Enhanced Tumor Targeting and Antitumor Activity of Methylated ß-Cyclodextrin-Threaded Polyrotaxanes by Conjugating Cyclic RGD Peptides.

We previously reported that acid-degradable methylated ß-cyclodextrins (Me-ß-CDs)-threaded polyrotaxanes (Me-PRXs) can induce autophagic cell death through endoplasmic reticulum (ER) stress-related autophagy, even in apoptosis-resistant cells. Hence, Me-PRXs show great potential as anticancer therapeutics. In this study, peptide-supermolecule conjugates were designed to achieve the targeted delivery of Me-PRX to malignant tumors. Arg-Gly-Asp peptides are well-known binding motifs of integrin αvß3, which is overexpressed on angiogenic sites and many malignant tumors. The tumor-targeted cyclic Arg-Gly-Asp (cRGD) peptide was orthogonally post-modified to Me-PRX via click chemistry. Surface plasmon resonance (SPR) results indicated that cRGD-Me-PRX strongly binds to integrin αvß3, whereas non-targeted cyclic Arg-Ala-Glu (cRGE) peptide conjugated to Me-PRX (cRGE-Me-PRX) failed to interact with integrins αvß3. In vitro, cRGD-Me-PRX demonstrated enhanced cellular internalization and antitumor activity in 4T1 cells than that of unmodified Me-PRX and non-targeted cRGE-Me-PRX, due to its ability to recognize integrin αvß3. Furthermore, cRGD-Me-PRX accumulated effectively in tumors, leading to antitumor effects, and exhibited excellent biocompatibility and safety in vivo. Therefore, cRGD conjugation to enhance selectivity for integrin αvß3-positive cancer cells is a promising design strategy for Me-PRXs in antitumor therapy.

Differences in the Intracellular Localization of Methylated ß-Cyclodextrins-Threaded Polyrotaxanes Lead to Different Cellular States.

Activation of autophagy represents a potential therapeutic strategy for the treatment of diseases that are caused by the accumulation of defective proteins and the formation of abnormal organelles. Methylated ß-cyclodextrins-threaded polyrotaxane (Me-PRX), a supramolecular structured polymer, induces autophagy by interacting with the endoplasmic reticulum. We previously reported on the successful activation of mitochondria-targeted autophagy by delivering Me-RRX to mitochondria using a MITO-Porter, a mitochondria-targeted nanocarrier. The same level of autophagy induction was achieved at one-twentieth the dosage for the MITO-Porter (Me-PRX) compared to the naked Me-PRX. We report herein on the quantitative evaluation of the intracellular organelle localization of both naked Me-PRX and the MITO-Porter (Me-PRX). Mitochondria, endoplasmic reticulum and lysosomes were selected as target organelles because they would be involved in autophagy induction. In addition, organelle injury and cell viability assays were performed. The results showed that the naked Me-PRX and the MITO-Porter (Me-PRX) were localized in different intracellular organelles, and organelle injury was different, depending on the route of administration, indicating that different organelles contribute to autophagy induction. These findings indicate that the organelle to which the autophagy-inducing molecules are delivered plays an important role in the level of induction of autophagy.

Structure-function relationships of cholesterol mobilization from the endo-lysosome compartment of NPC1-deficient human cells by ß-CD polyrotaxanes.

Niemann-Pick Type C is a rare metabolic disorder characterized by the cellular accumulation of cholesterol within endosomal and lysosomal compartments. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) containing polyrotaxanes represent an attractive approach for treating this disease due to their ability to circulate in the blood stream for longer periods of time as a prodrug form of HP-ß-CD. Once inside the cell, the macromolecular structure is thought to break down into the Pluronic precursor and the active cyclodextrin agent that promotes cholesterol mobilization from the aberrant accumulations within NPC-deficient cells. We now report that both cholesterol and decaarginine (R10) endcapped polyrotaxanes are able to remove cholesterol from NPC1 patient fibroblasts. R10 endcapped materials enter these cells and are localized within endosomes after 16 h. The cholesterol mobilization from endo-lysosomal compartments of NPC1 cells by the polyrotaxanes was directly related to their extent of endcapping and their threading efficiency. Incorporation of 4-sulfobutylether-ß-cyclodextrin (SBE-ß-CD) significantly improved cholesterol mobilization due to the improved solubility of the compounds. Additionally, in our efforts to scale-up the synthesis for preclinical studies, we prepared a library of polyrotaxanes using a solid phase synthesis method. These compounds also led to significant cholesterol mobilization from the cells, however, cytotoxicity studies showed that they were substantially more toxic than those prepared by the solvent-assisted method, thus limiting the therapeutic utility of agents prepared by this expedited method. Our findings demonstrate that complete endcapping of the polyrotaxanes and improved solubility are important design features for delivering high copy numbers of therapeutic ß-CD to promote enhanced sterol clearance in human NPC1-deficient cells.

