelectron spectroscopy and visualized by electron microscopy and light scattering. The generation of reactive oxygen species was increased following dual NIR irradiation at 650 nm and 808 nm, which was attributed to the increased liberation of Ce6. In vitro, dual NIR irradiation significantly enhanced the anticancer effect of Ce6-incorporating AuNP by increasing the population of apoptotic cells. In vivo, tumor xenografted animals exhibited much better tumor suppression when subjected to dual NIR irradiation. Thus, we propose the use of Ce6-incorporating AuNP coupled to dual NIR irradiation for future anticancer treatment of solid tumors.

Enhancement of myogenic differentiation was accomplished with nanohybrid hydrogels composed of fragmented electrospun nanofibrils (NFs) and gelatin/alginate hydrogels. Electrospun nanofibrous meshes were fragmented and hydrolyzed to short fragments of NFs and subsequently embedded in gelatin/alginate hydrogels and further crosslinked with C2C12 cells. Cell-incorporated NF-embedded hydrogels exhibited a lower swelling ratio and higher mechanical properties as compared to hydrogels without NF due to the rigidity of NF and increased matrix-to-cell interaction. Viability of C2C12 cells in the hydrogels gradually increased in proportion to the amount of NF in the hydrogels. Both confocal microscopy and myogenic gene expression analysis indicated that the formation and maturation of myotubes and the expression of muscle motor proteins were significantly accelerated when NF-embedded hydrogels were employed. Contraction behavior of matured myotubes under electrical stimulation revealed that myotubes in NF-embedded hydrogels exhibited a longer contraction length than that of myotubes in hydrogels without NF. Thus, the nanohybrid matrix demonstrates the potential of nanofiber-embedded hydrogels for engineering myotube formation with topographical cues.

Recently, the degradation and detection of 2,4-dinitrotoluene (2,4-DNT) capable of producing 2,4,6-trinitrotoluene (TNT) for environmental and human health risks have been developed. We prepared photoresponsive Au-decorated Fe

Herein, we fabricated a uniform and dispersible Ag/indium tin oxide (ITO) cosputtered film on a two-dimensional ordered polystyrene template and observed distinct localized surface plasmon resonance (LSPR) properties that can be tuned by changing the doping level. The increase in the optical band gap is due to the variation in the metallic Ag content, which can effectively change the accumulation of free electrons in the conduction band, in addition to the near-IR absorbance. Surface-enhanced Raman scattering (SERS) was used to monitor the variations in the band gap and transfer of electrons, which causes variations in the SERS intensity. The presented research provides new insights into the relationships between the carrier density and maximum absorption wavelength, band gap distribution, and charge transfer process. This is the first study on the influence of the carrier density on the properties of Ag/ITO cosputtered films and suggests practical applications of these films.

In this research, studies of mechanical properties, in vitro cytotoxicity, and in vivo biological safety of compounded rubber samples that contained various novel vulcanizing agents derived from poly(alkylphenol) disulfide were investigated to evaluate the possibility of clinical application. From tensile test results, every compounded rubber sample showed improved elongation properties compared to the reference. In MTT and ALP assays, every compounded rubber sample showed non-cytotoxic levels compared to the control sample containing inorganic sulfur as the vulcanizing agent. The cell viability and activity were comparable to those of the control sample even at extract concentrations up to 10%. In the animal model test, no compounded rubber sample showed any other biological response during implantation for 4 weeks, and the skin sensitization test results indicated lack of sensitivity response (Grades 0 and I).

Although nanofibrous meshes are considered promising cultivation beds for maintaining cell differentiation, 3D cultivation is not possible because their nanoporous structures impede cell infiltration. To facilitate transverse cell migration across nanofibrous meshes, electrospun nanofibers are prepared with structures that vary in response to red laser light. Polyoxalate (POX), composed of oxalate linkers and oligomeric caprolactone, is synthesized and electrospun into fibrous meshes with a photosensitizer (chlorin e6, Ce6). These meshes exhibit morphological and chemical changes upon laser irradiation, and mass erosion rates of the meshes are faster after laser irradiation. Cell cultivation on POX meshes reveals that red laser effectively facilitates traverse migration of the cells without affecting cell viability. The use of light-triggered change of meshes is envisioned to promote the migration of cells during 3D matrix cultivation.

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.

