New Biodegradable Drug Delivery System for Patients with Dry Eye.

PURPOSE: This study aimed to develop a new type of drug delivery system (DDS) for treatment of dry eye. METHODS: A new lens-type biodegradable DDS was manufactured using gelatin methacryloly, antibiotics, and conjunctival epithelial cells as bio-inks in a Bio X 3D Bioprinter. Gelatin methacryloly was used as a base, and the conditions were analyzed to maintain the overall shape by using a mixture of 0.1%, 0.15%, and 0.3% hyaluronic acid. In addition, an experiment was conducted to measure the appropriate concentration by evaluating its cytotoxicity according to the concentration of antibiotics mixed therein to prevent infection. The degree of degradation according to the storage temperature and post-processing of the new lens-type biodegradable DDS was measured. RESULTS: Optimal conditions were maintained when using a nozzle pressure of 80 kPa and speed of 4 mm/sec, nozzle pressure of 50 kPa and speed of 3 mm/sec, nozzle pressure of 60 kPa and speed of 8 mm/sec for 0.1%, 0.15%, and 0.3% hyaluronic acid concentrations, respectively. Degradation did not occur at 4°C and all the lenses were degraded at 37°C within 24 hours. In addition, the degradation rate was delayed according to the ultraviolet crosslink treatment time. Tobramycin 1% was used as an antibiotic during manufacture. CONCLUSIONS: A new lens-type biodegradable DDS that can control the degree of degradation was designed using a 3-dimentional bioprinter. This DDS will contribute to ease of treatment, protection of the cornea, and regeneration of the epithelium in patients with dry eye.

Oxidative stress-induced aberrant G9a activation disturbs RE-1-containing neuron-specific genes expression, leading to degeneration in human SH-SY5Y neuroblastoma cells.

Oxidative stress-induced neurodegeneration is one of several etiologies underlying neurodegenerative disease. In the present study, we investigated the functional role of histone methyltransferase G9a in oxidative stress-induced degeneration in human SH-SY5Y neuroblastoma cells. Cell viability significantly decreased on H2O2 treatment; however, treatment with the G9a inhibitor BIX01294 partially attenuated this effect. The expression of neuron-specific genes also decreased in H2O2- treated cells; however, it recovered on G9a inhibition. H2O2-treated cells showed high levels of H3K9me2 (histone H3 demethylated at the lysine 9 residue), which is produced by G9a activation; BIX01294 treatment reduced aberrant activation of G9a. H3K9me2 occupancy of the RE-1 site in neuron-specific genes was significantly increased in H2O2-treated cells, whereas it was decreased in BIX01294-treated cells. The differentiation of H2O2-treated cells also recovered on G9a inhibition by BIX01294. Consistent results were observed when used another G9a inhibitor UCN0321. These results demonstrate that oxidative stress induces aberrant activation of G9a, which disturbs the expression of neuron-specific genes and progressively mediates neuronal cell death. Moreover, a G9a inhibitor can lessen aberrant G9a activity and prevent neuronal damage. G9a inhibition may therefore contribute to the prevention of oxidative stress-induced neurodegeneration.

Analysis of oligomeric complexes of the amyloid-forming FYLLYY peptide by collision-induced dissociation with electrospray ionization mass spectrometry.

The monomeric and oligomeric structures of the "FYLLYY" ß2 microglobulin (ß2m) active sequence, formed in (DMSO/CH3CN) solution, were investigated using electrospray ionization (ESI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Dissociation of dimer and trimer ions was investigated by tandem mass spectrometry using collision induced dissociation (CID). The covalent bond fragmentation patterns were observed in the 21+ and 32+ MS/MS spectra (21+ = [dimer+H]1+ and 32+ = [trimer + 2H]2+). A π-π stacking geometry for the FYLLYY 21+ complex and partial parallel ß-sheet geometry for the 32+ complex are proposed to be stable structures. The observed covalent bond fragment ions in the MS/MS spectra of the 32+ complex are considered to have originated from the partial parallel ß-sheet moiety. The FYLLYY → AALLGY (or FYLLAA) substituted sequence was also investigated by CID-MS/MS. Our MS/MS analysis suggests that the π-π stacking interaction structures are important in dimer binding rather than the structures of a complete parallel or anti-parallel ß-sheet 21+ complex.

G9a inhibition promotes neuronal differentiation of human bone marrow mesenchymal stem cells through the transcriptional induction of RE-1 containing neuronal specific genes.

