γ-Cyclodextrin-based polypseudorotaxane hydrogels for ophthalmic delivery of flurbiprofen to treat anterior uveitis.

Anterior uveitis is a sight-threatening inflammation inside the eyes. Conventional eye drops for anti-inflammatory therapy need to be administered frequently owing to the rapid elimination and corneal barrier. To address these issues, polypseudorotaxane hydrogels were developed by mixing Soluplus micelles (99.4 nm) and cyclodextrins solution. The optimized hydrogels exhibited shear-thinning and sustained release properties. The hydrogels exhibited higher transcorneal permeability coefficient (Papp, 1.84 folds) than that of drug solutions. Moreover, animal study indicated that the hydrogels significantly increased the precorneal retention (AUC, 21.2 folds) and intraocular bioavailability of flurbiprofen (AUCAqueous humor, 17.8 folds) in comparison with drug solutions. Importantly, the hydrogels obviously boosted anti-inflammatory efficacy in rabbit model of endotoxin-induced uveitis at a reduced administration frequency. Additionally, the safety of hydrogels was confirmed by cytotoxicity and ocular irritation studies. In all, the present study demonstrates a friendly non-invasive strategy based on γ-CD-based polypseudorotaxane hydrogels for ocular drug delivery.

Supramolecular Nanomachines as Stimuli-Responsive Gatekeepers on Mesoporous Silica Nanoparticles for Antibiotic and Cancer Drug Delivery.

Major objectives in nanomedicine and nanotherapy include the ability to trap therapeutic molecules inside of nano-carriers, carry therapeutics to the site of the disease with no leakage, release high local concentrations of drug, release only on demand - either autonomous or external, and kill the cancer cells or an infectious organism. This review will focus on mesoporous silica nanoparticle carriers (MSN) with a large internal pore volume suitable for carrying anticancer and antibiotic drugs, and supramolecular components that function as caps that can both trap and release the drugs on-command. Caps that are especially relevant to this review are rotaxanes and pseudorotaxanes that consist of a long chain-like molecule threaded through a cyclic molecule. Under certain conditions discussed throughout this review, the cyclic molecule can be attracted to one end of the rotaxane and in the presence of a stimulus can slide to the other end. When the thread is attached near the pore opening on MSNs, the sliding cyclic molecule can block the pore when it is near the particle or open it when it slides away. The design, synthesis and operation of supramolecular systems that act as stimuli-responsive pore capping devices that trap and release molecules for therapeutic or imaging applications are discussed. Uncapping can either be irreversible because the cap comes off, or reversible when the cyclic molecule is prevented from sliding off by a steric barrier. In the latter case the amount of cargo released (the dose) can be controlled. These nanomachines act as valves. Examples of supramolecular systems stimulated by chemical signals (pH, redox, enzymes, antibodies) or by external physical signals (light, heat, magnetism, ultrasound) are presented. Many of the systems have been studied in vitro proving that they are taken up by cancer cells and release drugs and kill the cells when stimulated. Some have been studied in mouse models; after IV injection they shrink tumors or kill intracellular pathogens after stimulation. Supramolecular constructs offer fascinating, highly controllable and biologically compatible platforms for drug delivery.

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.

ER stress-mediated autophagic cell death induction through methylated ß-cyclodextrins-threaded acid-labile polyrotaxanes.

Autophagy plays a pivotal role in the development and prevention of numerous diseases, and the induction of autophagy is regarded as a potential therapeutic approach for intractable diseases. In this study, the induction of autophagy by methylated ß-cyclodextrins (Me-ß-CDs)-threaded acid-labile polyrotaxane (Me-PRX) that can release the threaded Me-ß-CDs in response to acidic pH in lysosomes was investigated. We hypothesized that the Me-ß-CDs released from the Me-PRX interact with the membrane of organelles and cause autophagy. The Me-PRX preferentially accumulated in endoplasmic reticulum (ER) and caused ER stress, which was confirmed by gene expression analysis and the expression of an ER stress-marker protein. Accompanying the ER stress, cells treated with Me-PRX showed autophagy, which was not observed in cells treated with non-labile Me-PRX, other chemically modified PRXs, or free Me-ß-CD. Furthermore, the Me-PRX treatment induced autophagic cell death and caused cell death even in apoptosis-resistant cells. Overall, this study demonstrates that the acid-labile Me-PRX induces ER stress-mediated autophagic cell death, and the Me-PRX would be a promising candidate to induce effective cell death in apoptosis-resistant malignant tumors.

