Poly(l-threonine-co-l-threonine Succinate) Thermogels for Sustained Release of Lixisenatide.

Negatively charged poly(l-Thr-co-l-Thr succinate) (PTTs) was developed as a new thermogel. Aqueous PTT solutions underwent thermogelation over a concentration range of 6.0-8.3 wt %. Dynamic light scattering, FTIR, 1H NMR, and COSY spectra revealed the partial strengthening of the ß-sheet conformation and the dehydration of PTTs during the transition. Extendin-4 was released from the PTTs thermogel with a large initial burst release, whereas positively charged lixisenatide significantly reduced its initial burst release to 25%, and up to 77% of the dose was released from the gel over 14 days. In vivo study revealed a high plasma concentration of lixisenatide over 5 days and hypoglycemic efficacy was observed for type II diabetic rats over 7-10 days. The biocompatible PTTs were degraded by subcutaneous enzymes. This study thus demonstrates an effective strategy for reducing the initial burst release of protein drugs from thermogels with the introduction of electrostatic interactions between the drug and the thermogel.

Drug-Releasing Thermogel for Osteoarthritis Induction in an Animal Model.

The induction of disease states in animal models is an essential step in new drug discovery procedures. In this study, osteoarthritis (OA) was induced in a mouse model using a polypeptide thermogel-based sustained drug release system. Hydrophilic lactobionic acids and hydrophobic n-butyric acids were grafted onto ε-poly(l-lysine) to prepare a thermogelling polymer of ε-poly(l-lysine) grafted with lactobionic acid and butyric acid (PLLB). The gel modulus of PLLB is about 1000 Pa at 37 °C. Collagenase, which causes OA, was slowly released from the PLLB thermogel over two weeks. The PLLB formulation containing collagenases ranging from 1-10 units was intra-articularly injected into the knee of mice. OA mouse models with Osteoarthritis Research Society International (OARSI) grades of 3-6 were developed depending on the amounts of collagenase incorporated in the PLLB thermogel formulation. This study suggests that thermogel-based drug release formulations can be a precise tool for developing animal disease models in a dose-dependent manner.

Oligonucleotides as Inhibitors of Ice Recrystallization.

Oligonucleotides of adenine (A20), guanine (G20), cytosine (C20), thymine (T20), cytosine-guanine ((CG)20), and adenine-thymine ((AT)20) were investigated as model compounds for ice recrystallization inhibition (IRI). Dehydroxy uracil (dU20), U20, and T20 were also compared to investigate the effect of minute changes in the hydrophobicity of the oligonucleotides on the IRI activity. Among the oligonucleotides considered in this study, T20 exhibited the best performance for IRI. In addition, the degree of polymerization of oligothymines varied over 5, 10, 20, 30, 50, and 100, and T20 was found to be the most effective for IRI. The IRI mechanism was investigated by comparing U20 and T20, which exhibited the lowest and highest IRI activity, respectively, among the oligonucleotides for their dynamic ice-shaping, thermal hysteresis, and ice nucleation inhibition. Little or no dynamic ice-shaping activity and small thermal hysteresis were observed for both nucleotides. All of the findings suggest that not the ice-polymer adhesion but the hydrophobic interactions of T20 in the interface layer might interfere with the water deposition on the ice crystal surfaces and contribute to the IRI activity of the T20 oligonucleotide.

Poly(l-alanine-co-l-lysine)-g-Trehalose as a Biomimetic Cryoprotectant for Stem Cells.

Poly(l-alanine-co-l-lysine)-graft-trehalose (PAKT) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAKT. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAKT and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAKT solution, followed by cryopreservation at -196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0-1.0 and 0.0-15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAKT solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAKT was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAKT. 1H NMR and FTIR spectra suggested that the intracellular PEG-PAKTs interact with water and stabilize the cells during cryopreservation.

Janus Nanosheets with Face-Selective Molecular Recognition Properties from DNA-Peptide Conjugates.

Chemical and functional anisotropy in Janus materials offer intriguing possibilities for constructing complex nanostructures and regulating chemical and biological reactions. Here, the authors report the fabrication of Janus nanosheets from molecular building blocks composed of two information-carrying biopolymers, DNA and peptides. Experimental and structural modeling studies reveal that DNA-peptide diblock conjugates assemble into Janus nanosheets with distinct DNA and peptide faces. The surprising level of structural control is attributed to the exclusive parallel ß-sheet formation of phenylalanine-rich peptides. This approach is extended to triblock DNA1-peptide-DNA2 conjugates, which assemble into nanosheets presenting two different DNA on opposite faces. The Janus nanosheets with independently addressable faces are utilized to organize an enzyme pair for concerted enzymatic reactions, where enhanced catalytic activities are observed. These results demonstrate that the predictable and designable peptide interaction is a promising tool for creating Janus nanostructures with regio-selective and sequence-specific molecular recognition properties.

