Supramolecular Surface Coatings with Acetylated Polyrotaxane-Based Triblock Copolymers for Thermal Regulation of Cell Adhesion and Fabrication of Cell Sheets.

Polyrotaxanes (PRXs) containing acetylated α-cyclodextrins exhibit a temperature-dependent phase transition in aqueous solutions across their lower critical solution temperature (LCST) of approximately 26.6 °C. To gain insights into the interactions of acetylated PRXs (Ac-PRXs) with biological components, thermoresponsive supramolecular surfaces were prepared by coating tissue culture polystyrene (TCPS) surfaces with Ac-PRX triblock copolymers, and their surface properties across the LCST were evaluated. The wettability and protein adsorption of Ac-PRX-coated surfaces changed significantly between 10 and 37 °C, whereas the uncoated TCPS and unmodified PRX-coated surfaces did not alter the wettability and protein adsorption at 10 and 37 °C. The adhesion, proliferation, morphology, and adhesion strength of NIH/3T3 cells on Ac-PRX-coated surfaces were found to be similar to those of the uncoated and unmodified PRX-coated surfaces. However, the adhesion strength of NIH/3T3 cells on Ac-PRX-coated surfaces decreased drastically at 10 °C. Consequently, the cells spontaneously detached from the Ac-PRX-coated surfaces without enzymatic treatment. Additionally, when incubating confluent cells at 10 °C, the cells detached from Ac-PRX-coated surfaces as cell sheets while retaining extracellular matrix proteins. The findings of this study provide new directions for the design of thermoresponsive supramolecular biointerfaces for applications in bioseparation and cell manipulation.

Acetylation of Cyclodextrin-Threaded Polyrotaxanes Yields Temperature-Responsive Phase Transition and Coacervate Formation Properties.

Stimuli-responsive materials have received considerable attention for their application in biomedical fields. Herein, the temperature-dependent phase transition behavior of acetylated polyrotaxanes (Ac-PRXs) consisting of acetylated cyclodextrins threaded onto a linear axle polymer in aqueous solution is investigated. The aqueous solutions of Ac-PRXs exhibit temperature-dependent transmittance changes when the degree of acetylation is 30-40%. Upon increasing the temperature, Ac-PRXs are dehydrated and acetyl groups are subsequently associated through hydrophobic interactions. Interestingly, the Ac-PRXs form coacervate droplets with a diameter of ≈2-3 µm above their cloud points. These temperature-responsivities of Ac-PRXs are observed for other PRXs with different axle polymer molecular weights and compositions. Consequently, the acetylation of PRXs is an attractive chemical modification for yielding temperature-responsivities, and Ac-PRXs can be applied as temperature-responsive supramolecular materials.

Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials.

Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.

Cografting of Zwitterionic Sulfobetaines and Cationic Amines on ß-Cyclodextrin-Threaded Polyrotaxanes Facilitates Cellular Association and Tissue Accumulation with High Biocompatibility.

ß-Cyclodextrins (ß-CDs) and ß-CD-containing polymers have attracted considerable attention as potential candidates for the treatment of cholesterol-related metabolic and intractable diseases. We have advocated the use of ß-CD-threaded acid-degradable polyrotaxanes (PRXs) as intracellular delivery carriers for ß-CDs. As unmodified PRXs are insoluble in aqueous solutions, chemical modification of PRXs is an essential process to improve their solubility and impart novel functionalities. In this study, we investigated the effect of the modification of zwitterionic sulfobetaines on PRXs due to their excellent solubility, biocompatibility, and bioinert properties. Sulfobetaine-modified PRXs were synthesized by converting the tertiary amino groups of precursor 2-(N,N-dimethylamino)ethyl carbamate-modified PRXs (DMAE-PRXs) using 1,3-propanesultone. The resulting sulfobetaine-modified PRXs showed high solubility in aqueous solutions and no cytotoxicity, while their intracellular uptake levels were low. To further improve this system, we designed PRXs cografted with zwitterionic sulfobetaine and cationic DMAE groups via partial betainization of the DMAE groups. Consequently, the interaction with proteins, intracellular uptake levels, and liver accumulation of partly betainized PRXs were found to be higher than those of completely betainized PRXs. Additionally, partly betainized PRXs showed no toxicity in vitro or in vivo despite the presence of residual cationic DMAE groups. Furthermore, partly betainized PRXs ameliorated the abnormal free cholesterol accumulation in Niemann-Pick type C disease patient-derived cells at lower concentrations than ß-CD derivatives and previously designed PRXs. Overall, the cografting of sulfobetaines and amines on PRXs is a promising chemical modification for therapeutic applications due to the high cholesterol-reducing ability and biocompatibility of such modified PRXs. In addition, modification with both zwitterionic and cationic groups can be used for the design of various polymeric materials exhibiting both bioinert and bioactive characteristics.

