Nucleic Acid-Scavenging Electrospun Nanofibrous Meshes for Suppressing Inflammatory Responses

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

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 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

Electrospun Nanofibrils Surface-Decorated with Photo-Cross-Linked Hyaluronic Acid for Cell-Directed Assembly

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

Korean Amberjack Skin-Inspired Hyaluronic Acid Bioink for Reconstruction of Human Skin

Decellularized extracellular matrix (dECM) has been extensively employed as tissue engineering scaffolds because its components can greatly enhance the migration and proliferation of cultivating cells. In this study, we decellularized Korean amberjack skin and incorporated soluble fractions in hyaluronic acid hydrogels with 3D-printed tissue engineering hydrogels to overcome any limitation of animal-derived dECM. The hydrolyzed fish-dECM was mixed with methacrylated hyaluronic acid and chemically crosslinked to 3D-printed fish-dECM hydrogels, where fish-dECM contents affected both printability and injectability of the hydrogels. Swelling ratios and mass erosion of the 3D-printed hydrogels were dependent on fish-dECM contents, where higher fish-dECM in the hydrogel increased swelling ratios and mass erosion rates. The higher content of fish-dECM considerably enhanced the viability of the incorporated cells in the matrix for 7 days. Artificial human skin was constructed by seeding human dermal fibroblasts and keratinocytes in the 3D-printed hydrogels, and a formation of a bilayered skin was visualized with tissue staining. Thus, we envision that 3D-printed hydrogels containing fish-dECM can be an alternative bioink composed of a non-mammal-derived matrix

Fluorescence-Shadowing Nanoparticle Clusters for Real-Time Monitoring of Tumor Progression

Monitoring tumor progression is important for elucidating appropriate therapeutic strategies in response to anticancer therapeutics. To fluorescently monitor the in vivo levels of tumor-specific enzymes, we prepared matrix metalloprotease (MMP)-responsive gold nanoparticle (AuNP) clusters to sense tumor microenvironments. Specifically, AuNPs and quantum dots (QDs) were surface-engineered with two poly­(ethylene glycol) [PEG] shells and cyclooctyne moieties, respectively, for the copper-free click reaction. Upon “peeling off” of the secondary shell from the double-PEGylated AuNPs under MMP-rich conditions, shielded azide moieties of the AuNPs were displayed toward the QD, and those two particles were clicked into nanoparticle clusters. This consequently resulted in a dramatic size increase and fluorescence quenching of QDs via fluorescence energy transfer (FRET) due to the molecular proximity of the particles. We observed that FRET efficiency was modulated via changes in MMP levels and exposure time. Cancer cell numbers exhibited a strong correlation with FRET efficiency, and in vivo studies that employed solid tumor models accordingly showed that FRET efficiency was dependent on the tumor size. Thus, we envision that this platform can be tailored and optimized for tumor monitoring based on MMP levels in solid tumors