Poly-ε-caprolactone/poly(rotaxane) seeded with human dental pulp stem cells or osteoblasts promotes angiogenesis: a chorioallantoic membrane assay.

Biomaterials used for tissue regeneration should ideally provide a favorable environment for cell proliferation and differentiation. Angiogenesis is crucial for supplying oxygen and nutrients necessary for cellular survival at implantation sites. The aim of this study was to evaluate the overall angiogenesis response of a poly ε-caprolactone/poly (rotaxane) blend (poly-blend) carried by human dental pulp stem cells (hDPSCs) or osteoblasts (OB) seeded in the chorioallantoic membranes (CAM) of fertilized chicken eggs on embryonic day 7. They were classified into the following intervention groups: (a) poly(polymeric blend disks free of cells); (b) hDPSC seeded onto CAM; (c) poly/hDPSC (where hDPSCs were seeded onto poly-blend); (d) poly/OB (where osteoblasts were seeded onto poly); (e) OB (where hDPSCs differentiated into osteoblasts were seeded onto CAM); and (f) a negative control when a sterilized silicone ring free of cells or polymer was inserted into CAM. On embryonic day 14, the quantitative and qualitative characteristics of the blood vessels in the CAMs were analyzed macroscopically and microscopically. Macroscopic examination showed that the Poly/hDPSC samples exhibited an increased medium vessel density. Additionally, microscopic observations showed that the Poly/hDPSC group and poly alone resulted in a large lumen area of vascularization. Thus, poly ε-caprolactone/poly (rotaxane) did not impair angiogenesis. Furthermore, poly-blend carried by stem cells of dental pulp origin shows a better vasculogenic potential, which is essential for regenerative therapies.

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.

Post-Cross-Linking of Collagen Hydrogels by Carboxymethylated Polyrotaxanes for Simultaneously Improving Mechanical Strength and Cell Proliferation.

To improve the mechanical properties of collagen hydrogels, which are widely utilized as biomaterials, post-cross-linking of collagen hydrogels was performed using polyrotaxane (PRX) as a cross-linker. Herein, carboxymethyl group-modified PRXs (CMPRs) composed of carboxymethylated α-cyclodextrins (α-CDs) threaded along poly(ethylene glycol) (PEG) capped with bulky stoppers were used to cross-link via reaction with the amino groups in the collagen. Four series of CMPRs with different α-CD threading ratios and axle PEG molecular weights were used for the post-cross-linking of the collagen hydrogels to verify the optimal CMPR chemical compositions. The post-cross-linking of the collagen hydrogels with CMPRs improved the swelling ratios and mechanical properties, such as viscoelasticity and tensile strength. Among the tested CMPRs, CMPRs with an axle PEG molecular weight of 35,000 (PEG35k) resulted in better mechanical properties than CMPRs with a PEG10k axis. Additionally, the cell adhesion and proliferation were greatly improved on the surface of the collagen hydrogels post-cross-linked with CMPRs with the PEG35k axle. These findings suggest that the molecular weight of an axle polymer in CMPRs is a more important parameter than the α-CD threading ratios. Accordingly, the post-cross-linking of hydrogels with PRXs is promising for improving the mechanical properties and biomaterial functions of collagen hydrogels.

Development of a multi-arm polyrotaxanes modified mesoporous silica-coated gold nanoplatform for protecting endothelial progenitor cells against high glucose environment.

