Min Kyung Joo

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.

Significance of secondary structure in nanostructure formation and thermosensitivity of polypeptide block copolymers

Well-defined nanostructural control from biological motifs is gaining attention among materials scientists. We are reporting that the β-sheet structure of L-polyalanine plays a critical role in developing a fibrous nanostructure as well as the sol-to-gel transition of amphiphilic poly(ethylene glycol)-L/or DL-polyalanine diblock copolymers. L-isomers underwent transitions from random coils to β-sheets, and to nanofibers as the polymer concentration increased, whereas the DL-isomer remained as a random coil structure without developing any specific nanostructure. At high polymer concentrations, the aqueous polymer solutions underwent a sol-to-gel transition as the temperature increased, a so called reverse thermal gelation. The L-isomer with a preassembled β-sheet secondary structure facilitates the sol-to-gel transition rather than the DL-isomer with a random coil structure. Thus, only the L-isomer showed a sol-to-gel transition in the physiologically important range of 20–40 °C. This report provides fundamental information on the relationship between hierarchical structures of polypeptides and the thermosensitive sol-gel transition of the polypeptide aqueous solution.

Cell therapy for skin wound using fibroblast encapsulated poly(ethylene glycol)-poly(L-alanine) thermogel.

As a new application of a thermogel, a poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA) gel encapsulating fibroblasts was investigated for wound healing. The fibroblasts were encapsulated by the temperature sensitive sol-to-gel transition of the polymer aqueous solution. Under the in vitro three-dimensional (3D) cell culture condition, the PEG-L-PA thermogel was comparable with Matrigel for cell proliferation and was significantly better than Matrigel for collagen types I and III formation. After confirming the excellent 3D microenvironment of the PEG-L-PA thermogel for fibroblasts, in vivo wound healing was investigated by injecting the cell-suspended polymer aqueous solution on incisions of rat skin, where the cell-encapsulated gel was formed in situ. Compared with the phosphate buffered saline treated system and the cell-free PEG-L-PA thermogel, the cell-encapsulated PEG-L-PA thermogel not only accelerated the wound closure but also improved epithelialization and the formation of skin appendages such as keratinocyte layer (epidermis), hair follicles, and sebaceous glands. The results demonstrate the potential of thermogels for cell therapy as an injectable tissue-engineering scaffold.

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.

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.

Sol–gel transition of PEG–PAF aqueous solution and its application for hGH sustained release

We report a poly(ethylene glycol)–poly(L-alanine-co-L-phenylalanine) (PEG–PAF) aqueous solution as a polypeptide-based thermogelling system and its application as an injectable sustained release system for human growth hormone (hGH). The PEG–PAF aqueous solution underwent sol-to-gel transition at 16–34 °C in a polymer concentration range of 6.0–14.0 wt% as the temperature increased. Dynamic light scattering, circular dichroism, FTIR, and 13C-NMR spectra indicated that the secondary structure of PAF was preserved, however, PEG was dehydrated in the sol-to-gel transition temperature range. A micelle aggregation model was suggested for the sol-to-gel transition of the current PEG–PAF, similar to previous polyesters. The polymer was quite stable in water, and therefore, the molecular weight of the polymer did not significantly change and pH of the aqueous polymer solution was maintained at 7.2–7.8 during the 1 month storage of the polymer as an aqueous solution at room temperature. This point is clearly distinguished from previous thermogelling polymers based on polyesters, polyorthoesters, polyphosphazenes, poly(β-aminoester urethane)s, and polyanhydrides, which generate acid degradation products or can be degraded during storage as an aqueous polymer solution. Therefore, the current system can be used as a ready-to-use injectable implant for biomedical applications. When the polymer aqueous solution (0.5 mL) was injected into the subcutaneous layer of rats, the gel was formed by temperature-sensitive sol-to-gel transition, and the gel was completely eliminated from the implanted site in 1–5 ng mL−1in vivo, suggesting that the system is promising as a once-per-week delivery system for the hGH.

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.

Thermogelling Chitosan-g-(PAF-PEG) Aqueous Solution As an Injectable Scaffold

The present study reports on a thermogelling poly(ethylene glycol)-poly(L-alanine-co-L-phenyl alanine) grafted chitosan (CS-g-(PAF-PEG)) system, focusing on phase diagram, transition mechanism, and in vivo gel duration. The sol-to-gel transition temperature decreased from 27 to 11 °C as the concentration increased from 4.0 wt % to 9.0 wt %. The polymer formed micelles with 10-50 nm in diameter at 10 °C and formed large aggregates ranging from hundreds to thousands of nanometers in size as the temperature increased from 10 to 35 °C, suggesting that an extensive molecular aggregation might be involved in the sol-to-gel transition. To study the transition mechanism on a molecular level, we investigated pH, circular dichroism spectra, and (13)C NMR spectra of the CS-g-(PAF-PEG) aqueous solution as a function of temperature. As the temperature increased, deprotonation of the chitosan and dehydration of the PEG were suggested, whereas the α-helical secondary structure of PAF was slightly changed in the sol-to-gel transition temperature range of 10-50 °C. A gel was formed in situ after injecting the CS-g-(PAF-PEG) aqueous solution into the subcutaneous layer of rats. About 60-70% of the gel was eliminated in 1 week, and the remaining gel was completely cleared from the implant site in 14 days. The results indicate the potential of CS-g-(PAF-PEG) as a promising short-term carrier for pharmaceutical agents.

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.

Multiple Sol-Gel Transitions of PEG-PCL-PEG Triblock Copolymer Aqueous Solution

An aqueous solution of a poly(ethylene glycol)-polycaprolactone-poly(ethylene glycol) (PEG-PCL-PEG) with a composition of EG(13) CL(23) EG(13) undergoes multiple transitions, from sol-to-gel (hard gel)-to-sol-to-gel (soft gel)-to-sol, in the concentration range 20.0∼35.0 wt.-%. Through dynamic mechanical analysis, UV-vis spectrophotometry, small angle X-ray scattering, differential scanning calorimetry, microcalorimetry and (13) C NMR spectroscopy, the mechanism of these transitions was investigated. The hard gel and soft gel are distinguished by the crystalline and amorphous state of the PCL. The extent of PEG dehydration and the molecular motion of each block also played a critical role in the multiple transitions. This paper suggests a new mechanism for these multiple transitions driven by temperature changes.