Surface initiated atom transfer radical polymerization (SI-ATRP) documented a simple but efficient technique to grow a dense polymer layer on any surface. Gold nanoparticles (AuNPs) give a broad surface to immobilize sulfhyryl group-containing initiators for SI-ATRP, in addition, AuNPs are the major nanoparticulate carriers for delivery of anti-cancer therapeutics, since they are biocompatible and bioinert. In this work, AuNPs with a disulfide initiator were polymerized with sulfoethyl methacrylate by SI-ATRP to decorate the particles with anionic corona, and branched polyethyeleneimine (PEI) and siRNA were sequentially layered onto the anionic corona of AuNP by electrostatic interaction. The in vitro anti-cancer effect confirmed that AuNP with anionic corona showed higher degrees of apoptosis as well as suppression of the oncogene expression in a siRNA dose-dependent manner. The in vivo study of tumor-bearing nude mice revealed that mice treated with c-Myc siRNA-incorporated AuNPs showed dramatically decreased tumor size in comparison to those with free siRNA for 4 weeks. Furthermore, histological examination and gene expression study revealed that the decorated AuNP significantly suppressed c-Myc expression. Thus, we envision that the layer-by-layer assembly on the anionic brushes can be potentially used to incorporate nucleic acids onto metallic particles with high transfection efficiency.

Cartilage defect is difficult to heal due to its avascular properties. Implantation of mesenchymal stem cell is one of the most promising approach for regenerating cartilage defects. Here we prepared polymeric nanofibrils decorated with cartilage-derived decellularized extracellular matrix (dECM) as a chondroinductive scaffold material for cartilage repair. To fabricate nanofibrils, eletrospun PCL nanofibers were fragmented by subsequent mechanical and chemical process. The nanofibrils were surface-modified with poly(glycidyl methacrylate) (PGMA@NF) via surface-initiated atom transfer radical polymerization (SI-ATRP). The epoxy groups of PGMA@NF were subsequently reacted with dECM prepared from bovine articular cartilage. Therefore, the cartilage-dECM-decorated nanofibrils structurally and biochemically mimic cartilage-specific microenvironment. Once adipose-derived stem cells (ADSCs) were self-assembled with the cartilage-dECM-decorated nanofibrils by cell-directed association, they exhibited differentiation hallmarks of chondrogenesis without additional biologic additives. ADSCs in the nanofibril composites significantly increased expression of chondrogenic gene markers in comparison to those in pellet culture. Furthermore, ADSC-laden nanofibril composites filled the osteochondral defects compactly due to their clay-like texture. Thus, the ADSC-laden nanofibril composites supported the long-term regeneration of 12 weeks without matrix loss during joint movement. The defects treated with the ADSC-laden PGMA@NF significantly facilitated reconstruction of their cartilage and subchondral bone ECM matrices compared to those with ADSC-laden nanofibrils, non-specifically adsorbing cartilage-dECM without surface decoration of PGMA.

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.

A free-standing, antibacterial hydrogel was fabricated using silver-nanoparticle-immobilized cellulose nanofibers (CNFs) and alginate. Surface hydroxyl groups of CNFs were oxidized to carboxylate groups using (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TCNF), followed by the treatment with silver nitrate solution for surface adsorption of silver ions. In situ reduction of silver ions to produce silver nanoparticles was performed for the silver-adsorbed CNFs. Electron microscopy, X-ray diffraction, and spectroscopic analysis revealed that higher amounts of silver nanoparticles were immobilized on the surface of TCNF than on the surface of native CNF. Silver-nanoparticle-immobilized TCNF was embedded in alginate gels and silver ions from the matrix were slowly released for 7 days. Silver-nanoparticle-loaded alginate gels showed comparable antibacterial activity to silver-ions-loaded alginate gels, although the former showed a significantly lower cytotoxicity against animal cells. Thus, the antibacterial gels can potentially be applied to various skin surfaces to prevent bacterial infection while minimizing skin damage.

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.

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.

Fibroblasts are a major cell type in the dermis. When skin is wounded in various ways such as by abrasions, cuts or diabetic ulcer, proliferation and migration of dermal fibroblasts is necessary for cutaneous wound healing. Numerous studies have shown that adult stem cells secrete paracrine factors and these are able to promote wound healing by activating migration and proliferation of effector cells such as dermal fibroblasts. However, the paracrine factors secreted from pluripotent stem cells and the effect of these on dermal fibroblast proliferation and migration have been poorly characterized. In this study we cultured human induced pluripotent stem cells without any animal-derived components including feeder cells, and investigated the effect of stem cell-conditioned medium (iPSC-CM) on dermal fibroblast proliferation and migration. Results showed that the proliferation of mouse embryonic fibroblasts (STO cells) and human dermal fibroblasts (HDFs) were significantly stimulated by iPSC-CM. We determined that the optimal concentration of iPSC-CM in promoting the proliferation of HDFs was a 75% dilution. Scratch wound assay and transwell migration assay also demonstrated the stimulatory effect of iPSC-CM on the migration of HDFs. iPSC-CM is believed to have advantages because of the unique capabilities of iPSCs, which include infinite self-renewal, pluripotency and variety of donor cells. Thus, iPSC-CM is anticipated to be a valuable source of paracrine factors which can potentially be used for wound healing applications.

