Bo Gyu Choi

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.

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.

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.

Synthesis and characterization of novel thermo-responsive F68 block copolymers with cell-adhesive RGD peptide

Thermosensitive poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer, Pluronic F68, containing a hydrophobic unit, oligo-(lactic acid)(oligo-LA) or oligo-caprolactone (oligo-CL), 2-META and RGD as side groups was successfully synthesized and characterized by (1)H NMR, FTIR, and elemental analysis. Their aqueous solution displayed special gel-sol-gel phase transition behavior with increasing temperature from 10 to 70°C, when the polymer concentration was above critical micelle concentration (CMC). The gel-sol phase diagram was investigated using tube inversion method, rheological measurement, and dynamic light scattering. Based on these results, the gelation properties of modified F68 were affected by several factors such as the composition of the substituents, chain length of oligo L-LA or oligo ε-CL, and the concentration of the polymer solutions. The unique phase transition behavior with temperature was observed by modified F68 triblock copolymer, composed of the PPO blocks core and the PEO blocks shell in aqueous solution. This phenomenon was elucidated using (1)H NMR data; the alteration of hydrophobic interaction and chain mobility led to the formation of transparent gel, coexistence of gel-sol, and opaque gel. These hydrogels may be useful in drug delivery and tissue engineering.

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.

Block sequence affects thermosensitivity and nano-assembly: PEG-L-PA-DL-PA and PEG-DL-PA-L-PA block copolymers

We are reporting triblock copolymers of (ethylene glycol)44–(L-alanine)9–(DL-alanine)9 (PEG–L-PA–DL-PA) with α-helical L-PA localized between flexible PEG and DL-PA, and (ethylene glycol)44–(DL-alanine)9–(L-alanine)9 (PEG–DL-PA–L-PA) with gradient flexibility in water. Aqueous solutions of PEG–L-PA–DL-PA underwent only sol-to-gel transition, whereas those of PEG-DL-PA-L-PA underwent sol-to-gel-to-squeezed gel transitions as the temperature increased. The L-PAs of both polymers have an α-helical secondary structure in water at low temperature. However, the α-helical structure of the PEG-DL-PA-L-PA changed into a random coil structure as the temperature increased above 40 °C, whereas the PEG-L-PA-DL-PA kept the α-helical secondary structure over the same investigated temperature range of 4 °C to 50 °C. Cryo-transmission electron microscopy images and dynamic light scattering suggested that the PEG-L-PA-DL-PA develops spherical micelles, whereas the PEG-DL-PA-L-PA develops cylindrical bundles as well as spherical micelles in water. Even though both block copolymers have a similar composition of (ethylene glycol)44, (L-alanine)9, and (DL-alanine)9, they showed significantly different temperature-sensitivities as well as different nano-assemblies in water. This report suggests that the block sequence of a polymer is very important in developing a specific nano-structure as well as in controlling thermosensitivity of the polymer, thus providing useful molecular information in designing a biomaterial.

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.

Chondrocyte 3D-culture in RGD-modified crosslinked hydrogel with temperature-controllable modulus

AbstractBased on the thermosensitivity of Pluronic® F127 and the cytointeractability of Arg-Gly-Asp (RGD) sequences, RGD-modified F127 dimethacrylate (FM-RGD) hydrogel was investigated as a 3-dimensional culture matrix for chondrocytes. Chondrocytes were encapsulated in the hydrogel by radical copolymerization of FM-RGD and FM in the presence of cells. The FM-RGD hydrogel modulus could be modulated by temperature variation from 15 °C (1,300 Pa) to the culture temperature of 37 °C (8,900 Pa), at which the hydrogel provided a condrocyte-compatible microenvironment. Compared with the hydrogel prepared from F127 dimethacrylate (FM) without RGD, the RGD-modified hydrogel produced significant (p<0.01) improvements in cell proliferation, DNA production, and viability while allowing the chondrocytes to maintain their original spherical phenotypes.