Hypothermic Stem Cell Storage Using a Polypeptide Thermogel.

We report a polypeptide-based thermogel as a new tool for hypothermic storage of stem cells at ambient temperature (25 °C). Stem cells were suspended in the sol state (10 °C) of an aqueous poly(ethylene glycol)-poly(l-alanine) (PEG-PA) solution (4.0 wt %) in phosphate-buffered saline (PBS), which turned into a stem cell-incorporated gel by a heat-induced sol-to-gel transition. The cell harvesting procedure from the thermogels was simply performed through a gel-to-sol transition by diluting and cooling the system. More than 99% of stem cells died in PBS and Pluronic F127 thermogel (control thermogel) when the cells were stored at 25 °C for 7 days. The cell recovery rate from the PEG-PA thermogel (64%) was significantly greater than that from the commercially available HypoThermosol FRS preservation solution (HTS) (26%). Additionally, the surviving stem cells from the PEG-PA thermogel were healthier than those from HTS in terms of (1) expression of stemness biomarkers (NANOG, OCT4, and SOX2), (2) proliferation rate, and (3) differentiation potentials into osteogenic, chondrogenic, and adipogenic lineages. Membrane stabilization was suggested as a cell protection mechanism in the cytocompatible PEG-PA thermogel. The PEG-PA thermogel provides a convenient cytocompatible way for the storage and recovery of cells and thus is a promising tool for the transportation and short-term banking of cells.

Visible-Light Photocatalytic Conversion of Carbon Dioxide by Ni(II) Complexes with N4S2 Coordination: Highly Efficient and Selective Production of Formate.

The efficient and selective light-driven conversion of carbon dioxide to formate is a scientific challenge for green chemistry and energy science, especially utilizing visible-light energy and earth-abundant catalytic materials. In this report, two mononuclear Ni(II) complexes of pyridylbenzimidazole (pbi) and pyridylbenzothiazole (pbt), such as Ni(pbt)(pyS)2 (1) and Ni(pbi)(pyS)2 (2) (pyS = pyridine-2-thiolate), were prepared and their reactivities studied. The two Ni complexes were examined for CO2 conversion using eosin Y as a photosensitizer upon visible-light irradiation in a H2O/ethanol solvent. The photoreaction of CO2 catalyzed by complexes 1 and 2 selectively affords formate with a high efficiency (14 000 turnover number) and a high catalytic selectivity of ∼99%. Undesirable proton reduction pathways were completely suppressed in the photocatalytic reactions with these sulfur-rich Ni catalysts under CO2. Hydrogen photoproduction was also studied under argon. Their kinetic isotope effects and influence of solution pH for formate and H2 production in the photocatalytic reactions are described in relation to the reaction mechanisms. These bioinspired Ni(II) catalysts with N/S ligation in relation to [NiFe]-hydrogenases are the first examples of early transition metal complexes affording such high selectivity and efficiencies, providing a future path to design solar-to-fuel processes for artificial photosynthesis.

ROS-Sensitive Degradable PEG-PCL-PEG Micellar Thermogel.

A reactive oxygen species (ROS)-sensitive degradable polymer would be a promising material in designing a disease-responsive system or accelerating degradation of polymers with slow hydrolysis kinetics. Here, a thermogelling poly(ethylene glycol)-polycaprolactone-poly(ethylene glycol) (PEG-PCL-PEG or EG12 -CL20 -EG12 ) triblock copolymer with an oxalate group at the middle of the polymer is reported. The polymers form micelles with an average size of 100 nm in water. Thermogelation is observed in a concentration range of 8.0-37.0 wt%. In particular, the aqueous PEG-PCL-PEG triblock copolymer solutions are in a gel state at 37 °C in a concentration range of 25.0-37.0 wt%, whereas the aqueous PEG-PCL diblock copolymer solutions are in a sol state in the same concentration range at 37 °C. Thus, the gel depot could dissolve out once degradation of the triblock copolymers occurs at the oxalate group as confirmed by the in vitro experiment. In vivo gel formation is confirmed by injecting an aqueous PEG-PCL-PEG solution (36.0 wt%) into the subcutaneous layer of rats. The gel completely disappears in 21 d. A model polypeptide drug (cyclosporine A) is released over 21 d from the in situ formed gel. The micelle-based thermogel of PEG-PCL-PEG with ROS-triggering degradability is a promising injectable material for biomedical applications.

