Acid-Induced Intracellular Dissociation of Î²-Cyclodextrin-Threaded

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX-XXX

pubs.acs.org/molecularpharmaceutics

Acid-Induced Intracellular Dissociation of β‑Cyclodextrin-Threaded Polyrotaxanes Directed toward Attenuating Phototoxicity of Bisretinoids through Promoting Excretion Atsushi Tamura, Moe Ohashi, Kei Nishida, and Nobuhiko Yui* Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda, Tokyo 101-0062, Japan S Supporting Information *

ABSTRACT: In the retinal pigment epithelium of patients with agerelated macular degeneration (AMD), excess N-retinylidene-N-retinylethanolamine (A2E), a dimer of all-trans-retinal, accumulats to induce inflammatory cytokine secretion and phototoxic effects. Therefore, the reduction of intracellular A2E is a promising approach for the prevention and treatment of AMD. In this study, acid-labile β-cyclodextrin (β-CD)threaded polyrotaxanes (PRXs) were synthesized and investigated their effects on the removal of A2E accumulated in retinal pigment epithelium cells (ARPE-19) in comparison to nonlabile PRXs and 2-hydroxypropyl β-CD (HP-β-CD) were examined. GC-MS and HPLC studies strongly suggest that the acid-labile PRXs dissociated into their constituent molecules in cells by lysosomal acidification and threaded β-CDs were considered to be released from the PRXs. The released β-CDs formed an inclusion complex with A2E, which promoted the excretion of A2E. Indeed, the acid-labile PRXs effectively reduced intracellular A2E level at approximately a 10-fold lower concentration than HP-βCD. Accompanied with A2E removal, the toxicity and phototoxicity of A2E were attenuated by treatment with acid-labile PRXs. Because the nonlabile PRX failed to reduce intracellular A2E level and attenuate phototoxicity, intracellular release of threaded βCDs from the acid-labile PRX might contribute to reducing intracellular A2E. We conclude that acid-labile PRXs are promising candidates for the treatment of macular diseases through the removal of toxic metabolites. KEYWORDS: polyrotaxane, cyclodextrin, triphenylmethyl group, retinoid, phototoxicity, age-related macular degeneration

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To further improve the therapeutic efficacy of β-CDs in API applications, we have developed stimuli-labile β-CD-threaded polyrotaxanes (PRXs),17−19 CD-based supramolecular polymers composed of multiple β-CDs threaded with a linear polymer capped with bulky stopper molecules.20−25 Our previous study revealed that these PRXs can be more efficiently internalized into cells compared with HP-β-CD, because the interlocked β-CDs in the PRXs can prevent interaction with cholesterol in the plasma membrane, leading to increased efficiency of PRX endocytosis.17 Most importantly, when cleavable linkages are introduced in the axle of the PRXs, the threaded β-CDs in the PRXs can be released in response to intracellular stimuli, such as pH reduction in lysosomes, reaction with reductive molecules, or enzymatic reactions.25 We found that the intracellular release of β-CDs from the acid-labile PRXs contributes to improving the accumulation of cholesterol in lysosomes of NPC model cells at approximately a 100-fold lower concentration than HP-β-

INTRODUCTION

Cyclodextrins (CDs) have long been the focus of attention in the pharmaceutical field and their use as pharmaceutical excipients has been extensively studied, because CDs can encapsulate various low molecular weight compounds into their hydrophobic cavity, depending on their cavity size.1−3 Recently, the application of CDs as active pharmaceutical ingredients (APIs) has received considerable attention, because CDs have been shown to have therapeutic effects in various intractable diseases, such as Alzheimer’s disease, atherosclerosis, and Niemann-Pick type C (NPC) disease.4−8 In particular, the treatment of NPC disease with hydroxypropyl β-CD (HP-β-CD) is a promising therapeutic approach and clinical trials are ongoing to validate therapeutic efficacy in patients with NPC disease.9,10 However, the β-CD derivatives preferentially interact with cholesterol in the plasma membrane and remove it from the membrane.11,12 Because of this strong interaction with the plasma membrane, as well as hydrophilic character, the cellular internalization efficiency of β-CD derivatives via endocytosis is typically low.13,14 This is one of the reasons why a high dose of β-CD derivatives is required.5−7,15,16 © XXXX American Chemical Society

