ABSTRACT
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.
Subject(s)
Gels , Peptides , Animals , Peptides/chemistry , Peptides/pharmacology , Rats , Gels/chemistry , Diabetes Mellitus, Experimental/drug therapy , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacokinetics , Male , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/administration & dosage , Rats, Sprague-Dawley , Drug Liberation , Diabetes Mellitus, Type 2/drug therapy , Glucagon-Like Peptide-2 ReceptorABSTRACT
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.
ABSTRACT
We synthesized a series of [(l-Ala)x-co-(l-Thr succinate)y] (PATs), which are analogous to natural antifreezing glycoprotein with the structure of [l-Ala-l-Ala-l-Thr disaccharide]n, by varying the composition and degree of succinylation while fixing their molecular weight (Mn) and Ala/Thr ratio at approximately 10-12 kDa and 2:1, respectively. We investigated their ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), dynamic ice shaping (DIS), thermal hysteresis (TH), and protein cryopreservation activities. Both IRI and INI activities were greater for PATs with higher l-Ala content (PATs-3 and PATs-4) than those with lower l-Ala content (PATs-1 and PATs-2). DIS activity with faceted crystal growth was clearly observed in PATs-2 and PATs-4 with a high degree of succinylation. TH was small with <0.1 °C for all PATs and slightly greater for PATs with a high l-Ala content. Except for PATs-1, the protein (lactate dehydrogenase, LDH) stabilization activity was excellent for all PATs studied, maintaining LDH activity as high as that of fresh LDH even after 15 freeze-thaw cycles. To conclude, the cryo-active biomimetic PATs were synthesized by controlling the l-Ala content and degree of succinylation. Our results showed that PATs with an l-Ala content of 65-70% and degree of succinylation of 12-19% exhibited the cryo-activities of IRI, INI, and DIS, and particularly promising properties for the cryoprotection of LDH protein.
Subject(s)
Ice , Succinic Acid , Threonine , Biomimetics , Cryoprotective Agents/pharmacology , Cryoprotective Agents/chemistry , SuccinatesABSTRACT
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.
Subject(s)
Osteoarthritis , Polylysine , Mice , Animals , Drug Liberation , Osteoarthritis/drug therapy , Disease Models, Animal , CollagenasesABSTRACT
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.
Subject(s)
Ice , Oligonucleotides , Thymine , Crystallization , Water , Antifreeze Proteins/chemistryABSTRACT
Hydrogels are a three-dimensional network material with a high equilibrium water content where chemical, physical, or biomolecular crosslinking systems have been used for the network formation. In this study, we report a thermosensitive cytogel of lactobionic acid/butanoic acid-conjugated poly(ε-l-lysine) (PKLC4). The thermogelation of the aqueous PKLC4 solution (3.5 wt %) was induced by partial dehydration accompanying a random coil-to-ß-sheet transition of the polymer. During the sol-to-gel transition, the modulus increased from <0.05 Pa at <10 °C to 1300-1360 Pa at 37 °C. When HepG2 cells were incorporated into the PKLC4 solution, the gel modulus at 37 °C increased to 2300-2670 Pa. Moreover, the gel modulus was significantly affected by the cell type, population of the HepG2 cells, and live/dead states of the HepG2 cells. The cells proliferate better in the biointeractive PKLC4 thermogel than in the bioinert PEG-PA thermogel. To conclude, by combining thermosensitivity and specific binding of the receptor to the substrate, the hydrogel attained a high modulus without delay in gel time. This study provides new insights into hydrogel preparation in that substrate-receptor binding can be utilized as a crosslinking system to control the hydrogel modulus as well as a design principle for three-dimensional cache that improves cytocompatibility for cells.
Subject(s)
Peptides , Polyethylene Glycols , Peptides/chemistry , Polyethylene Glycols/chemistry , Polymers , Hydrogels/pharmacology , Hydrogels/chemistryABSTRACT
BACKGROUND: A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at -196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required. METHOD: As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at -196 °C for 7 days. Then, cell recovery was assayed. RESULTS: We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants. CONCLUSIONS: PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.