Emerging Nanoassembly of Polyrotaxanes Comprising Acetylated α-Cyclodextrins and High-Molecular-Weight Axle Polymer.

Acetylated α-cyclodextrin (α-CD)/poly(ethylene glycol) (PEG)-based polyrotaxanes (Ac-PRXs) with varying degrees of acetylation (DA) and molecular weight of axle PEG were synthesized and their solubility in aqueous solutions was investigated. Ac-PRXs with low DA (less than 35%) were dissolved in aqueous solutions without considering the molecular weight of axle PEG, whereas Ac-PRXs with high DA (more than 40%) and low molecular weight of axle PEG (less than 35000) were precipitated into the solutions. Interestingly, Ac-PRXs with high DA and high molecular weight of axle PEG (100000) exhibited a colloidal dispersion in aqueous solutions. It is considered that the threaded acetylated α-CDs formed hydrophobic microenvironments via hydrophobic interactions and the noncovered segments of axle PEGs provided colloidal stability. Furthermore, the potential application of Ac-PRX100k as a drug carrier was examined and it was established that Ac-PRX100k can encapsulate a hydrophobic drug. Accordingly, acetylation of PRXs is a viable approach to promote solubility in aqueous solutions and prepare self-assembled nanoparticles.

Polyelectrolyte Complexes between Polycarboxylates and BMP-2 for Enhancing Osteogenic Differentiation: Effect of Chemical Structure of Polycarboxylates.

Bone morphogenetic protein 2 (BMP-2) has received considerable attention because of its osteoinductivity, but its use is limited owing to its instability and adverse effects. To reduce the dose of BMP-2, complexation with heparin is a promising approach, because heparin enhances the osteoinductivity of BMP-2. However, the clinical use of heparin is restricted because of its anticoagulant activity. Herein, to explore alternative polymers that show heparin-like activity, four polycarboxylates, poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(aspartic acid) (PAsp), and poly(glutamic acid) (PGlu), were selected and their capability to modulate the osteoinductivity of BMP-2 was evaluated. Dynamic light scattering indicated that these polycarboxylates formed polyelectrolyte complexes with BMP-2. The osteogenic differentiation efficiency of MC3T3-E1 cells treated with the polycarboxylate/BMP-2 complexes was investigated in comparison to that of the heparin/BMP-2 complex. As a result, PGlu/BMP-2 complex showed the highest activity of alkaline phosphatase, which is an early-stage marker of osteogenic differentiation, and rapid mineralization. Based on these observations, PGlu could serve as an alternative to heparin in the regenerative therapy of bone using BMP-2.

Cellular Orientation on Repeatedly Stretching Gelatin Hydrogels with Supramolecular Cross-Linkers.

The cytocompatibility of biological and synthetic materials is an important issue for biomaterials. Gelatin hydrogels are used as biomaterials because of their biodegradability. We have previously reported that the mechanical properties of gelatin hydrogels are improved by cross-linking with polyrotaxanes, a supramolecular compound composed of many cyclic molecules threaded with a linear polymer. In this study, the ability of gelatin hydrogels cross-linked by polyrotaxanes (polyrotaxane-gelatin hydrogels) for cell cultivation was investigated. Because the amount of polyrotaxanes used for gelatin fabrication is very small, the chemical composition was barely altered. The structure and wettability of these hydrogels are also the same as those of conventional hydrogels. Fibroblasts adhered on polyrotaxane-gelatin hydrogels and conventional hydrogels without any reduction or apoptosis of adherent cells. From these results, the polyrotaxane-gelatin hydrogels have the potential to improve the mechanical properties of gelatin without affecting cytocompatibility. Interestingly, when cells were cultured on polyrotaxane-gelatin hydrogels after repeated stress deformation, the cells were spontaneously oriented to the stretching direction. This cellular response was not observed on conventional hydrogels. These results suggest that the use of a polyrotaxane cross-linking agent can not only improve the strength of hydrogels but can also contribute to controlling reorientation of the gelatin.

