An injectable thermoresponsive-hydrogel for lamellar keratoplasty: In-situ releases celastrol and hampers corneal scars.

Corneal stromal fibrosis is a common cause of visual impairment resulting from corneal injury, inflammation and surgery. Therefore, there is an unmet need for inhibiting corneal stromal fibrosis. However, bioavailability of topical eye drops is very low due to the tear and corneal barriers. In situ delivery offers a unique alternative to improve efficacy and minimize systemic toxicity. Herein, a drug delivery platform based on thermoresponsive injectable hydrogel/nano-micelles composite with in situ drug-controlled release and long-acting features is developed to prevent corneal scarring and reduce corneal stromal fibrosis in lamellar keratoplasty. The in-situ gelation hydrogels enabled direct delivery of celastrol to the corneal stroma. In vivo evaluation with a rabbit anterior lamellar keratoplasty model showed that hydrogel/micelles platform could effectively inhibit corneal stromal fibrosis. This strategy achieves controlled and prolonged release of celastrol in the corneal stroma of rabbit. Following a single corneal interlamellar injection, celastrol effectively alleviated fibrosis via mTORC1 signal promoting autophagy and inhibiting TGF-ß1/Smad2/3 signaling pathway. Overall, this strategy demonstrates promise for the clinical application of celastrol in preventing corneal scarring and reducing corneal stromal fibrosis post-lamellar keratoplasty, highlighting the potential benefits of targeted drug delivery systems in ocular therapeutics.

Acetylation of histone 3 promotes miR-29a expression and downregulates STAT3 in sepsis.

BACKGROUND: MiR-29a targets signal transducers and activators of transcription 3 (STAT3) and negatively regulates its expression. Both miR-29a and STAT3 have been implicated in sepsis and upregulated miR-29a was associated with sepsis. However, the regulation of miR-29a in sepsis is not well elucidated. METHODS: We treated TC-1 cells with interleukin (IL)-6 and the expression of miR-29a and STAT3 was measured. We pre-treated TC-1 cells with histone deacetylase inhibitor Trichostatin A, DNA methylation inhibitor 5-Azacytidine or histone acetyltransferase inhibitor A-485, then treated cells with IL-6 and analyzed the expression of miR-29a and STAT3. We measured the expression of histone deacetylases and histone acetyltransferase, and glycolysis in IL-6-treated TC-1 cells. We administrated miR-29a inhibitor or STAT3 inhibitor to septic mice and the survival rate and expression of anti-apoptotic factors were measured. RESUTLS: IL-6 promoted miR-29a expression while suppressed STAT3 expression. Upregulation of miR-29a was associated with sepsis. Histone acetylation promoted miR-29a expression. IL-6 promoted glycolysis in TC-1 cells, which resulted in Acetyl-CoA accumulation. Inhibition of miR-29a promoted survival rate in septic mice while inhibiting STAT3 exacerbated death in mice. The protection of miR-29a inhibition against sepsis was abolished when STAT3 was inhibited. CONCLUSION: Histone acetylation promoted miR-29a expression, resulting in downregulation of STAT3 and exacerbation of sepsis.

Kinetic Control of Parallel versus Antiparallel Amyloid Aggregation via Shape of the Growing Aggregate.

By combining atomistic and higher-level modelling with solution X-ray diffraction we analyse self-assembly pathways for the IFQINS hexapeptide, a bio-relevant amyloid former derived from human lysozyme. We verify that (at least) two metastable polymorphic structures exist for this system which are substantially different at the atomistic scale, and compare the conditions under which they are kinetically accessible. We further examine the higher-level polymorphism for these systems at the nanometre to micrometre scales, which is manifested in kinetic differences and in shape differences between structures instead of or as well as differences in the small-scale contact topology. Any future design of structure based inhibitors of the IFQINS steric zipper, or of close homologues such as TFQINS which are likely to have similar structures, should take account of this polymorphic assembly.

Supramolecular Assembly of C3 -Peptides into Helical Fibers Stabilized through Dynamic Covalent Chemistry.

The supramolecular assembly of C3 -peptides carrying glycine-cysteine (Gly-Cys) dipeptide pendants into helical fibers is described. A Gly-Cys dipeptide sequence was selected to ensure the chirality, and at the same time, to provide a chance to crosslink the structure through oxidation of the thiol into dynamic covalent disulfide, and transfer supramolecular helical fibers into covalent ones. Supramolecular assembly in aprotic solvents affords long helical fibers with left-handedness, which can be stabilized through formation of disulfide. Transformation of supramolecular helical fibers into covalent ones was examined by NMR and FTIR spectroscopies as well as atom force microscopy.

Self-immolative colorimetric, fluorescent and chemiluminescent chemosensors.