ABSTRACT
Nanocomposites of hydrophobic organo-clay/polypropylene (organo-clay/PP) were efficiently developed through a solution-blending technique. For this, we utilized various smectite clays as host agents; namely, Na-montmorillonite (Mt, ~1000 nm), Na-fluorine mica (Mica, ~1500 nm), and Na-hectorite (Ht, ~60 nm) with varied sizes, layer charges, and aspect ratios. Such clays were functionalized with cetyltrimethylammonium (CTA) bromide via an intercalation technique to obtain hydrophobic organic clays. The as-made clay particles were further mixed with a PP/xylene solution; the latter was removed to obtain the final product of the CTA-clay/PP nanocomposite. An X-ray diffraction (XRD) analysis confirmed that there were no characteristic (001) diffraction peaks for CTA-Mica in the PP nanocomposites containing CTA-Mica, assuring the fact that the Mica layers could be completely exfoliated and thereby homogenously composited within the PP. On the other hand, the CTA-Mt and CTA-Ht incorporated composites had broader (001) peaks, which might have been due to the partial exfoliation of CTA-Mt and CTA-Ht in the composites. Among the three CTA-clay/PP nanocomposites, the CTA-Mica nanohybrid showed an enhanced thermal stability by ~42 °C compared to the intact host polymer matrix. We also noted that when the CTA-Mica content was ~9 mass % in the nanocomposites, the Young's modulus was drastically maximized to 69%. Our preliminary results therefore validated that out of the three tested clay-PP nanocomposites, the CTA-Mica nanofiller served as the best one to improve both the thermal and mechanical properties of the PP nanocomposites.
ABSTRACT
Thermogelling behavior of aqueous polymer solutions comes from the delicate balance between hydrophilic and hydrophobic moieties of the polymer. Typically, poly(ethylene glycol) (PEG) has been used as a hydrophilic block in most thermogels reported to date. However, recent papers have suggested the potential immunogenicity of PEG-conjugated compounds. Here, we report that aqueous solutions of dl-polyalanine (DL-PA) with a specific molecular weight can exhibit thermogelling behavior. In particular, DL-PA with a molecular weight (Mn) of 6690 Da, DL-PA67, exhibited sol-to-gel transition at the physiologically important temperature range of 30-40 °C. 1H NMR and FTIR data indicated that the mechanism of thermogelation is related to dehydration and conformational changes of DL-PA67 from random coil to ß-sheet structures. Subcutaneous injection of an aqueous DL-PA67 solution into rats confirmed the gel formation and its histocompatibility with mild tissue irritation.
Subject(s)
Peptides , Polyethylene Glycols , Animals , Hydrogels/chemistry , Peptides/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , Rats , Water/chemistryABSTRACT
Hyaluronic acid-graft-poly(propylene glycol) (HA-g-PPG) was prepared to induce hydrophobic interactions between HA-g-PPG and F127 PPGs (poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol)) and consequent increases in gel stability of F127 gel. Molecular weights of 340, 1000, and 2500 Da were used for PPG, and grafting ratios of HA-g-PPG varied over 3%, 12%, and 50%. Using rheology measurements, 1H NMR spectra, lower critical solution temperature measurements, dynamic light scattering, and transmission electron spectroscopy, hydrophobic crosslinking and intermicellar bridge formation were suggested in the aqueous HA-g-PPG/F127 hybrid solutions. In particular, the gel stability of the HA-g-PPG/F127 hybrid thermogel increased from 2 days (F127 only) to 6 days, thus the hybrid thermogel can provide longer delivery of an incorporated drug. The HA-g-PPG/F127 thermogel exhibited tissue compatibility in the subcutaneous layer of rats. The protein drug release from the gel indicated that interactions between negative charged HA-g-PPG and positive charged drug (calcitonin) reduced initial burst release.
