Min Hee Park

Block length affects secondary structure, nanoassembly and thermosensitivity of poly(ethylene glycol)-poly(L-alanine) block copolymers

Poly(ethylene glycol)-conjugated polypeptides have been drawing attention as a biomaterial as well as a pharmaceutical agent. In this paper, we synthesized a series of poly(ethylene glycol)-poly(L-alanine) block copolymers (PEG-L-PA) and investigated the block length effect on (1) the secondary structure of the PA, (2) the nanostructure of the self-assembled amphiphilic PEG-L-PA, and (3) the thermosensitivity of the PEG-L-PA aqueous solution. First, the molecular weight of the L-PA was fixed at 700–760 Daltons and that of the conjugated PEG varied over 1,000, 2,000, and 5,000 Daltons. L-PA with an antiparallel β-sheet structure in water transformed into an α-helical structure, and the self-assembled nanostructure of PEG-L-PA changed from a fibrous structure to a spherical micellar structure as the molecular weight of conjugated PEG increased. Then, when the molecular weight changed from 700 to 1,500 Daltons at a fixed molecular weight of PEG at 2,000, similar transitions involving antiparallel β-sheets changing to α-helices, and fibers to spherical micelles were observed. The polymer aqueous solution underwent a sol-to-gel transition as the temperature increased in a high polymer concentration range of 3–14 wt%. Interestingly, the transition temperature did not follow the simple rule that a more hydrophobic polymer has a lower transition temperature. This paper suggests that the control of PEG molecular weight in PEG-conjugated polypeptide biomaterials is important in that it affects the secondary structure of the polypeptide, the nanoassembled morphology, and the thermosensitivity of the polymer.

3D Culture of Adipose-Tissue-Derived Stem Cells Mainly Leads to Chondrogenesis in Poly(ethylene glycol)-Poly(l-alanine) Diblock Copolymer Thermogel

Poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA)s with L-PA molecular weights of 620, 1100, and 2480 Da and a fixed molecular weight of PEG at 5000 Da were synthesized to compare the thermosensitive behavior, and to investigate their potential as a three-dimensional (3D) culture matrix of adipose-tissue-derived stem cells (ADSCs). The sol-to-gel transition temperature and the concentration ranges where the transition was observed decreased as the L-PA molecular weight increased. ADSCs were cultured in the 3D matrixes of in situ formed PEG-L-PA hydrogels, which were produced by increasing the temperature of cell-suspended PEG-L-PA aqueous solutions. The spherical morphology was maintained in the PEG-L-PA hydrogel, while the cells underwent fibroblastic morphological changes in the Matrigel over 14 days of incubation. ADSCs exhibited high expression of type II collagen in the PEG-L-PA thermogel. In addition, they also moderately expressed the biomarker of myogenic differentiation factor 1 as the same mesodermal lineages, as well as the type III β-tubulin as a cross-differentiation biomarker. Similar to the in vitro study, the ADSCs predominantly exhibited chondrogenic biomarkers in the in vivo study. The study demonstrates that the polypeptide thermogel of PEG-L-PA is promising as a 3D culture matrix of ADSCs and as an injectable tissue engineering biomaterial.

Enzymatically degradable thermogelling poly(alanine-co-leucine)-poloxamer-poly(alanine-co-leucine).

In the search for an enzymatically degradable thermogelling system, we are reporting poly(alanine-co-leucine)-poloxamer-poly(alanine-co-leucine) (PAL-PLX-PAL) aqueous solution. As the temperature increased, the polymer aqueous solution underwent sol-to-gel transition at 20-40 °C in a polymer concentration range of 3.0-10.0 wt %. The amphiphilic polymers of PAL-PLX-PAL form micelles in water, where the hydrophobic PALs form a core and the hydrophilic PLXs form a shell of the micelle. FTIR, circular dichroism, and (13)C NMR spectra suggest that the α-helical secondary structure of PAL is preserved; however, the molecular motion of the PLX significantly decreases in the sol-to-gel transition range of 20-50 °C. The polymer was degraded by proteolytic enzymes such as matrix metalloproteinase and elastase, whereas it was quite stable against cathepsin B, cathepsin C, and chymotrypsin or in phosphate-buffered saline (control). The in situ formed gel in the subcutaneous layer of rats showed a duration of ∼ 47 days, and H&E staining study suggests the histocompatibility of the gel in vivo with a marginal inflammation response of capsule formation. A model drug of bovine serum albumin was released over 1 month by the preset-gel injection method. The thermogelling PAL-PLX-PAL can be a promising biocompatible material for minimally invasive injectable drug delivery.

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.

Reverse thermal gelation of PAF-PLX-PAF block copolymer aqueous solution.