Thermogelling Inclusion Complex System for Fine-Tuned Osteochondral Differentiation of Mesenchymal Stem Cells.

How to control osteochondral differentiation of mesenchymal stem cells at a proper stage is a key issue for articular cartilage regeneration. To solve this problem, injectable scaffolds with different chemical functional groups were designed by introducing one equivalent of α-cyclodextrin (α-CD) carboxylate and α-CD phosphate along poly(ethylene glycol)-poly(l-alanine) (PEG-L-PA) block copolymers. Dynamic light scattering, transmission electron microscopy images, and two-dimensional NMR spectra indicated that the PEG-L-PA block copolymers formed inclusion complexes with α-CD derivatives. Aqueous solutions of PEG-L-PA block copolymers (P), α-CD carboxylate/PEG-L-PA block copolymers (PCC), and α-CD phosphate/PEG-L-PA block copolymers (PCP) underwent sol-to-gel transition as the temperature increased. The storage moduli of P, PCC, and PCP gels ranged from 1000 to 1300 Pa at 37 °C. Tonsil-derived mesenchymal stem cells (TMSCs) were incorporated in situ in the gel during thermogelation of P, PCC, and PCP, which became the three-dimensional cell culture systems with different functional groups. After 21 days of incubation of TMSCs in the P, PCC, and PCP systems, the chondrogenic differentiation biomarker of type II collagen significantly increased in the P system, whereas the osteogenic biomarkers of osteocalcin and runt-related transcription factor 2 significantly increased in the PCP system. Both chondrogenic and osteogenic biomarkers were highly expressed in the PCC system. This study proved that thermogelling inclusion complex systems consisting of PEG-L-PA block copolymers and α-CD derivatives could be an excellent injectable matrix for fine-controlling osteochondral differentiation of mesenchymal stem cells.

Iron Ion-Releasing Polypeptide Thermogel for Neuronal Differentiation of Mesenchymal Stem Cells.

A poly(ethylene glycol)-based thermogel can capture an iron ion (Fe3+) through a crown ether-like coordination bond between the oxygen atom and metal ions, thus, providing a sustained Fe3+-releasing system. Poly(ethylene glycol)-l-poly(alanine) thermogel was used in this study. The polypeptide forms a rather robust gel, and the degradation products are a neutral amino acid, which provides cyto-compatible neutral pH environments during the cell culture. During the heat-induced sol-to-gel transition at 37 °C, tonsil-derived mesenchymal stem cells (TMSCs) and iron ions were incorporated, leading to the formation of a three-dimensional matrix toward neuronal differentiation of the incorporated TMSCs. The initial concentration of the iron ions was varied between 0, 15, 30, and 60 mM. About 10% of the loaded iron ions was released over 21 days, which continuously supplied iron ions to the cells. The incorporation of iron ions not only increased the gel modulus at 37 °C from 107 to 680 Pa, but also promoted cell aggregation with a significant secretion of the cell adhesion signal of FAK. Expression of biomarkers related to the neuronal differentiation of TMSCs, including NFM, MAP2, GFAP, NURR1, NSE, and TUBB3, increased 4-35-fold at the mRNA level in the Fe3+-containing system compared to that of the system without Fe3+. Immunofluorescence studies also confirmed pronounced cell aggregation and a significant increase in neuronal biomarkers at the protein level. This study suggests that an iron ion-releasing thermogelling system can be a promising injectable scaffold toward neuronal differentiation of stem cells.

Fast Degradable Polycaprolactone for Drug Delivery.

Polycaprolactone (PCL) was reported a long time ago; however, its biomedical applications has not been extensively investigated in comparison with poly(lactide- co-glycolide) (PLGA) due to its too slow degradation profile. Here, we are reporting an oxalate-connected oligocaprolactone multiblock copolymer (PCL-OX) as a fast degradable PCL while maintaining its crystalline properties and low melting point of PCL. The in vivo application of the paclitaxel-loaded PCL-OX microspheres provided a steady plasma drug concentration of 6-9 µg/mL over 28 days, similar to that of the PLGA microspheres. Both PCL and PLGA microspheres were completely cleared two months after in vivo implantation. The PCL-OX microspheres showed a similar tissue compatibility to that of PLGA microspheres in the subcutaneous layer of rats. These findings suggest that PCL-OX is a useful biomaterial that solves the slow degradation problems of PCL and, thus, may find uses in other biomedical applications as an alternative to PLGA.