Subject(s)
Hyaluronic Acid , Polyethylene Glycols , Animals , Drug Liberation , Hyaluronic Acid/chemistry , Micelles , Polyethylene Glycols/chemistry , Propylene Glycols , RatsABSTRACT
BACKGROUND: Thermosensitive micelles with rigid cores that exhibit a reversible lower critical solution temperature at 30-35 °C can be applied for drug delivery. METHOD: Hydrophilic monomethoxy poly(ethylene glycol) was conjugated to hydrophobic deoxycholic acid to prepare monomethoxy poly(ethylene glycol)-deoxycholic acid (mPEG-DC). Micelle formation and thermosensitive solution behavior were studied using various methods, including hydrophobic dye solubilization, transmission electron microscopy, dynamic light scattering, turbidity measurement, microcalorimetry, and 1H-NMR spectroscopy. Drug release from the thermosensitive micelles was demonstrated using estradiol, a model drug. RESULTS: The mPEG-DC formed micelles with a critical micelle concentration of 0.05 wt.% and an average size of 15 nm. Aqueous mPEG-DC solutions exhibit a lower critical solution temperature (LCST) that is independent of concentration and reversible over heating and cooling cycles. The LCST transition is an entropically driven process involving dehydration of the PEG shell. The thermosensitive mPEG-DC micelles with rigid DC cores were applied as an estradiol delivery system in which estradiol was released, without initial burst, over the 16 days in a diffusion-controlled manner. CONCLUSIONS: This study suggests that mPEG-DCs form thermosensitive micelles with rigid cores that can function as an excellent diffusion-controlled hydrophobic drug delivery system without initial burst release. Thermosensitive core-rigid micelles of monomethoxy poly(ethylene glycol)-deoxy cholic acid.
ABSTRACT
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.
Subject(s)
Mesenchymal Stem Cells , Trehalose , Alanine , Biomimetics , Cell Survival , Cryopreservation/methods , Cryoprotective Agents/pharmacology , Dimethyl Sulfoxide , Lysine , Trehalose/pharmacologyABSTRACT
Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor ß subunit (CBFß) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFß and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.
Subject(s)
Aminopterin/analogs & derivatives , Biocompatible Materials/pharmacology , Mesenchymal Stem Cells/drug effects , Peptides/chemistry , Temperature , Aminopterin/chemistry , Aminopterin/pharmacology , Biocompatible Materials/chemistry , Cell Differentiation/drug effects , Chondrogenesis/drug effects , Gels/chemistry , Humans , Materials TestingABSTRACT
Dietary uptake of folic acid (FA) improves cartilage regeneration. In this work, we discovered that three days of FA treatment is highly effective for promoting chondrogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs). In a three-dimensional pellet culture, the levels of typical chondrogenic biomarkers, sulfated glycosaminoglycan, proteoglycan, type II collagen (COL II), SRY box transcription factor 9 (SOX 9), cartilage oligomeric matrix protein (COMP), and aggrecan (ACAN) increased significantly in proportion to FA concentration up to 30 µM. At the mRNA expression level, COL II, SOX 9, COMP, and ACAN increased 3.6-6.0-fold with FA treatment at 30 µM compared with the control system that did not receive FA treatment, and the levels with FA treatment were 1.6-2.5 times greater than those in the kartogenin-treated positive control system. FA treatment did not increase type I collagen α1 (COL I α1), an osteogenic biomarker which is a concern with most chondrogenic promoters. At the high FA concentration of 100 µM, significant decreases in chondrogenic biomarkers were observed, which might be related to DNA methylation. A thermogel system incorporating TMSCs and FA provided sustained release of FA over several days, similar to the FA treatment. The thermogel system confirmed the efficacy of FA in promoting chondrogenic promotion of TMSCs. The increased nuclear translocation of core-binding factor ß subunit (CBFß) and the runt-related transcription factor 1 (RUNX1) expression after FA treatment, together with molecular docking studies, suggest that the chondrogenic enhancement mechanism of FA is mediated by CBFß and RUNX1.