Hybrid composites composed of nanofibrils and hydrogels were fabricated for cell cultivation in a cell-friendly environment mimicking the extracellular matrix. Electrospun fibrous mats composed of polycaprolactone (PCL) were digested to nanofibrils (NF) under optimized conditions and subsequently incorporated into hydrogels composed of gelatin and alginate. The hydrogel hybrids containing nanofibrils (NF/hydrogel) were calcium-crosslinked and solidified with or without cells. The mechanical properties of the cell-incorporated NF/hydrogels were dramatically increased in proportion to the NF content in the NF/hydrogels after 7 days of cultivation. Mass erosion of the NF/hydrogel was significantly attenuated when NF content increased. Confocal microscopy revealed that fibroblasts cultivated in NF/hydrogels showed superior adhesion behavior onto the matrix compared to those without NF, and the formation of spindle and stress fibers was distinct in NF/hydrogels with a high NF content. Cells on NF/hydrogels showed higher levels of collagen synthesis as NF content increased. Thus, NF/hydrogels are expected to serve as a cell culture matrix facilitating cell–matrix interactions by combining supportive nanofibrils and hydrogels.

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.

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.

Charged phospholipids are employed to formulate liposomes with different surface charges to enhance the permeation of active ingredients through epidermal layers. Although 3D skin tissue is widely employed as an alternative to permeation studies using animal skin, only a small number of studies have compared the difference between these skin models. Liposomal delivery strategies are investigated herein, through 3D skin tissue based on their surface charges. Cationic, anionic, and neutral liposomes are formulated and their size, zeta-potential, and morphology are characterized using dynamic light scattering and cryogenic-transmission electron microscopy (cryo-TEM). A Franz diffusion cell is employed to determine the delivery efficiency of various liposomes, where all liposomes do not exhibit any recognizable difference of permeation through the synthetic membrane. When the fluorescence liposomes are applied to 3D skin, considerable fluorescence intensity is observed at the stratum cornea and epithelium layers. Compared to other liposomes, cationic liposomes exhibit the highest fluorescence intensity, suggesting the enhanced permeation of liposomes through the 3D skin layers. Finally, the ability of niacinamide (NA)-incorporated liposomes to suppress melanin transfer in pigmented 3D skin is examined, where cationic liposomes exhibit the highest degree of whitening effects.

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.

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.

Multilayered assembly of hydrophilic polymers is used to decorate electrospun nanofibrous mats for the air–liquid interface cultivation of epidermal cells. e-Caprolactone is ring-opening polymerized into carboxyl-terminated poly(e-caprolactone) (PCL) and subsequently conjugated with poly(ethyleneimine) (PEI) in order to prepare the PCL–PEI block copolymers. The electrospun PCL–PEI nanofibers are chemically conjugated with multivalent poly(ethyeleneglycol) (PEG) chains, and PEI is chemically tethered to the PEG layers in order to further decorate the surface with multiple layers of PEG. The layered PEG is characterized using both thermal properties and direct visualization of the whole mats with fluorescence labeling, which exhibit escalating levels of PEG content with increasing numbers of PEG layering processes. The PCL–PEI nanofibers with PEG multilayers exhibit superior water swelling rates compared with unmodified mats and have high water retaining behaviors for prolonged periods without additional water supply in a PEG layer-dependent manner. When epidermal cells are cultivated on the PEG-multilayered mats without a continuous supply of the cell culture medium, the viability is maintained for 48 h and gradually decreases after 48 h. The epidermis-specific genes are highly expressed in cells cultivated on the PEG-multilayered mats in comparison with those on an insert well or unmodified mats, which is confirmed by qualitative real-time polymerase chain reaction and immunocytochemical staining of total keratin. Thus, PEG-multilayered nanofibrous mats can be a novel cell culture matrix for cell culture systems where the cell cultivation conditions require air–liquid interfaces.