Recent study reported that endothelial progenitor cells (EPCs) have potential to treat diabetic macroangiopathy. High glucose environment of diabetes can affect the adhesion of EPCs by decreasing the expression of CXC chemokine receptor 4 (CXCR4) and affect the proliferation of EPCs by decreasing the expression of miR-126. The results showed that the cytotoxicity of GNR@MSNs@PEI to EPCs was significantly lower than PEI; the temperature of GNR@MSNs@PEI solution can be controlled between 38-40°C under 808 nm laser irradiation. 25.67 µg of pcDNA3.1-GFP-CXCR4 and 5.36 µg of FITC-miR-126 could be loaded in 1 mg of GNR@MSNs@PEI; GNR@MSNs@PEI has gene transfection almost the same as Lipofectamine 3000. Subsequent in vitro studies showed that pcDNA3.1-GFP-CXCR4 and miR-126 loaded GNR@MSNs@PEI can significantly increase the adhesion and proliferation and decrease the apoptosis of EPCs treated with high glucose under 808 nm laser irradiation. In conclusion, nano-carriers (GNR@MSNs@PEI) with high pcDNA3.1-CXCR4 and miR-126 loading capacity, high biocompatibility, well cell internalization, and controllable release ability were constructed to transfer CXCR4 expression plasmid (pcDNA3.1-CXCR4) and miR-126 into EPCs efficiently. Further in vitro studies indicated that pcDNA3.1-CXCR4 and miR-126-loaded GNR@MSNs@PEI could protect EPCs against high glucose-induced injury.

A Self-Regulating DNA Rotaxane Linear Actuator Driven by Chemical Energy.

Nature-inspired molecular machines can exert mechanical forces by controlling and varying the distance between two molecular subunits in response to different inputs. Here, we present an automated molecular linear actuator composed of T7 RNA polymerase (T7RNAP) and a DNA [2]rotaxane. A T7 promoter region and terminator sequences are introduced into the rotaxane axle to achieve automated and iterative binding and detachment of T7RNAP in a self-controlled fashion. Transcription by T7RNAP is exploited to control the release of the macrocycle from a single-stranded (ss) region in the T7 promoter to switch back and forth from a static state (hybridized macrocycle) to a dynamic state (movable macrocycle). During transcription, the T7RNAP keeps restricting the movement range on the axle available for the interlocked macrocycle and prevents its return to the promotor region. Since this range is continuously depleted as T7RNAP moves along, a directional and active movement of the macrocycle occurs. When it reaches the transcription terminator, the polymerase detaches, and the system can reset as the macrocycle moves back to hybridize again to the ss-promoter docking site. The hybridization is required for the initiation of a new transcription cycle. The rotaxane actuator runs autonomously and repeats these self-controlled cycles of transcription and movement as long as NTP-fuel is available.

A twin-axial pseudorotaxane for phosphorescence cell imaging.

A twin-axial pseudorotaxane is constructed using a phenylpyridine salt with diethanolamine (DA-PY) and cucurbit[8]uril (CB[8]), and it not only displays phosphorescence in aqueous solution but it can also be used for targeted cell-imaging.

A Complex Comprising a Cyanine Dye Rotaxane and a Porphyrin Nanoring as a Model Light-Harvesting System.

A nanoring-rotaxane supramolecular assembly with a Cy7 cyanine dye (hexamethylindotricarbocyanine) threaded along the axis of the nanoring was synthesized as a model for the energy transfer between the light-harvesting complex LH1 and the reaction center in purple bacteria photosynthesis. The complex displays efficient energy transfer from the central cyanine dye to the surrounding zinc porphyrin nanoring. We present a theoretical model that reproduces the absorption spectrum of the nanoring and quantifies the excitonic coupling between the nanoring and the central dye, thereby explaining the efficient energy transfer and demonstrating similarity with structurally related natural light-harvesting systems.

Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates.

Understanding protein folding under conditions similar to those found in vivo remains challenging. Folding occurs mainly vectorially as a polypeptide emerges from the ribosome or from a membrane translocon. Protein folding during membrane translocation is particularly difficult to study. Here, we describe a single-molecule method to characterize the folded state of individual proteins after membrane translocation, by monitoring the ionic current passing through the pore. We tag both N and C termini of a model protein, thioredoxin, with biotinylated oligonucleotides. Under an electric potential, one of the oligonucleotides is pulled through a α-hemolysin nanopore driving the unfolding and translocation of the protein. We trap the protein in the nanopore as a rotaxane-like complex using streptavidin stoppers. The protein is subjected to cycles of unfolding-translocation-refolding switching the voltage polarity. We find that the refolding pathway after translocation is slower than in bulk solution due to the existence of kinetic traps.

Construction of pH sensitive smart glutathione peroxidase (GPx) mimics based on pH responsive pseudorotaxanes.