Subject(s)
Core Binding Factor Alpha 2 Subunit , Folic Acid , Biomarkers/metabolism , Cell Differentiation , Cells, Cultured , Chondrocytes/metabolism , Chondrogenesis , Core Binding Factor Alpha 2 Subunit/metabolism , Folic Acid/metabolism , Molecular Docking SimulationABSTRACT
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.
Subject(s)
Chondrogenesis , Peptides , Cell Differentiation , Peptides/pharmacology , Polyethylene Glycols , Stem CellsABSTRACT
Precise control over the size and shape of ice crystals is a key factor to consider in designing antifreezing and cryoprotecting molecules for cryopreservation of cells. Here, we report that a poly(ethylene glycol)-poly(l-alanine) (PEG-PA) block copolymer exhibits excellent cryoprotecting properties for stem cells and antifreezing properties for water. As the molecular weight of PA increased from 500, 760, and 1750 Da (P1, P2, and P3) at the same PEG molecular weight of 5000 Da, the ß-sheet content decreased and α-helix content increased. Comparing P2 (PEG-PA; 5000-760) and P4 (PEG-PA: 1000-750), ß-sheets increased as the PEG block length decreased. The critical micelle concentration of the PEG-PA block copolymers was in a range of 0.5-3.0 mg/mL and was proportional to the hydrophobicity of the PEG-PA block copolymers. The P1, P2, and P3 self-assembled into spherical micelles, whereas P4 formed micelles with cylindrical morphology. The difference in the block copolymer structure affected ice recrystallization inhibition (IRI) activity and cryopreservation of cells. IRI activity was assayed via mean largest grain size (MLGS), and interactions between polymers and ice crystal surfaces were studied by dynamic ice-shaping studies. The MLGS decreased to 58 â 53 â 45 â 35 â 23% of that of PBS, as the polymer (PEG-PA 5000-500) concentration increased from 0.0 (PBS; control) â 1.0 â 5.0 â 10 â 30 â 50 mg/mL. The MLGS of PEG 5k solutions (negative control) decreased to 74 â 71 â 64 â 44 â 37% of that of PBS in the same concentration range. P3 and P4 with a longer hydrophobic PA block developed elongated ice crystals at above 30 mg/mL. The dynamic ice-shaping study exhibited that ice crystals became needle-shaped, as the hydrophobicity of the polymer increased as in P2-P4. The cell recovery in the P1 system after cryopreservation at -196 °C for 7 days was 87% of that of the dimethyl sulfoxide (DMSO) 10% system (positive control). The cell recovery was 48% for the P2 system and drastically decreased to less than 30% of that of the DMSO 10% system in the P3, P4, PEG 5k, PEG 1k, PVA 80H, and PVA 100H systems. Current studies suggest that IRI activity, round ice crystal shaping, and membrane stabilization activity of P1 cooperatively provide excellent cell recovery among the candidate systems. Recovered stem cells exhibited excellent proliferation and multilineage differentiation into osteocytes, chondrocytes, and adipocytes. To conclude, the PEG-PA (5000-500) block copolymer is suggested to be a promising antifreezing cryoprotectant for stem cells.
Subject(s)
Cryoprotective Agents/pharmacology , Ice , Mesenchymal Stem Cells/drug effects , Peptides/pharmacology , Polyethylene Glycols/pharmacology , Water/chemistry , Cell Proliferation/drug effects , Child , Cryoprotective Agents/chemistry , Humans , Male , Palatine Tonsil/cytology , Particle Size , Peptides/chemistry , Polyethylene Glycols/chemistryABSTRACT
Cancer progression and migration in the tumor microenvironment are related to cell types and three-dimensional (3D) matrices. Therefore, developing biomimetic tumor models, including co-culture systems and a tunable 3D matrix, could play an essential role in understanding the cancer environment. Here, multicellular spheroids using human adipose-derived mesenchymal stem cells (hADSCs) and breast cancer cells (MDA-MB-231) within the 3D matrix were used as a tumor microenvironment (TME) mimicking platform. The amphiphilic peptide block copolymer and hyaluronic acid (HA) formed a self-assembled structure, which provides a biocompatible 3D environment for the cells. Multicellular spheroids were formed on the optimized plate and were observed as cell migration from a spheroid within a 3D matrix, such as the invasive and metastatic cancer of TME. This study suggests a new 3D platform using polymer complexes and the importance of tumor complexities, including various cell types and microenvironments.