Recent studies have shown that epigenomic modifications are significantly associated with neuronal differentiation. Many neuronal specific genes contain the repressor element-1 (RE-1), which recruits epigenetic modulators, such as the histone methyltransferase G9a and interrupts the expression of neuronal genes in non-neuronal cells. This study investigated the functional role of G9a during neuronal differentiation of human bone marrow mesenchymal stem cells (BM-MSCs). Human BM-MSCs treated with the G9a inhibitor BIX01294 showed an increased expression of various neuronal-lineage genes. Using genomic sequence analysis, we identified RE-1 consensus sequences in the proximal region of several neuronal-specific genes. Chromatin immunoprecipitation (ChIP) assay results have showed that H3K9me2 (dimethylation of lysine 9 on histone 3) occupancy at RE-1-containing sequences from neuronal-specific genes was significantly decreased in BIX01294-MSCs. When BIX01294-MSCs were differentiated with neuronal induction medium, cells differentiated more effectively into neuron-like cells, complete with a cell body and dendrites. Expression of neuronal-specific genes containing the RE-1 sequences was significantly increased in differentiated BIX01294-MSCs, as confirmed by immunocytochemical staining and immunoblotting. Thus, this study shows that BIX01294 pretreated human BM-MSCs can be effectively differentiated into neuron-like cells by induced expression of neuronal-specific genes containing RE-1 sequences.

Investigation of the copper binding site on the human islet amyloid polypeptide hormone.

The metal ion binding sites of human islet amyloid polypeptide (hIAPP) have been investigated to explain the biological activity difference in the fibril formation process. The structures of [hIAPP...Cu (or Al)](n+) and [hIAPP17-30...Cu]2+ complex were investigated by electrospray ionization-mass spectrometry (ESI-MS). The fragmentation patterns of [hIAPP...Cu [or Al)](n+) and [hIAPP17-30...Cu]2+ complex were analyzed by tandem mass spectrometry (MS/MS) and multi-stage mass spectrometry (MS3) spectra. The [hIAPP+Cu+H]3+, [hIAPP+Al+H]4+ and [hIAPP17-30+Cu]2+ complexes were observed in MS spectra. The Cu binding site of hIAPP is suggested to be the N22-F-G-A-I26 part for the [hIAPP+Cu+H]3+ gas-phase complex. The original hIAPP conformation was supposed to be changed by the interaction between the Cu ion and the N22-F-G-A-I26 part in the [hIAPP+Cu+H]3+ gas-phase complex.

Enzymatically degradable temperature-sensitive polypeptide as a new in-situ gelling biomaterial.

We are reporting a poly (ethylene glycol)-block-poly(alanine-co-phenyl alanine) (PEG-PAF) aqueous solution that undergoes sol-to-gel transition as the temperature increases. The sol-to-gel transition was observed at as low a concentration as 3.0-7.0 wt.%. Micellar aggregation accompanying small conformational changes of the peptide from random coils to beta-sheets is suggested as the sol-to-gel transition mechanism of the PEG-PAF aqueous solution. The PEG-PAF is stable in phosphate buffered saline, however, it degraded in the subcutaneous layer of rats. In vitro study showed that proteolytic enzymes such as cathepsin B, cathepsin C, and elastase that are present in the subcutaneous layer of the mammalian tissue might be responsible for the degradation of the polymer in rats. As a feasibility study of this material, a single shot of an aqueous insulin formulation (13.8 mg insulin/kg) showed a hypoglycemic effect over 18 days in rats. The current functional polypeptide may be very promising as an in-situ gelling system for tissue engineering, cell/stem cell therapy, and drug delivery.

Determination of a binding site of Cu and Ni metal ions with oxytocin peptide by electrospray tandem mass spectrometry and multiple mass spectrometry.

The structures of a [Ni(II), Cu(II)- - -oxytocin] complex were investigated by electrospray ionization-mass spectrometry in positive mode. The fragmentation patterns of the [Ni(II), Cu(II) + OT](2+) complex were analyzed by tandem mass spectrometry and multiple mass spectrometry in the gas-phase. Conformations of metalII ion binding to oxytocin (OT) have been studied to explain the biological activity difference in the physiological solution. The [Ni(II) + OT](2+) and [Cu(II) + OT](2+) complexes were observed as the main ions in MS spectra. The Cys1-Tyr2-Ile3-Gln4 sequence of oxytocin is suggested to be a binding site for the [Ni(II) + OT](2+) gas-phase complex and Ile3-Gln4-Asn5-Cys6 sequence for the [Cu(II) + OT](2+) gas-phase complex. The specific binding site of CuII ion in the [Cu(II) + OT](2+) complex is explained as a reason of the negligible effect on the [Cu(II)- - -oxytocin] biological activity in aqueous solution.