Liquid crystal (LC) emulsions composed of multi-lamellar structures were formulated to enhance the percutaneous absorption of retinyl palmitate across skin barriers. The LC emulsions were prepared by hot process emulsification of an aqueous phase and an oil phase containing retinyl palmitate. Percutaneous absorption levels across synthetic membranes were determined by Franz cell diffusion study. The diffusional absorption level of the oil phase in the LC emulsions was also visualized by fluorescence imaging of Nile red-containing emulsions on synthetic membranes. Transport of retinyl palmitate across pig skin was fluorescently visualized. In vitro cell culture study showed that the LC emulsions enhanced cellular metabolism and proliferation of fibroblasts in comparison to the plain emulsion. The multi-lamellar structures composed of aligned fatty alcohol chains were revealed using freezefracture scanning electron microscopy and polarized light microscopy. The LC emulsions showed increased retention at the membrane as well as at the acceptor in comparison to plain oil-in-water emulsions. Total fluorescence intensity at the membrane with the LC emulsions was higher compared to plain emulsions. Confocal laser scanning microscopy (CLSM) of cryo-cut cross-sections revealed that Nile red was highly localized in the epidermis layers, particularly in the stratum corneum in comparison to the plain emulsions. LC emulsions also enhanced transport of retinyl palmitate at cellular levels, in addition to facilitating percutaneous absorption across dermal layers.

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 (e-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.

Ovalbumin-incorporated antigen carriers were prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of iron oxide nanoparticles for magnetic sorting of antigen-presenting cells. Iron oxide nanoparticles were surface-decorated with cationic shells by SI-ATRP, and the primary amines on the surface were further tri-methylated. Surface decoration of the nanoparticles was characterized by Fourier transform infrared spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and transmission electron microscopy with energy-dispersive spectrometry. Ovalbumin-loaded nanoparticles showed higher incorporation in comparison to non-decorated nanoparticles, and the loaded ovalbumin was released faster at low pH than at neutral pH. Ovalbumin-loaded nanoparticles were endocytosed by macrophages, and macrophages with nanoparticles were easily harvested by magnetic separation. Magnetically sorted macrophages showed higher release of cytokines including TNF-α, MIP-1α, and IL-1β than unsorted macrophages. These results suggest that ovalbumin-loaded nanoparticles can potentially increase the efficiency of immune therapy during the antigen-presenting pathway.

A two-step strategy for coaxial electrospinning and postelectrospinning is an effective method for fabricating superfine nanofibers composed of highly swellable hydrogels. Alginate and poly(e-caprolactone) [PCL] were coelectrospun via fibrous meshes with a coaxial nozzle; alginate at the core was subsequently cross-linked in calcium chloride solution. The PCL sheath was removed from the meshes by repeated organic-phase washing. The peeling process was monitored by scanning electron microscopy, transmission electron microscopy, and differential scanning calorimetry, and the complete removal of the PCL outer layers was confirmed by the thinning of the fiber volume. The obtained alginate hydronanofiber showed extreme water-swellability and mass erosion depending on the degree of cross-linking. We also measured the nanoscale and macroscale mechanical properties of a single nanofiber and of the whole mesh by atomic force microscopy and rheometry. Quantitative analysis of nanomechanical properties indicated that the hydronanofiber with higher cross-linking density had higher stiffness and Derjaguin–Muller–Toporov modulus. Cells laid on the mesh and the vertical infiltration distance were visualized and quantified by confocal laser scanning microscopy. Cells on the mesh with higher cross-linking density infiltrated deeply to the bottom of the mesh. Thus, hydrogel-like nanofibrous meshes are versatile matrices allowing for deep infiltration of cells throughout the mesh via manipulation of the mechanical properties of the nanofiber.

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.

Due to a lack of proper drug carriers to deliver treatments for mucositis, many cancer patients suffer from oral mucositis caused by chemotherapy and radiotherapy. We prepared a double-layered electrospun nanofibrous sheets composed of Eudragit and chitosan to accelerate the healing rate of oral mucous ulcer. Human growth hormone (hGH) and Eudragit in a mixture of dimethylacetamide and ethanol were co-electrospun to nanofibrous sheets. The electrospun fibrous mat was subsequently layered with chitosan by a dip-coating method. Chitosan-layered sheets showed attenuated mass erosion while uncoated sheets were instantly melted at the physiological condition. The released hGH was trapped on the chitosan layer by the ionic interaction between positively charged chitosan and negatively charged hGH, and a large number of entrapped proteins remained on the SIS membrane due to the muco-adhesive properties of chitosan. hGH-incorporated sheets significantly increased proliferation of human dermal fibroblasts. In vivo study employing oral ulcers in dogs, the ulcers dressed with chitosan-layered sheets showed enhanced wound recovery and the chitosan layers on the sheet greatly assisted prolonged recovery. Therefore, chitosan-layered Eudragit nanofibrous sheets can be potentially applied to developing muco-adhesive wound dressing materials with pH-dependent drug release by adjusting the thickness of chitosan sheath on the sheets. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1396-1406, 2016.