α,ω-Diphenylalanine-End-Capping of PEG-PPG-PEG Polymers Changes the Micelle Morphology and Enhances Stability of the Thermogel.

Pluronics F127 (P, PEG-PPG-PEG triblock copolymer) was coupled with diphenylalanine (FF) to prepare FF-end-capped Pluronics (FFPFF). With increasing temperature from 10 to 60 °C, the FFPFF self-assembled to vesicles in water. The unimer-to-vesicle transition accompanies endothermic enthalpy of 53.9 kcal/mol. Aqueous P and FFPFF solutions exhibited thermogelation in 15.0-24.0 wt %. The gel phase of FFPFF was stable up to 90 °C, whereas that of P turned into a sol again at 55-86 °C, indicating that end-capping with FF improved the gel stability against heat. In addition, the carboxylic acids of the FF end-groups can form coordination bonds with metal ions, and the gel modulus at 37 °C increased from 15-21 KPa (P) to 20-25 KPa (FFPFF) to 24-28 KPa (FFPFF-Zn), and the duration of gel against water-erosion increased from 24 h (P) to 60 h (FFPFF-Zn), leading to a useful biomaterial for sustained drug delivery. The FFPFF-Zn gels implanted in the rats' subcutaneous layer induced a mild inflammatory responses. Contrary to the previous end-capping of Pluronics by poly(lactic acid), polycarprolactone, carboxylic acid, and so on that weakened the gel stability, the diphenylalanine end-capping strengthened the stability of Pluronics gel against heat and water-erosion. This paper suggests that the control of polymer nanoassemblies directed by FF end-groups improves the mechanical properties and stability of the resulting thermogel and, thus, provides a useful drug delivery carrier with prolonged durability.

EF-Hand Mimicking Calcium Binding Polymer.

There are four EF-hand polypeptides in calmodulin, a natural ubiquitous calcium binding protein that activates the enzymes involved in Ca(2+)-mediated signal transduction. An EF-hand polypeptide has six carboxylate functional groups in the middle loop region between two rigid polypeptides. In this study, a calcium binding polymer (CBP) with a structure of poly(L-alanine)-poly(L-alanine-co-L-glutamic acid)-poly(ethylene glycol)-poly(L-alanine-co-L-glutamic acid)-poly(L-alanine) (PA-PAE-PEG-PAE-PA; A11.1-A3.4E3.2-EG40.1-A3.4E3.2-A11.1) was synthesized by mimicking the EF-hand polypeptide. The 6-7 carboxylate functional groups from PAE are expected to form a binding site for Ca(2+). As the Ca(2+) bound to CBP, small changes in the circular dichroism spectra and (13)C NMR spectra were observed, indicating that Ca(2+) binding to CBP induced changes in the conformation of CBP. The binding constant of CBP to Ca(2+) was investigated by using the competitive binding of 2,2',2″,2‴-{ethane-1,2-diylbis[oxy(4-bromo-2,1-phenylene)nitrilo]} tetraacetic acid (5,5-Br2-BAPTA). The binding constant obtained with a CaLigator program by least-squares fitting of the absorbance profile as a function of Ca(2+) concentration was 5.1 × 10(5) M(-1), which was similar to that of calmodulin. The selectivity of CBP for metal ion binding was compared among Ca(2+), Cu(2+), and Zn(2+). The binding constant was obtained through a similar competitive binding study with murexide. The binding constants for Ca(2+), Cu(2+), and Zn(2+) were 7.0 × 10(5), 4.2 × 10(5), and 1.7 × 10(5) M(-1), respectively, indicating 2-4-fold higher selectivity of CBP for Ca(2+) compared to Cu(2+) and Zn(2+). The CBP has selectivity for Ca(2+), and binding affinity for Ca(2+) was similar to the biological Ca(2+) binding motif of calmodulin.

Feasibility of injectable thermoreversible gels for use in intramuscular injection of parathyroid autotransplantation.

