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.

Chiro-Optical Modulation for NURR1 Production from Stem Cells.

Nuclear receptor related 1 (NURR1) is an essential protein for maintenance of dopaminergic neurons in adult midbrain of which deficiency leads to Parkinson's disease. To enhance the NURR1 production of neural cells, various approaches are under investigation. Here we report that NURR1 is highly expressed in stem cells by exposure to an L-polarized blue light emitting diode (LED). Compared to stem cells cultured in the absence of a LED, under polarized green and red LEDs, the stem cells exposed to a polarized blue LED significantly enhanced neuronal biomarkers such as neurofilament M (NFM) and neuron specific enolase (NSE) at both mRNA and protein levels. In particular, NURR1 was selectively enhanced by the stem cells exposed to the L-polarized blue LED. Stem cells exposed to the L-polarized blue LED increased mitochondrial ATP and intracellular calcium ions, which support neuronal differentiation of the stem cells. This study suggests that chiro-optical treatments by using polarized light with a specific wavelength can be used for engineering of stem cells with enhanced specific biochemicals, which may open a new method for a specific disease.

Hepatogenic Supported Differentiation of Mesenchymal Stem Cells in a Lactobionic Acid-Conjugated Thermogel.

To investigate the effect of receptor substrate of target cells on stem cell differentiation, lactobionic acid-conjugated poly[(propylene glycol)-b-(ethylene glycol)-b-(propylene glycol)]-poly(l-alanine) (LB-PLX-PA) was synthesized, and then thermogelling systems consisting of LB-PLX-PA and PLX-PA in a ratio of 0/100 (LB-0), 5/95 (LB-5), and 20/80 (LB-20) were constructed as an injectable three-dimensional scaffold toward hepatogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs). Modulus of LB-0, LB-5, and LB-20 increased to 500-800 Pa at 37 °C (gel) due to the heat induced sol-to-gel transition of the systems during which TMSCs were incorporated into the gel. Based on biomarker expressions and hepatic biofunctions of the differentiated cells, the receptor substrate (LB)-conjugated bioactive thermogel provides compatible microenvironments for the differentiated cells, and thus gives pronounced positive results on the differentiation of the stem cells into target cells during three-dimensional culture, compared with a passive thermogel.

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.

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.

Nanocomposite versus Mesocomposite for Osteogenic Differentiation of Tonsil-Derived Mesenchymal Stem Cells.

Injectable inorganic/organic composite systems consisting of well-defined mesocrystals (4-8 µm) of calcium phosphate and polypeptide thermogel significantly enhance the osteogenic differentiation of the tonsil derived mesenchymal stem cells (TMSCs). Compared to composite systems incorporating nanoparticles (10-100 nm) or pure hydrogel systems, osteogenic biomarkers including alkaline phosphatase (ALP), bone morphogenetic protein 2, and osteocalcin are highly expressed at both the mRNA level and the protein level in the mesocrystal composite systems. ALP activity of differentiated cells is also significantly higher in the mesocomposite systems compared to the nanocomposite systems or the pure hydrogel systems. The mesocomposite systems provide not only hard surfaces for binding the cells/proteins by the inorganic mesocrystals but also a soft matrix for holding the cells by the hydrogel. Through the current research, (1) a novel method of preparing mesocrystals is developed, (2) TMSCs are proved as a new resource of stem cells, and (3) the mesocomposite systems are proved to be a promising tool in controlling stem cell differentiation. (4) Finally, the research emphasizes the significance of mesoscience as a new perspective of science in controlling cell and material interfaces.

Microsphere-Incorporated Hybrid Thermogel for Neuronal Differentiation of Tonsil Derived Mesenchymal Stem Cells.

Neuronal differentiation of tonsil-derived mesenchymal stem cells (TMSCs) is investigated in a 3D hybrid system. The hybrid system is prepared by increasing the temperature of poly(ethylene glycol)-poly(l-alanine) aqueous solution to 37 °C through the heat-induced sol-to-gel transition, in which TMSCs and growth factor releasing microspheres are suspended. The in situ formed gel exhibits a modulus of 800 Pa at 37 °C, similar to that of brain tissue, and it is robust enough to hold the microspheres and cells during the 3D culture of TMSCs. The neuronal growth factors are released over 12-18 d, and the TMSCs in a spherical shape initially undergo multipolar elongation during the 3D culture. Significantly higher expressions of the neuronal biomarkers such as nuclear receptor related protein (Nurr-1), neuron specific enolase, microtubule associated protein-2, neurofilament-M, and glial fibrillary acidic protein are observed in both mRNA level and protein level in the hybrid systems than in the control experiments. This study proves the significance of a controlled drug delivery concept in tissue engineering or regenerative medicine, and a 3D hybrid system with controlled release of growth factors from microspheres in a thermogel can be a very promising tool.

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.

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.

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 ß-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.

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 ß-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.

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.

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 ß-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.

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.

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.

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 ß-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.

Temperature-responsive compounds as in situ gelling biomedical materials.

Aqueous solutions that undergo sol-to-gel transition as the temperature increases have been extensively studied during the last decade. The material can be designed by controlling the hydrophilic and hydrophobic balance of the material. Basically, the molecular weight of the hydrophilic block and hydrophobic block of a compound should be fine-tuned from the synthetic point of view. In addition, stereochemistry, microsequence, topology, and nanostructures of the compound also affect the transition temperature, gel window, phase diagram, and modulus of the gel. From a practical point of view, biodegradability, biocompatibility, and interactions between the material and drug or cell should be considered in designing a thermogelling material. The interactions are particularly important in that they control drug release profile and initial burst release of the drug in the drug delivery system, and affect cell proliferation, differentiation, and biomarker expression in three-dimensional cell culture and tissue engineering application. This review provides an in-depth summary of the recent progress of thermogelling systems including polymers, low molecular compounds, and nanoemulsions. Their biomedical applications were also comparatively discussed. In addition, perspectives on future material design of a new thermogelling material and its application are suggested.

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.

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.