ABSTRACT
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.
Subject(s)
DNA , Nanostructures , Peptides , PhenylalanineABSTRACT
The control of ice recrystallization is very important in cryo-technological fields such as the food industry, biopharmaceuticals, and cell storage. Ice recrystallization inhibition (IRI) compounds are therefore designed to limit the growth of ice crystals, decrease the crystal size, and control the crystal shape. To improve the IRI activity of cryo-systems, various synthetic polymers such as biomimetic polypeptides from polar fish, facially amphiphilic polymers, polyampholytes, poly(vinyl alcohol) derivatives, and block copolymers with hydrophilic-hydrophobic balance have been developed. Except for graphene oxide, poly(vinyl alcohol) has thus far exhibited the best performance among these polymers. Herein, poly(l-alanine-co-l-lysine) (PAK) was shown to exhibit a similar IRI activity to that of poly(vinyl alcohol). Moreover, in contrast to the needle-shaped ice crystals generated by the aqueous PVA solution, the PAK solution was shown to generate cubic-to-spherical shaped ice crystals. Furthermore, neither poly(l-alanine-co-l-aspartic acid) (PAD) nor poly(ethylene glycol) (PEG) with a similar molecular weight provided any significant IRI activity. Examination by FTIR and circular dichroism spectroscopies indicated that the PAK forms α-helices, whereas the PAD forms random coils in water. Further, a dynamic ice shaping study suggested that PAK strongly interacts with ice crystals, whereas PAD and PEG only weakly interact. These results suggest that PAK is an important compound with superior IRI activity and that this activity is dependent upon the functional groups and secondary structure of the polypeptides.
Subject(s)
Antifreeze Proteins , Ice , Alanine , Antifreeze Proteins/chemistry , Crystallization , Lysine , Peptides , Polymers/chemistry , Polyvinyl Alcohol/chemistry , WaterABSTRACT
BACKGROUND: The aqueous solution behavior of thermosensitive PEG-PA block copolymers as well as secondary structure of PA is expected to significantly change through modification of the hydrophobic PA by long chain alkyl (C18) groups with different configurations. METHOD: Oleoyl and stearoyl (C18) groups were conjugated to poly(ethylene glycol)-poly(L-alanine) (PEG-PA; EG45A16) diblock copolymers to compare their conjugation effect on nano-assemblies and corresponding aqueous solution behavior of the polymers. RESULTS: Due to the nature of a hydrophilic PEG block and a hydrophobic PA or C18-modified PA, PEG-PA, oleoyl group-conjugated PEG-PA (PEG-PAO), and stearoyl group-conjugated PEG-PA (PEG-PAS) block copolymers form micelles in water. Compared with PEG-PA, the micelle size of PEG-PAO and PEG-PAS increased. Circular dichroism and FTIR spectra of aqueous polymer solutions showed that ß sheet content increased, whereas α helix content decreased by C18 modification of PEG-PA. PEG-PAS showed better performance in ice crystallization inhibition than PEG-PAO. The sol-to-gel transition temperatures of aqueous PEG-PAO solutions were 25-37 °C higher than those of aqueous PEG-PA solutions, whereas aqueous PEG-PAS solutions remained as gels in the temperature range of 0-80 °C. 1H-NMR spectra indicated that the oleoyl groups increased core mobility, whereas stearoyl groups decreased the core mobility of the micelles in water. The difference in micromobility between PAO and PAS interfered or promoted gelation of the aqueous polymer solutions, respectively. CONCLUSIONS: This study suggests that a hydrophobic C18-modification of polypeptide induces α helix-to-ß sheet transition of the polypeptide; however, aqueous solution behaviors including ice recrystallization inhibition and gelation are significantly affected by the nature of the hydrophobic molecule.