Du Young Ko

3D Culture of Tonsil-Derived Mesenchymal Stem Cells in Poly(ethylene glycol)-Poly(l-alanine-co-l-phenyl alanine) Thermogel

Poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) (PEG-PAF) aqueous solutions undergo sol-to-gel transition as the temperature increases. The transition is driven by the micelle aggregation involving the partial dehydration of the PEG block and the partial increase in β-sheet content of the PAF block. Tonsil-tissue-derived mesenchymal stem cells (TMSCs), a new stem cell resource, are encapsulated through the sol-to-gel transition of the TMSC-suspended PEG-PAF aqueous solutions. The encapsulated TMSCs are in vitro 3D cultured by using induction media supplemented with adipogenic, osteogenic, or chondrogenic factors, where the TMSCs preferentially undergo chondrogenesis with high expressions of type II collagen and sulfated glycosaminoglycan. As a feasibility study of the PEG-PAF thermogel for injectable tissue engineering, the TMSCs encapsulated in hydrogels are implanted in the subcutaneous layer of mice by injecting the TMSC suspended PEG-PAF aqueous solution. The in vivo studies also prove that TMSCs undergo chondrogenesis with high expression of the chondrogenic biomarkers. This study suggests that the TMSCs can be an excellent resource of MSCs, and the thermogelling PEG-PAF is a promising injectable tissue engineering scaffold, particularly for chondrogenic differentiation of the stem cells.

Composite System of Graphene Oxide and Polypeptide Thermogel As an Injectable 3D Scaffold for Adipogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

As two-dimensional (2D) nanomaterials, graphene (G) and graphene oxide (GO) have evolved into new platforms for biomedical research as biosensors, imaging agents, and drug delivery carriers. In particular, the unique surface properties of GO can be an important tool in modulating cellular behavior and various biological sequences. Here, we report that a composite system of graphene oxide/polypeptide thermogel (GO/P), prepared by temperature-sensitive sol-to-gel transition of a GO-suspended poly(ethylene glycol)-poly(L-alanine) (PEG-PA) aqueous solution significantly enhances the expression of adipogenic biomarkers, including PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL, compared to both a pure hydrogel system and a composite system of G/P, graphene-incorporated hydrogel. We prove that insulin, an adipogenic differentiation factor, preferentially adhered to GO, is supplied to the incorporated stem cells in a sustained manner over the three-dimensional (3D) cell culture period. On the other hand, insulin is partially denatured in the presence of G and interferes with the adipogenic differentiation of the stem cells. The study suggests that a 2D/3D composite system is a promising platform as a 3D cell culture matrix, where the surface properties of 2D materials in modulating the fates of the stem cells are effectively transcribed in a 3D culture system.

Polypeptide Thermogels as a Three Dimensional Culture Scaffold for Hepatogenic Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells

Tonsil-derived mesenchymal stem cells (TMSCs) were investigated for hepatogenic differentiation in the 3D matrixes of poly(ethylene glycol)-b-poly(l-alanine) (PEG-L-PA) thermogel. The diblock polymer formed β-sheet based fibrous nanoassemblies in water, and the aqueous polymer solution undergoes sol-to-gel transition as the temperature increases in a concentration range of 5.0-8.0 wt %. The cell-encapsulated 3D matrix was prepared by increasing the temperature of the cell-suspended PEG-L-PA aqueous solution (6.0 wt %) to 37 °C. The gel modulus at 37 °C was about 1000 Pa, which was similar to that of decellularized liver tissue. Cell proliferation, changes in cell morphology, hepatogenic biomarker expressions, and hepatocyte-specific biofunctions were compared for the following 3D culture systems: TMSC-encapsulated thermogels in the absence of hepatogenic growth factors (protocol M), TMSC-encapsulated thermogels where hepatogenic growth factors were supplied from the medium (protocol MGF), and TMSC-encapsulated thermogels where hepatogenic growth factors were coencapsulated with TMSCs during the sol-to-gel transition (protocol GGF). The spherical morphology and size of the encapsulated cells were maintained in the M system during the 3D culture period of 28 days, whereas the cells changed their morphology and significant aggregation of cells was observed in the MGF and GGF systems. The hepatocyte-specific biomarker expressions and metabolic functions were negligible for the M system. However, hepatogenic genes of albumin, cytokeratin 18 (CK-18), and hepatocyte nuclear factor 4α (HNF 4α) were significantly expressed in both MGF and GGF systems. In addition, production of albumin and α-fetoprotein was also significantly observed in both MGF and GGF systems. The uptake of cardiogreen and low-density lipoprotein, typical metabolic functions of hepatocytes, was apparent for MGF and GGF. The above data indicate that the 3D culture system of PEG-L-PA thermogels provides cytocompatible microenvironments for hepatogenic differentiation of TMSCs. In particular, the successful results of the GGF system suggest that the PEG-L-PA thermogel can be a promising injectable tissue engineering system for liver tissue regeneration after optimizing the aqueous formulation of TMSCs, hepatogenic growth factors, and other biochemicals.