Self-immolative chemistry features a cascade of disassembly reactions in response to external stimuli, which provides great opportunities to design new self-immolative chemosensors with advanced performance and/or functions. Self-immolative spacers in these chemosensors not only facilitate the linkage of designed triggers to various chromophores or fluorophores, but can also be used to solve inherent problems associated with native chemosensors, such as low reactivities, poor stabilities and slow response times. Their capacity for stimuli-responsive release through operation of a self-immolative reaction further enables integration of sophisticated functions into chemosensors, including signal amplification, enzyme activity localization, and drug monitoring. Significant advances have been made in the field of self-immolative chemosensors, leading to intriguing applications to sensitive detection of analytes, bioimaging and cancer theranostics. This tutorial review summarizes this recent progress with a focus on their design strategies and sensing mechanisms.

Use of a thermoresponsive polymer in ethanol fermentation carried out in a cadmium-containing medium.

In this study, a new thermoresponsive polymer, PG1-co-PHEDTA, was used as a reagent for the detoxification and recovery of cadmium from ethanol fermentation carried out in a cadmium-containing medium. We found that the polymer, PG1-co-PHEDTA, had an important role in ethanol production. In the absence of PG1-co-PHEDTA, ethanol fermentation was severely inhibited by cadmium. However, the inhibitory effect of cadmium could be significantly alleviated by the addition of PG1-co-PHEDTA, and the rates of glucose consumption and ethanol production were similar to those reported for cadmium-free fermentation processes. The investigation into the key units of PG1-co-PHEDTA showed that HEDTA was the contributing factor for the positive effect on ethanol fermentation. However, the effect of HEDTA that was incorporated into PG1 was higher than that of the free HEDTA. Three-fold higher concentration of free HEDTA was required to obtain similar results as that with PG1-co-PHEDTA additive. Glutathione (GSH) and cadmium assays demonstrated that the transport of cadmium into the cell could be prevented by PG1-co-PHEDTA via the formation of a chelated structure with the HEDTA groups in PG1-co-PHEDTA. By applying the unique phase transition of PG1-co-PHEDTA, cadmium of more than 90% could be removed from fermentation broths with a simple centrifugation step.

Thermoresponsive dendronized polyprolines via the "grafting to" route.

The first and second generations of dendronized polyprolines P3G1, P3G2, and P4G1 are prepared via the "grafting to" route, and their thermoresponsive properties and helical conformations investigated. High molar masses of polyproline main chains carrying azido groups are achieved first by polycondensation of peptide precursors through activated ester strategy. Oligoethylene glycol dendrons cored with alkyne are then attached onto the main chains through click reaction. These polymers are found to be thermoresponsive. Circular dichroism spectroscopy investigation indicates, in contrast to P3G2 and P4G1 which adopt the expected PPII conformation in aqueous conditions, P3G1 prefers to adopt PPI helical conformation, and this conformation is stable within the measured time period and temperature range.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties.

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

Twofold pH and temperature stimuli-responsive behaviour in block copolypeptide-decorated single wall carbon nanotubes.

Temperature- and pH-responsive dispersions of carbon nanotubes in water have been obtained by using di- and tri-block copolypeptides, in which the temperature responsive behaviour is provided by a poly(N-isopropylacrylamide) block, while the pH responsiveness is provided by two different polypeptides with an ampholytic behaviour, poly(L-glutamic acid) and poly(L-lysine). The molecular architectures of the block copolypeptides can be engineered to fine-tune the stimuli-responsive behaviour.

Low toxic, thermoresponsive dendrimers based on oligoethylene glycols with sharp and fully reversible phase transitions.

Novel first (G1) and second (G2) generation dendrimers based on three-fold branched oligoethylene glycol dendrons are efficiently synthesized which show characteristic thermoresponsive behavior and negligible cytotoxicity (for G2).

Stimuli-responsive zwitterionic block copolypeptides: poly(N-isopropylacrylamide)-block-poly(lysine-co-glutamic acid).

Synthesis of novel zwitterionic block copolypeptides, poly(N-isopropylacrylamide)-block-poly(L-glutamic acid-co-L-lysine) [PNiPAM(n)(PLG(x)-co-PLLys(y))m , where n is the number-average degree of polymerization (DP(n)) of PNiPAM block, x and y are the mole fraction of glutamic acid and lysine residues, respectively, and m is the total DP(n) of the peptide block], and their stimuli-responsiveness to temperature and pH variation in aqueous solutions are described. Initiated with the amino-terminated poly(N-isopropylacrylamide) (PNiPAM(n)-NH2), ring-opening polymerization (ROP) of a mixture of gamma-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), and Boc-L-lysine N-carboxyanhydride (BLLys-NCA) afforded the block copolypeptides PNiPAM(n)(PBLG(x)-co-PBLLys(y))m, with a poly(N-isopropylacrylamide) block together with a random copolypeptide block, which was then deprotected with HBr/trifluoroacetic acid into the double hydrophilic block copolypeptides, PNiPAM(n)(PLG(x)-co-PLLys(y))m. Their block ratios and lengths, as well as the amino acid residue ratios in the random copolypeptide block are varied (n = 360, x = 0.4-0.5, y = 0.4-0.6, and m = 220-252). The secondary structures of the copolypeptides in aqueous solution at different pH conditions were examined. Phase transitions in aqueous solutions induced by both pH and temperature variation were investigated by (1)H NMR spectroscopy. The transitions induced by temperature were also explored by turbidity measurements using UV/vis spectroscopy for their lower critical aggregation temperature (LCAT) determination. Furthermore, these aggregation processes were followed by dynamic light scattering measurements.