Scavenger Receptor A-Mediated Targeting of Carboxylated Polyrotaxanes to Macrophages and the Impacts of Supramolecular Structure.

Because macrophages are involved in the pathology of many diseases, targeting delivery of therapeutic molecules to macrophages is important issue. Polyrotaxanes (PRXs) composed of multiple cyclodextrins threaded with a linear polymer were utilized as a therapeutic agent for metabolic disease and for regulating cellular metabolism. For targeting delivery of PRXs to macrophages, carboxyethyl ether group-modified PRXs (CEE-PRXs) are designed for promoting interaction to macrophage scavenger receptor class A (SR-A). The cellular internalization of anionic CEE-PRXs in SR-A-positive macrophage-like cells (RAW264.7) is remarkably higher than that of nonionic PRX, whereas the cellular internalization efficiency in SR-A-negative cells is comparable between anionic and nonionic PRX. Furthermore, the molecular weight of axle polymer and the number of CEE groups modified on PRX are found to be the predominant factors governing cellular internalization efficiency in SR-A-positive RAW264.7 cells. Thus, CEE-PRXs are a promising design for targeting delivery of PRXs to macrophages.

Potentiating bioactivity of BMP-2 by polyelectrolyte complexation with sulfonated polyrotaxanes to induce rapid bone regeneration in a mouse calvarial defect.

Bone reconstruction is a challenging issue in the regeneration of surgically removed bone and disease-related bone defects. Although bone morphogenetic protein-2 (BMP-2) has received considerable attention as a bone regeneration inducer, a high dose of BMP-2 is typically required due to its short life-time under in vivo conditions. We have proposed a method to enhance the osteogenetic differentiation ability of BMP-2 in vitro that is based on supramolecular polyelectrolyte complexation with sulfonated polyrotaxanes (PRXs) consisting of sulfopropyl ether (SPE)-modified α-cyclodextrins threaded along a poly(ethylene glycol) chain capped with terminal bulky stopper molecules. In this study, we evaluated the in vivo bone regeneration ability of the SPE-PRX/BMP-2 complexes in a mouse calvarial defect model in comparison to free BMP-2 and heparin/BMP-2 complexes. The regenerated bone area was determined by X-ray computed microtomography, and the mice implanted with sulfonated PRX/BMP-2 complexes exhibited rapid and significant bone regeneration compared to those implanted with free BMP-2 and heparin/BMP-2 complexes. We concluded that the sulfonated PRX/BMP-2 complexes are a promising candidate for clinical bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1355-1363, 2017.

A designed room-temperature triplet ligand from pyridine-2,6-diyl bis(tert-butyl nitroxide).

The magnetic study on newly developed 4-mesitylpyridine-2,6-diyl bis(tert-butyl nitroxide) shows that almost the whole population has a ground triplet state at room temperature, and the ability of complex formation as a tridentate triplet ligand was proven with a diamagnetic yttrium(iii) ion.

Supramolecular inclusion complexation of simvastatin with methylated ß-cyclodextrins for promoting osteogenic differentiation.

Statins are recognized as a potential candidate to induce the regeneration of bone. However, statins are a strongly hydrophobic drug and it is difficult to administer at the local sites. In this study, the inclusion complexes of simvastatin (SV) with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) and randomly methylated ß-cyclodextrin (RM-ß-CD) were prepared to improve the water-solubility and the osteogenic differentiation ability of the inclusion complexes in MC3T3-E1 cells was investigated. The water-solubility of SV increased linearly upon the addition of both HP-ß-CD and RM-ß-CD, due to the formation of inclusion complexes. The osteogenic differentiation ability of the inclusion complexes were evaluated by the production of alkaline phosphatase (ALP) and late stage osteogenetic gene expression in MC3T3-E1 cells after 14 days of culture. As a result, the RM-ß-CD/SV inclusion complexes showed significantly higher ALP production and the expression of bone sialoprotein (BSP) and osteocalcin (OCN) than the untreated and free SV-treated cells. Additionally, the production of bone morphogenetic protein-2 (BMP-2) from MC3T3-E1 cells after the treatment with RM-ß-CD/SV inclusion complexes was significantly higher than the untreated and free SV-treated cells. Accordingly, it is concluded that the inclusion complexation of SV with RM-ß-CD is a potential formulation for bone regenerative therapy to improve water-solubility and osteodifferentiation efficiency.