Polyrotaxane-based systemic delivery of ß-cyclodextrins for potentiating therapeutic efficacy in a mouse model of Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a fatal metabolic disorder characterized by the lysosomal accumulation of cholesterol. Although 2-hydroxypropyl ß-cyclodextrin (HP-ß-CD) promotes the excretion of cholesterol and prolongs the life span in animal models of NPC disease, it requires extremely high dose. We developed acid-labile ß-CD-based polyrotaxanes (PRXs) comprising multiple ß-CDs threaded along a polymer chain capped with acid-cleavable stopper molecules for potentiating therapeutic efficacy of ß-CD in NPC disease. The acid-labile PRXs dissociate under the acidic lysosomes and release threaded ß-CDs in lysosomes, which promotes cholesterol excretion in NPC disease model cells at lower concentration than HP-ß-CD. In this study, the therapeutic effect of the PRXs in a mouse model of NPC disease was investigated. Weekly administration of the PRXs significantly prolonged the life span and suppressed neurodegeneration in mice, even at a dose of 500mg/kg, a markedly lower dose than previously reported for HP-ß-CD. Detailed analysis of tissue cholesterol revealed that PRX treatment markedly suppressed the tissue accumulation of cholesterol in the NPC mouse model, but did not alter cholesterol content in wild-type mice. Acid-labile PRX is therefore a promising candidate for potentiating the efficacy of ß-CD in the treatment of NPC disease.

A Supramolecular Substance, [2] Rotaxane, Induces Apoptosis in Human Molt-3 Acute Lymphoblastic Leukemia Cells.

The antitumor effects of a supramolecular substance, the [2] rotaxane (TRO-A0001), and its molecular mechanisms were investigated. TRO-A0001 suppressed the proliferation of cultured human Molt-3 acute lymphoblastic leukemia cells for 12-72 h in a dose-dependent manner. Based on flow cytometry, TRO-A0001 clearly induced apoptosis after 24 h. The mitochondrial membrane potential disappeared after treatment with 1.0 µM of TRO-A0001. Expression of the cleaved forms of capase-9 and caspase-3 was significantly increased in cells exposed to TRO-A0001, whereas the expression of XIAP, a type of inhibitor of apoptosis family, was decreased. These results suggest that [2] rotaxane TRO-A0001 may be a highly promising new antitumor medicine.

Supramolecular chemistry of p-sulfonatocalix[n]arenes and its biological applications.

CONSPECTUS: Developments in macrocyclic chemistry have led to supramolecular chemistry, a field that has attracted increasing attention among researchers in various disciplines. Notably, the discoveries of new types of macrocyclic hosts have served as important milestones in the field. Researchers have explored the supramolecular chemistry of several classical macrocyclic hosts, including crown ethers, cyclodextrins, calixarenes, and cucurbiturils. Calixarenes represent a third generation of supramolecular hosts after cyclodextrins and crown ethers. Easily modified, these macrocycles show great potential as simple scaffolds to build podand-like receptors. However, the inclusion properties of the cavities of unmodified calixarenes are not as good as those of other common macrocycles. Calixarenes require extensive chemical modifications to achieve efficient endo-complexation. p-Sulfonatocalix[n]arenes (SCnAs, n = 4-8) are a family of water-soluble calixarene derivatives that in aqueous media bind to guest molecules in their cavities. Their cavities are three-dimensional and π-electron-rich with multiple sulfonate groups, which endow them with fascinating affinities and selectivities, especially toward organic cations. They also can serve as scaffolds for functional, responsive host-guest systems. Moreover, SCnAs are biocompatible, which makes them potentially useful for diverse life sciences and pharmaceutical applications. In this Account, we summarize recent work on the recognition and assembly properties unique to SCnAs and their potential biological applications, by our group and by other laboratories. Initially examining simple host-guest systems, we describe the development of a series of functional host-guest pairs based on the molecular recognition between SCnAs and guest molecules. Such pairs can be used for fluorescent sensing systems, enzymatic activity assays, and pesticide detoxification. Although most macrocyclic hosts prevent self-aggregation of guest molecules, SCnAs can induce self-aggregation. Researchers have exploited calixarene-induced aggregation to construct supramolecular binary vesicles. These vesicles respond to internal and external stimuli, including temperature changes, redox reactions, additives, and enzymatic reactions. Such structures could be used as drug delivery vehicles. Although several biological applications of SCnAs have been reported, this field is still in its infancy. Continued exploration of the supramolecular chemistry of SCnAs will not only improve the existing biological functions but also open new avenues for the use of SCnAs in the fields of biology, biotechnology, and pharmaceutical research. In addition, we expect that other interdisciplinary research efforts will accelerate developments in the supramolecular chemistry of SCnAs.