PEG‐Poly(l‐alanine) Thermogel As a 3D Scaffold of Bone‐Marrow‐Derived Mesenchymal Stem Cells

Bone-marrow-derived mesenchymal stem cells (BMSCs) were cultured in three-dimensional (3D) scaffolds formed by temperature-sensitive sol-to-gel transition of BMSC-suspended poly(ethylene glycol)-poly(L-alanine) (PEG-PA) aqueous solutions. A commercialized thermogelling 3D scaffold of Matrigel™ was used for the comparative study. The cells maintained their spherical shapes in the PEG-PA thermogel, whereas fibrous cell morphologies were observed in the Matrigel™. Type II collagen and myogenic differentiation factor 1 were dominantly expressed in the PEG-PA thermogel. On the other hand, a significant extent of type III β-tubulin was expressed in the Matrigel™ in addition to type II collagen and myogenic differentiation factor 1. After confirming the dominant chondrogenic differentiation of the BMSCs in the PEG-PA thermogel in in vitro study, in vivo study was performed for injectable tissue engineering application of the BMSCs/PEG-PA system. The cell-growing implant was formed in situ by subcutaneous injection of the BMSC-suspended PEG-PA aqueous solution to mice. In vivo study also proved the excellent expressions of chondrogenic biomarkers including collagen type II and sulfated glycosaminoglycan in the mouse model. This paper suggests that the PEG-PA thermogel is a very promising as a 3D culture matrix as well as an injectable tissue-engineering system for preferential chondrogenic differentiation of the BMSCs.

Control of rhGH Release Profile from PEG–PAF Thermogel

Poly(ethylene glycol)-poly(l-alanine-co-l-phenyl alanine) diblock copolymers (PEG-PAF) of 2000-990 Da (P2K) and 5000-2530 Da (P5K) with the different molecular weights of PEGs, but having a similar molecular weight ratio of hydrophobic block to hydrophilic block were synthesized to compare their solution behavior and corresponding protein drug release profiles from their in situ formed thermogels. The PEG-PAF aqueous solutions underwent heat-induced sol-to-gel transition in a concentration range of 18.0-24.0 wt % and 8.0-12.0 wt % for P2K and P5K, respectively. P5K formed bigger micelles than P2K, of a broad distribution, whereas the PAF blocks of P5K developed richer in α-helix than those of P2K in the core of the micelles. As the temperature increased, the micelles underwent dehydration of the PEG, which led to the aggregation of micelles, while the secondary structure of PAF was slightly affected during the sol-to-gel transition. The P5K exhibited higher tendency to aggregate and formed a tighter gel than P2K. Upon injection into the subcutaneous layer of rats, both polymer aqueous solutions formed a biocompatible gel with typical mild inflammatory tissue responses. Recombinant human growth hormone (rhGH) maintained its stability without forming any aggregates in both sol (4 °C) and gel (37 °C) states of the PEG-PAFs. Even though P2K and P5K have a similar molecular weight ratio of hydrophobic block to hydrophilic block, the P5K system exhibited a reduced initial burst release, improved bioavailability, and prolonged therapeutic duration of the rhGH, compared to the P2K system. The current research suggests that a drug release profile is a complex function of self-assembling carriers and incorporated drugs, and thus, a promising protein delivery system could be designed by adjusting the molecular parameters of a thermogel.

Incorporation of D-alanine into poly(ethylene glycol) and L-poly(alanine-co-phenylalanine) block copolymers affects their nanoassemblies and enzymatic degradation