Surgical transplantation of parathyroid tissue into the forearm muscle is one of the most commonly used surgical techniques. While simple, the procedure suffers from drawbacks. This study evaluated the feasibility of thermoreversible gel as an injectable carrier for parathyroid autotransplantation. Polyethyleneglycol-polyalanine-co-phenylalanine (PEG-PAF) thermoreversible gel (sol form at 4 °C, gel form at 37 °C) were manufactured. Thirty-eight Sprague-Dawley rats were divided into two groups (19 control, C group; 19 experimental, P group). The parathyroid glands of rats were excised. Parathyroid tissues were transplanted into the muscle pocket in sternocleidomastoid muscle in the C group. In the P group, the tissues were injected into the same muscle mixed with 0.3 ml thermoreversible gel. The serum levels of parathyroid hormone (PTH), ionized calcium, and phosphorous were measured before surgical procedure, on 7, 21, 56, and 70 days after surgery. Histology and immunohistochemistry were performed. Preoperative median PTH level of the C and the P group were 60.80 and 43.85 pg/ml, respectively (p = 0.641). Seventy days after surgery, median PTH level was 32.8 and 25.61 pg/ml, respectively. On day 70, the PTH level was restored by 54 % in the C group and 56 % in the P group compared to the preoperative value (p = 0.620). There were no significant intergroup differences in the ionized calcium/phosphorous level. Histology and immunohistochemistry revealed the successful transplantation of parathyroid tissues into the muscles in both groups. In conclusion, the PEG-PAF-based thermoreversible gel is a good candidate carrier material for intramuscular parathyroid autotransplantation.

Phosphorylcholine-Based Zwitterionic Biocompatible Thermogel.

Zwitterionic polymers have been investigated as surface-coating materials due to their low protein adsorption properties, which reduce immunogenicity, biofouling, and bacterial adsorption of coated materials. Most zwitterionic polymers, reported so far, are based on (meth)acrylate polymers which can induce toxicity by residual monomers or amines produced by degradation. Here, we report a new zwitterionic polymer consisting of phosphorylcholine (PC) and biocompatible poly(propylene glycol) (PPG) as a new thermogelling material. The PC-PPG-PC polymer aqueous solution undergoes unique multiple sol-gel transitions as the temperature increases. A heat-induced unimer-to-micelle transition, changes in ionic interactions, and dehydration of PPG are involved in the sol-gel transitions. Based on the broad gel window and low protein adsorption properties, the PC-PPG-PC thermogel is proved for sustained delivery of protein drugs and stem cells over 1 week.

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.

Differentiation of tonsil-tissue-derived mesenchymal stem cells controlled by surface-functionalized microspheres in PEG-polypeptide thermogels.

Poly(ethylene glycol)-poly(l-alanine) diblock copolymer (PEG-L-PA; molecular weight of each block of 1000-1080 Da) aqueous solutions undergo sol-to-gel transition in a 3.0-8.0 wt % concentration range as the temperature increases. By incorporating the polystyrene microspheres with different functional groups with a size of 100-800 µm in in situ formed PEG-L-PA thermogels, the differentiation of tonsil-tissue-derived mesenchymal stem cells (TMSCs) was investigated. The mRNA expression and immunohistochemical assays suggested that the TMSCs preferentially undergo adipogenesis in the ammonium (-NH3(+))- or thiol (-SH)-functionalized microsphere incorporated thermogels; chondrogenesis in the thiol-, phosphate (PO3(2-))-, or carboxylate (-COO(-))-functionalized microsphere incorporated thermogels; and osteogenesis in the phosphate-, carboxylate-functionalized, or neat polystyrene microsphere incorporated thermogels. This paper provides a new TMSC 3D culture system of a sol-gel reversible matrix and suggests that the surface-functional groups of microspheres in the thermogel can control the preferential differentiation of stem cells into specific cell types during the 3D culture.

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.

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.

Low-Molecular-Weight PEGs for Cryopreservation of Stem Cell Spheroids.

Stem cell spheroids (SCSs) are a valuable tool in stem cell research and regenerative medicine. SCSs provide a platform for stem cell behavior in a more biologically relevant context with enhanced cell-cell communications. In this study, we investigated the recovery of SCSs after cryopreservation at -196 °C for 7 days. Prior to cryopreservation, the SCSs were preincubated for 0 h (no preincubation), 2 h, 4 h, and 6 h at 37 °C in the presence of low-molecular-weight poly(ethylene glycol) (PEG) with molecular weights of 200, 400, and 600 Da. The recovery rate of SCSs was markedly affected by both the PEG molecular weight and the preincubation time. Specifically, when SCSs were preincubated with a PEG200 solution for 2 to 6 h, it significantly enhanced the recovery rate of the SCSs. Internalization of PEG200 through simple diffusion into the SCSs may be the cryoprotective mechanism. The PEG200 diffuses into the SCSs, which not only suppresses osmotic pressure development inside the cell but also inhibits ice formation. The recovered SCSs demonstrated both fusibility and capabilities for proliferation and differentiation comparable to SCSs recovered after dimethyl sulfoxide 10% cryopreservation. This study indicates that PEG200 serves as an effective cryoprotectant for SCSs. A simple preincubation procedure in the presence of the polymer greatly improves the recovery rate of SCSs from cryopreservation.