Syringeable self-assembled cyclodextrin gels for drug delivery.

The design of syringeable cyclodextrin (CD) gels is a developing area in the drug delivery and tissue engineering fields, since they offer the possibility of being administered with minimally invasive maneuvers to form depots that can remain for prolonged time in the implantation site. Two different supramolecular systems can be obtained exploiting the capability of CDs to form inclusion complexes. (i) The threading of free CDs on certain blocks or side chains of copolymers leads to polypseudorotaxanes, which can assembly via regular stacking of the threaded CDs. The resultant assemblies can be reversible broken under a certain shear stress and reformed at rest, exhibiting thixotropy that enables the flow through the syringe and the gel recovery in the implantation site. (ii) CDs grafted to polymer chains can develop their ability to form inclusion complexes with complementary guest moieties in other polymeric structures. The result is a ladder- or zipper-like arrangement, which can be also broken and reformed under certain stress conditions. Both types of CDsupramolecular gels can load and stabilize a variety of drugs via interaction with available polymer functional groups or with the CDs that are not participating in other complexes. Moreover, since the complex formation depends on various external and internal variables of the body, the syringeable CD gels can also provide stimuli-responsive drug release. This review focuses on the two main types of syringeable CD gels, prepared via self-aggregation of poly(pseudo)rotaxanes and via zipper-like assembly of CD-functionalized and guest-functionalized macromolecules, and analyzes the mechanisms and variables involved in the gelling processes and the most recent applications in the drug delivery field.

Competitive displacement of drugs from cyclodextrin inclusion complex by polypseudorotaxane formation with poloxamer: implications in drug solubilization and delivery.

The competitive interactions between the poly-[propylene oxide] (POO)-poly-[ethylene oxide] (PEO) block copolymer poloxamer 407 (Pluronic F127) and two drugs, triamcinolone acetonide and ciclopirox olamine, by the formation of inclusion complexes with two cyclodextrin hydrophilic derivatives, hydroxypropyl-ß-cyclodextrin (HPßCD; molar substitution (MS) 0.65) and partially methylated-ß-cyclodextrin (MßCD; MS 0.57), were studied by means of one-dimensional (1)H NMR, 2D ROESY experiments, solubility studies and drug release studies. 1D and 2D NMR and solubility studies indicate that both triamcinolone acetonide and ciclopirox olamine form stable inclusion complexes with the cyclodextrin derivatives. In the case of ciclopirox olamine the complex was more stable at pH 1. Effective complexation of poloxamer with the two cyclodextrins (CDs) was also evidenced by NMR analysis, and competitive displacement of the drugs from the CD cavity by the polymer was observed. Drug solubility in CD solutions was not modified by the addition of polymers, indicating that a decrease in solubility due to the competitive displacement is probably compensated by the solubilizing effect of polymer micellization. Finally, polypseudorotaxanes formation has a significant influence on the release of the drugs studied. Changes in the release rate depend on the stability of drug-CD inclusion complex and on cyclodextrin concentration in the bulk solution; so polypseudorotaxane formation can be employed to modulate drug controlled release from thermosensitive hydrogels.

Biocleavable polyrotaxane-plasmid DNA polyplex for enhanced gene delivery.

A biocleavable polyrotaxane, having a necklace-like structure consisting of many cationic alpha-cyclodextrins (alpha-CDs) and a disulfide-introduced poly(ethylene glycol) (PEG), was synthesized and examined as a nonviral gene carrier. The polyrotaxane formed a stable polyplex having positively charged surface even at low charge ratio. This is likely to be due to structural factors of the polyrotaxane, such as the mobile motion of alpha-CDs in the necklace-like structure. Rapid endosomal escape was observed 90 min after transfection. The positively charged surface and the good buffering capacity are advantageous to show the proton sponge effect. The pDNA decondensation occurred through disulfide cleavage of the polyrotaxane and subsequent supramolecular dissociation of the noncovalent linkages between alpha-CDs and PEG. Transfection of the DMAE-SS-PRX polyplex is independent of the amount of free polycation. Those properties played a key role for delivery of pDNA clusters to the nucleus. Therefore, the polyplex nature and the supramolecular dissociation of the polyrotaxane contributed to the enhanced gene delivery.