High stability of the polyproline II helix in polypeptide bottlebrushes.

Polymer bottlebrushes with monodisperse oligoproline side chains were efficiently synthesized, and the conformation of the peptide side chains in different solvents was investigated. Polymers with number-average degrees of polymerization (DPn) of 89 and 366 were obtained by polymerization of the macromonomer in iPrOH/MeCN (1:1) and hexafluoroisopropanol, respectively. Circular dichroism (CD) spectra of the bottlebrush polymers in the neutral and charged states reveal that the oligoproline side chains attain stable polyproline II (PPII) helical conformations not only in aqueous solution, but also in aliphatic alcohol solutions. Dense attachment of oligopeptides onto a linear polymer chain did not lead to an increase in helix content. The possible effects of the main-chain length on the conformational stability were examined. The switching between the polyproline I (PPI) and PPII helical conformations for the oligoproline side chains in aliphatic alcohol solutions is believed to be inhibited by the overcrowded structure in the polymer bottlebrushes.

Synthesis and characterization of thermo- and pH-responsive double-hydrophilic diblock copolypeptides.

Synthesis of novel double-hydrophilic diblock copolypeptides (BCPs), poly(l-glutamic acid)-block-poly(N-isopropylacrylamide) (PLGnPNm), and their thermoresponsive properties in aqueous solutions at different pH values are described. The diblock copolypeptides were synthesized by a combination of ring-opening polymerization (ROP) of gamma-benzyl-l-glutamate N-carboxyanhydrides (BLG-NCA) and reversible addition-fragmentation chain transfer (RAFT) polymerization of N-isopropylacrylamide (NiPAM). A new class of RAFT agents (CTA-2 and CTA-3) with amino-functional groups was designed for this purpose. Two different strategies, i.e., macrochain transfer agent (CTA) and macroinitiator routes, were utilized and compared on the control of the chemical structures of the resulting BCPs. Their block ratios and lengths are broadly varied (n = 21-600 and m =180-442). Their thermally switchable aggregation behaviors in aqueous solutions were investigated at the microscopic level by 1H NMR spectroscopy and at the macroscopic level by turbidity measurements using UV/vis spectroscopy. The latter was also utilized for their lower critical aggregation temperature (LCAT) determination. The effects of block lengths and ratios as well as solution pH values on the collapse of NiPAM chain and aggregation process of BCPs were examined. This aggregation process was also followed by dynamic light scattering (DLS) measurements, and the thermally induced aggregate structures were investigated by transmission electron microscopy (TEM).

Multigram solution-phase synthesis of three diastereomeric tripeptidic second-generation dendrons based on (2S,4S)-, (2S,4R)-, and (2R,4S)-4-aminoprolines.

Three diastereomeric second-generation (G2) dendrons were prepared by using (2S,4S)-, (2S,4R)-, and (2R,4S)-4-aminoprolines on the multigram scale with highly optimized and fully reproducible solution-phase methods. The peripheral 4-aminoproline branching units of all the dendrons have the 2S,4S configuration throughout, whereas those units at the focal point have the 2S,4S, 2S,4R, and 2R,4S configurations. These latter configurations led to the dendrons being named (2S,4S)-1, (2S,4R)-1, and (2R,4S)-1, respectively. The 4-aminoproline derivatives used in this study are new, although many closely related compounds exist. Their syntheses were optimized. The dendron assembly involved amide coupling, the efficiency of which was also optimized by employing the following well-known reagents: EDC/HOBt, DCC/HOSu, TBTA/HOBt, TBTU/HOBt, BOP/HOBt, pentafluorophenol, and PyBOP/HOBt. It was found that the use of PyBOP is by far the best for dendrons (2S,4S)-1 and (2R,4S)-1, and pentafluorophenol active ester is best for (2S,4R)-1. Because of their multigram scale, all couplings were done in solution instead of by solid-phase procedures. Purifications were, nevertheless, easy. The optical purities of the key intermediates as well as the three G2 dendrons were analyzed by chiral HPLC analysis. These novel, diastereomeric second-generation dendrons have a rather compact and conformationally highly rigid structure that makes them interesting candidates for applications, for example, in the field of dendronized polymers and in organocatalysis.