Two organoselenium compounds, both of which were modified with two primary amine groups, were designed and synthesized to mimic the catalytic properties of glutathione peroxidase (GPx). It was demonstrated that the catalytic mechanism of the diselenide organoselenium compound (compound 1) was a ping-pong mechanism while that of the selenide organoselenium compound (compound 2) was a sequential mechanism. The pH-controlled switching of the catalytic activities was achieved by controlling the formation and dissociation of the pseudorotaxanes based on the organoselenium compounds and cucurbit[6]uril (CB[6]). Moreover, the switching was reversible at pH between 7 and 9 for compound 1 or between 7 and 10 for compound 2.

[Intracellularly Degradable Polyrotaxanes for Therapeutic Applications].

Recently, the application of ß-cyclodextrins (ß-CDs) as therapeutic agents has received considerable attention. ß-CDs have been reported to have therapeutic effects on various diseases, such as Niemann-Pick type C (NPC) disease, a family of lysosomal storage disorders characterized by the lysosomal accumulation of cholesterol. To further improve the therapeutic efficacy of ß-CDs, the use of ß-CD-threaded polyrotaxanes (PRXs) has been proposed as a carrier of ß-CDs for NPC disease. PRXs are supramolecular polymers composed of many CDs threaded onto a linear polymer chain and capped with bulky stopper molecules. In this review, the design of PRXs and their therapeutic applications are described. To achieve the intracellular release of threaded ß-CDs from PRXs, stimuli-cleavable linkers are introduced in an axle polymer of PRXs. The stimuli-labile PRXs can dissociate into their constituent molecules by a cleavage reaction under specific stimuli, such as pH reduction in lysosomes. The release of the threaded ß-CDs from acid-labile PRXs in acidic lysosomes leads to the formation of an inclusion complex with the cholesterol that has accumulated in NPC disease patient-derived fibroblasts, thus promoting the extracellular excretion of the excess cholesterol. Moreover, the administration of PRXs to a mouse model of NPC disease caused significant suppression of the tissue accumulation of cholesterol, resulting in a prolonged life span in the model mice. Additionally, the induction of autophagy by the methylated ß-CD-threaded PRXs (Me-PRXs) is described. Accordingly, the stimuli-labile PRXs are expected to be effective carriers of CDs for therapeutic applications.

An Artificial Molecular Shuttle Operates in Lipid Bilayers for Ion Transport.

Inspired by natural biomolecular machines, synthetic molecular-level machines have been proven to perform well-defined mechanical tasks and measurable work. To mimic the function of channel proteins, we herein report the development of a synthetic molecular shuttle, [2]rotaxane 3, as a unimolecular vehicle that can be inserted into lipid bilayers to perform passive ion transport through its stochastic shuttling motion. The [2]rotaxane molecular shuttle is composed of an amphiphilic molecular thread with three binding stations, which is interlocked in a macrocycle wheel component that tethers a K+ carrier. The structural characteristics enable the rotaxane to transport ions across the lipid bilayers, similar to a cable car, transporting K+ with an EC50 value of 1.0 µM (3.0 mol % relative to lipid). We expect that this simple molecular machine will provide new opportunities for developing more effective and selective ion transporters.

Allosteric Control of Oxidative Catalysis by a DNA Rotaxane Nanostructure.

DNA is a versatile construction material for the bottom-up assembly of structures and functional devices in the nanoscale. Additionally, there are specific sequences called DNAzymes that can fold into tertiary structures that display catalytic activity. Here we report the design of an interlocked DNA nanostructure that is able to fine-tune the oxidative catalytic activity of a split DNAzyme in a highly controllable manner. As scaffold, we employed a double-stranded DNA rotaxane for its ability to undergo programmable and predictable conformational changes. Precise regulation of the DNAzyme's oxidative catalysis can be achieved by external stimuli (i.e., addition of release oligos) that modify the spatial arrangement within the system, without interfering with the catalytic core, similar to structural rearrangements that occur in allosterically controlled enzymes. We show that multiple switching steps between the active and inactive conformations can be performed consistent with efficient regulation and robust control of the DNA nanostructure.

Oligo(ethylene glycol)-modified ß-cyclodextrin-based polyrotaxanes for simultaneously modulating solubility and cellular internalization efficiency.