The aqueous solution of poly(L-Ala-co-L-Phe)-poly(propylene glycol)-poly(ethylene glycol)-poly(propylene glycol)-poly(L-Ala-co-L-Phe) block copolymers (PAF-PLX-PAF) in a concentration range of 6.0-10.0 wt % underwent sol-to-gel transition as the temperature increased from 10 to 50 degrees C. Circular dichroism spectra, hydrophobic dye solubilization, dynamic light scattering, and transmission electron microscopy image of the polymer suggest that the polymers form micelles in water, where the hydrophilic (PLX) blocks form a shell and the hydrophobic (PAF) blocks form a core of the micelle. Circular dichroism, FTIR, and (13)C NMR spectra suggest that sol-to-gel transition accompanies partial strengthening of the beta-sheet structure of PAF and a decrease in molecular motion of the PLX. The sol-to-gel transition temperature could be controlled by varying the molecular weight of PAF and PLX blocks, the ratio of Ala to Phe, and the corresponding secondary structure of the polypeptide.

Thermal gelling polyalanine-poloxamine-polyalanine aqueous solution for chondrocytes 3D culture: Initial concentration effect

Three dimensional (3D) cell culturing in an artificial matrix needs understanding of the dynamic microenvironments of the extracellular matrix and the cells. In this paper, we investigated a thermal gelling polyalanine–poloxamer–polyalanine (PA–PLX–PA) aqueous solution for chondrocyte 3D culture, focusing on the initial concentration of the polymer aqueous solution. As the polymer concentration increased from 7.0 wt. % to 10.0 wt. % and 15.0 wt. %, moduli of the in situ formed gels at 37 °C were increased from 350–380 Pa to 2100–2300 Pa and 5300–5700 Pa, respectively. In addition, the population and thickness of the nanofibers in the gel were increased. Chondrocytes kept their spherical phenotypes in the 3D environment of the in situ formed PA–PLX–PA hydrogel. They showed excellent cell viability, increased production of sGAG and type II collagen in PA–PLX–PA gel prepared from initial polymer concentration of 7.0 wt. % and 10.0 wt. %, emphasizing the significance of the micromechanical environments for the 3D cell culture.

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.

pH/temperature sensitive chitosan-g-(PA-PEG) aqueous solutions as new thermogelling systems

As a new thermal gelling polymer aqueous solution, we are reporting a poly(ethylene glycol)-poly(alanine) grafted chitosan (CS-g-(PA-PEG)) system. The sol–gel transition temperature and the modulus of the in situ-formed thermal gel at 37 °C changed 17 → 27 →32 °C and 396 → 241 → 43 Pa, respectively, as the pH increased from 3.0 to 6.5 and to 9.0. The mechanism of such a pH/temperature sensitive behaviour of the CS-g-(PA-PEG) aqueous solution was investigated by studying changes in the conformation of chitosan, polyalanine and PEG of the CS-g-(PA-PEG). As the temperature increased, ammonium groups of the chitosan partially deprotonated to a neutral form, the α-helical content of the polyalanine increased and molecular motion of the PEG decreased. Such changes cooperatively increase the hydrophobicity and viscosity of CS-g-(PA-PEG), resulting in the sol–gel transition of the polymer aqueous solution with increasing temperature. As the pH increased, ammonium groups of the chitosan deprotonated to a neutral form and the α-helical content of polyalanine decreased, which induce a change in the nanoassembly of the polymer. CS-g-(PA-PEG) significantly increased the gel modulus of a previously reported PEG grafted chitosan (CS-g-PEG) thermal gel by incorporating the α-helical polyalanine moieties between CS and PEG. The CS-g-(PA-PEG) could be a promising biomaterial as a new robust thermogel with pH and temperature sensitivity.

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.

CO2- and O2-Sensitive Fluorophenyl End-Capped Poly(ethylene glycol)

Pentafluorophenyl end-capped poly(ethylene glycol) (PF-PEG-PF) aqueous solution shows a lower critical solution temperature (LCST), which is sensitive to the type of gases dissolved in the solution. LCST increases from 24.5 to 26 °C when dissolved carbon dioxide is replaced by oxygen. The transparent-to-turbid transition is reversibly observed when the dissolved carbon dioxide in the PF-PEG-PF aqueous solution is exchanged with oxygen, and vice versa, at 24.5 °C. (19) F NMR and (1) H NMR spectra of the PF-PEG-PF in D2 O suggest that 1) dehydration of PEG is the main reason of developing LCST of the PF-PEG-PF aqueous solution, 2) minute differences in the intermolecular interactions, as demonstrated by changes in the chemical shift of the PF-PEG-PF peaks, induce such a difference in LCST. This paper provides a new insight in designing a stimuli-responsive polymer in that fine tuning of a phase transition can be controlled by the type of dissolved gas.