Focal adhesion between substrates and cells greatly controls cytoskeletal changes as well as differentiation of the cultivating cells. We here investigated focal adhesion behaviors and differentiation profiles of adipose-derived stem cells on the nano-scaled pillar arrays with diverse pillar-to-pillar distances.

Manipulated roughness has been etched on nanofibers to modulate adsorption of serum proteins for serum-free cells cultivation. Mixtures of Polycaprolactone and Pluronic with various blending ratios are electrospun to nanofibers and Pluronic is subsequently leached out by methanol. Electron microscopy reveals that surface roughness of the nanofibers is changed according to the contents of Pluronic. Both weight loss monitoring and NMR spectroscopy confirm that all Pluronic is leached out after methanol treatment. Water swelling ratio and protein adsorption of rougher nanofibers are higher than those of smoother ones. Also, when serum incorporation on the nanofibers is estimated in 0.01-10% serum solution, rougher nanofibers show higher serum incorporation and those soaked in 10% serum solution are employed for serum-free cell cultivation. Viability of the cells cultivated on rougher nanofibers is much higher after 24 h. Thus, Pluronic-induced leaching-out strategy can be potentially employed for fabricating roughness on nanofibers for enhancing protein adsorption for serum-free cell cultivation.

Cancer cells overexpressing folate receptors have been targeted using a folate decorated carriers for anti-cancer drugs in aims to overcome the tissue non-specificity of anti-cancer agents. We here prepared magnetic nanoparticles and surface-decorated them with different amounts of folate to optimize the number of the immobilized folate on the carriers for superior targeting effects. Magnetic nanoparticles were prepared by oxidizing ferric or ferrous chloride solution to iron oxide in the presence of poly(vinyl alcohol). The magnetic nanoparticles were functionalized with primary amines for subsequent reactions with the different feed ratios of the activated folate. The magnetization degree of the folate magnetic magnetization were slightly decreased when the folate on the particles were increased. Intracellular uptakes of the nanoparticles were shown to be increased and become saturated dependent on the amounts of the surface-immobilized folate. The folate-decorated magnetic nanoparticles showed negligible cytotoxicity against KB cells from 5 μg to 35 μg of the nanoparticle weights.

Fragmented nucleic acids are potent stimulators for inflammatory responses provoking pathological outcomes by activating adaptive immunity. In this study, highly cationic surfaces were prepared on electrospun nanofibrous meshes to scavenge nucleic acids to the surfaces. Poly(ε-caprolactone) [PCL]-poly(ethylenimine) [PEI] block copolymers were synthesized by coupling the carboxyl-terminated PCL to the primary amines of branched PEI. Polymeric solutions composed of PCL-PEI and PCL were electrospun to nanofibrous mats, and the surfaces were further methylated to prepare highly cationic surfaces on the mats. Raman spectroscopy revealed that the presence of increased methylated amines on the surfaces of the mats compared to unmodified mats. The methylated surfaces showed significant increases of wettability after methylation, suggesting highly charged surfaces caused by methylation of the primary amines. When the blend ratio of PCL-PEI was increased, the scavenged DNA was also increased, and the methylation further strengthened the scavenging ability of the mats. Fluorescently labeled oligodeoxynucleic acids were significantly adsorbed on the surface of the mats depending on the amounts of PCL-PEI and the degree of methylation. In the presence of the methylated nanofibrous mats, inflammatory responses induced by CpG oligonucleotides in murine macrophages were significantly reduced, which was confirmed by measuring inflammatory cytokine levels including TNF-α and IFN-γ.

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.

Monomeric catechols are displayed on the surface of polymeric nanofibers by robust catechol-thiol interactions to enhance cell adhesion and migration. Dihydroxyphenyl propionic acid is chemically conjugated to primary amine groups of poly(ϵ-caprolactone)-poly(ethylene glycol)-amine (PCL-PEG) nanofibers to display catechol moieties on the surface. At basic pH, catecholized nanofibers incorporate thiol groups at a five-fold higher rate than at acidic pH, while catechol-coated surfaces do not show any pH-dependent binding. Live/dead cell staining indicates that the catecholized nanofibers do not exert any cytotoxic effects. Also, NIH 3T3 cells cultured on the catecholized nanofibers show increased attachment and migration that is proportional to the amount of the immobilized catechol moieties on the surface. These results clearly indicate that 6 nmol of monomeric catechols on the surface of nanofiber can promote cell adhesion and migration by thiol-catehol interactions.