We developed stimuli-labile polyrotaxanes (PRXs) composed of ß-cyclodextrin (ß-CD), Pluronic as an axle polymer, and acid-cleavable N-triphenylmethyl groups as bulky stopper molecules, and found that the PRXs are potent therapeutics for Niemann-Pick type C disease, because the PRX can effectively reduce intracellular cholesterol through the intracellular release of threaded ß-CDs. In general, the PRXs need to be chemically modified with hydrophilic functional groups because PRXs are not soluble in aqueous media. Herein, four series of oligo(ethylene glycol)s (OEGs) with different ethylene glycol repeating unit (2 or 3) and chemical structure of OEG terminal (hydroxy or methoxy) were modified onto the threaded ß-CDs in PRX. The effects of the structure of OEG on the aqueous solubility, toxicity, and cellular internalization efficiency of OEG-modified PRXs were investigated to optimize the chemical structure of OEG. The hydroxy-terminated OEG-modified PRXs showed excellent solubility in aqueous media and no toxicity, regardless of the number of ethylene glycol repeating units. In the case of the methoxy-terminated OEG-modified PRXs, sufficient solubility in aqueous media and negligible toxicity were observed when the number of ethylene glycol repeating units was 3, while low solubility and toxicity were observed when the ethylene glycol repeating unit was 2. Additionally, cellular uptake levels of methoxy-terminated OEG-modified PRXs in RAW264.7 cells were higher than those of hydroxy-terminated OEG-modified PRXs. Consequently, the chemical structure of the OEG strongly affects the chemical and biological properties of the PRXs, and that a methoxy-terminated OEG with 3 ethylene glycol repeating units is the most preferable modification of PRXs, since the resultant PRX is sufficiently soluble in aqueous media, non-toxic, and possesses high cellular internalization efficiency.

Crosslinked Network with Rotatable Binding Sites Based on Monocarboxylated α-Cyclodextrin [2]Rotaxane Capable of Angiotensin†III Recognition.

Synthetic receptors selective for target peptides or proteins have received attention because of their potential applications in the separation of biomolecules and biomedical diagnostics. Herein, a [2]rotaxane-based functional monomer containing monocarboxylated α-cyclodextrin (α-CD) was synthesized, and its crosslinked polymers were evaluated to determine their binding ability to a model peptide, angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe), containing an arginine (Arg) residue. The binding ability of the resulting polymers toward angiotensin III, angiotensin IV (Val-Tyr-Ile-His-Pro-Phe), and FMRF-amide (Phe-Met-Arg-Phe) was examined by the batch-binding assay and compared with that of control polymers, in which maleic acid-introduced α-CD was chemically crosslinked. The results suggest that the [2]rotaxane-based functional monomer in the crosslinked polymer contributes to the high affinity toward angiotensin III. The α-CD motion and rotation within the [2]rotaxane-based crosslinked polymer may be applicable for designing molecular recognition materials.

Stabilizing Effects for Antibody Formulations and Safety Profiles of Cyclodextrin Polypseudorotaxane Hydrogels.

Antibodies often have poor physicochemical stability during storage and transport, which is a serious drawback for the development of antibody-based drugs. In this study, we prepared polypseudorotaxane (PPRX) hydrogels consisting of cyclodextrins (CyDs) and polyethylene glycol, and evaluated them as stabilizers for commercially available antibody-based drugs. α-CyD and γ-CyD formed PPRX hydrogels with polyethylene glycol (molecular weight 20,000 Da) in the presence of antibody-based drugs such as omalizumab, palivizumab, panitumumab, and ranibizumab. Importantly, both α- and γ-CyD PPRX hydrogel formulations provided high stabilizing effects (ca. 100%) to the all antibody-based drugs used in this study. Furthermore, approximately 100% of the binding activity of omalizumab to the immunoglobulin E receptor was retained after the release from the hydrogels. Plasma levels of omalizumab after subcutaneous injection of the γ-CyD PPRX hydrogel to rats were equivalent to those of omalizumab alone. According to the results of blood chemistry tests, the weights of organs and histological observations α- and γ-CyD PPRX hydrogels induced no serious adverse effects. These results suggest that CyD PPRX hydrogels are useful as safe and promising stabilizing formulations for antibody-based drugs.

Dual substrate-controlled kinase activity leads to polyphosphorylated lasso peptides.

Lasso peptides are characterized by their peculiar lariat knot-like structure. Except for maturation of this fold, post-translational modifications of lasso peptides are rare. However, we recently delineated the biosynthetic pathway of a post-translationally phosphorylated lasso peptide, paeninodin. In this study, further investigation of two kinases revealed their ability to transfer multiple phosphate groups onto precursor peptide substrates, ultimately leading to polyphosphorylated lasso peptides. We found that this polyphosphorylating activity depended on the identity of the phosphate donor and the sequence of the precursor peptide. Our investigations provide new insight into the remarkable strategies for chemical diversification employed by the lasso peptide biosynthetic machinery.