Poly(ethylene glycol)–poly(alanine-co-phenylalanine) (PEG–PAF) block copolymers with similar molecular weights were synthesized to investigate the effect of the partial incorporation of D-alanine into PEG-L-PAF. The ratio of L-alanine to D-alanine of the polymer varied over 100/0, 80/20, 60/40, 50/50, and 0/100. Circular dichroism and FTIR spectra indicated that the PEG–PAFs with the mixed composition of L-alanine and D-alanine exhibited dominantly random coil structures, whereas the PEG–PAF with the enantiomeric alanine (100/0 or 0/100) exhibited right-handed or left-handed α-helical structures as well as β-sheet structures, respectively. Dynamic light scattering of the polymer aqueous solution indicated that the size of nanoassemblies significantly decreased as a result of the partial incorporation of D-alanine into PEG-L-PAF. The most probable diameters were 20–40 nm and 80–105 nm for the PEG–PAFs with a mixed composition (L-alanine/D-alanine ratios of 80/20, 60/40, and 50/50) and an enantiomeric composition (L-alanine/D-alanine ratios of 0/100 and 100/0), respectively. As the temperature increased, the relative β-sheet content decreased for PEG–PAF with the enantiomeric alanine, while there was no significant change in random coil structures of PEG–PAFs with a mixed composition. 13C-NMR spectra suggest the dehydration and decrease in molecular motion of PEG during the sol-to-gel transition of PEG–PAF. Through the partial incorporation of D-alanine into PEG-L-PAF, thermogelling behaviour was also observed at much higher temperatures and concentrations due to the difference in the secondary structure and nanoassembly of the polymers. The enzymatic degradability of PEG–PAF also decreased as a result of the partial incorporation of D-alanine into PEG-L-PAF. The paper suggests that partial incorporation of D-alanine into an L-polypeptide block copolymer can be a useful method in designing a biodegradable material as well as controlling nanoassemblies of the polymer.

Ion and Temperature Sensitive Polypeptide Block Copolymer

A poly(ethylene glycol)/poly(L-alanine) multiblock copolymer incorporating ethylene diamine tetraacetic acid ([PA-PEG-PA-EDTA(m)) was synthesized as an ion/temperature dual stimuli-sensitive polymer, where the effect of different metal ions (Cu(2+), Zn(2+), and Ca(2+)) on the thermogelation of the polymer aqueous solution was investigated. The dissociation constants between the metal ions and the multiblock copolymer were calculated to be 1.2 × 10(-7), 6.6 × 10(-6), and 1.2 × 10(-4) M for Cu(2+), Zn(2+), and Ca(2+), respectively, implying that the binding affinity of the multiblock copolymer for Cu(2+) is much greater than that for Zn(2+) or Ca(2+). Atomic force microscopy and dynamic light scattering of the multiblock copolymer containing metal ions suggested micelle formation at low temperature, which aggregated as the temperature increased. Circular dichroism spectra suggested that changes in the α-helical secondary structure of the multiblock copolymer were more pronounced by adding Cu(2+) than other metal ions. The thermogelation of the multiblock copolymer aqueous solution containing Cu(2+) was observed at a lower temperature, and the modulus of the gel was significantly higher than that of the system containing Ca(2+) or Zn(2+), in spite of the same concentration of the metal ions and their same ionic valence of +2. The above results suggested that strong ionic complexes between Cu(2+) and the multiblock copolymer not only affected the secondary structure of the polymer but also facilitated the thermogelation of the polymer aqueous solution through effective salt-bridge formation even in a millimolar range of the metal ion concentration. Therefore, binding affinity of metal ions for polymers should be considered first in designing an effective ion/temperature dual stimuli-sensitive polymer.

Temperature-sensitive polypeptide nanogels for intracellular delivery of a biomacromolecular drug

Temperature sensitive nanogels prepared from ionic complexes of positively charged poly(ethylene glycol)-poly(l-lysine)-poly(l-alanine) (PEG-PK-PA) and negatively charged hyaluronic acid (HA) were investigated as intracellular delivery vehicles of a biomacromolecular drug of fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA). By varying the weight ratio of the polymer to hyaluronic acid from 100/0 to 19/81, the zeta potential of the nanogel could be controlled from +47 mV (100/0), 0 mV (67/33), and -47 mV (35/65). In particular, the nanogels prepared from 67/33 exhibited 0 mV, and the size was reversibly changed from 220 nm at 20 °C to 160 nm at 37 °C with a narrow size distribution. The internalization of the FITC-BSA loaded nanogel was significantly affected by the zeta potential. In particular, the nanogel with zero zeta potential was very effective in internalizing the model drug. The cells treated with chlorpromazine significantly reduced the internalization efficiency, suggesting that clathrin mediated endocytosis is the main mechanism of the internalization of the nanogel. Cytotoxicity measured by the MTT assay suggested that the PEG-PK-PA/HA ionic complex nanogel is significantly less cytotoxic than PEG-PK-PA itself. This paper suggests that (1) the PEG-PK-PA/HA nanogel could be tightened by heat-induced shrinkage, (2) the internalization efficiency of the nanocarrier could be controlled by modulating the size and zeta potential of the nanogel, and (3) cytotoxicity of the positively charged nanogel was significantly improved by the formation of the ionic complex with negatively charged hyaluronic acid.