Received: September 30, 2017 Revised: October 26, 2017 Accepted: November 1, 2017

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DOI: 10.1021/acs.molpharmaceut.7b00859 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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CD.17−19 It is expected that the released β-CDs form an inclusion complex with cholesterol in lysosomes and accelerate the excretion of cholesterol. Therefore, the use of stimuli-labile PRXs is beneficial for potentiating the therapeutic efficacy of βCDs in NPC disease. Moreover, the function of the PRXs to release threaded β-CDs may also be effective at removing other intracellular biochemicals and metabolites that are abnormally accumulated in cells. To verify the potential use of the PRXs in other diseases or metabolites, we focused on age-related macular degeneration (AMD). AMD is a progressive chronic disease of the central retina and is regarded as a major cause of irreversible blindness in people over 50 years of age.26,27 AMD is clinically classified into an atrophic form (dry AMD) and an exudative form (wet AMD).28,29 Atrophic AMD is characterized by a progressive degeneration of retinal pigment epithelium (RPE) cells and retinal photoreceptors. Exudative AMD is characterized be the involvement of choroid neovascularization (CNV), which leads to hemorrhage and fluid leakage.28,29 Although antiangiogenic therapy has been established for exudative AMD,30−33 the treatment of atrophic AMD remains a challenging issue.28 Preventing the main causes of these diseases, such as local inflammation and oxidative stress, is considered to be effective for the prevention and treatment of both types of AMD. The intracellular accumulation of lipofuscin, a yellow-brown pigment granule composed of partially digested lipids and proteins, in RPE cells is one of the most characteristic features of both types of AMD. 28,29,34−36 N-Retinylidene-N-retinylethanolamine (A2E), a dimer of all-trans-retinal generated in RPE cells, is a main component of lipofuscin and the accumulation of A2E in RPE cells is observed in AMD and other macular degenerative disorders, such as Stargardt’s disease.37−41 The intracellular deposition of A2E shows phototoxicity via the generation of reactive oxygen species,42 stimulation of the complement system,43 and the secretion of inflammatory cytokines and vascular endothelial growth factor (VEGF).44,45 Because these effects of A2E are related to the progression of AMD, the inhibition of A2E generation in RPE cells is a potential therapeutic strategy for AMD. Indeed, the inhibition of A2E production by chemical reagents significantly improves AMD pathologies associated with the deposition of A2E.46,47 As an alternative approach, Nociari and co-workers have reported that methylated β-CDs (Me-β-CDs) promote the excretion of A2E from RPE cells through inclusion complexation, leading to improved AMD symptoms.48 Therefore, the removal of intracellularly deposited A2E is also considered to be a promising approach for inhibiting the progression of AMD. In this study, acid-labile PRXs capped with acid-cleavable Ntriphenylmethyl (N-Trt) groups were investigated for the removal of A2E from cultured RPE cells. To elucidate the contribution of the intracellular dissociation of PRXs to release threaded β-CDs on the excretion of A2E, nonlabile PRXs capped with noncleavable C-Trt groups were designed as negative controls. To obtain direct evidence on the intracellular dissociation of the acid-labile PRXs, qualitative and quantitative analyses were performed using gas chromatography−mass spectrometry (GC-MS). Comparative studies between acidlabile and nonlabile PRXs provide important insights into the design of supramolecular therapeutic agents and the potential efficacy of acid-labile PRXs for the treatment of AMD.

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EXPERIMENTAL SECTION Materials. 2-(2-Hydroxyethoxy)ethyl (HEE) carbamatemodified acid-labile PRX (HEE-PRX) composed of HEEmodified β-CDs as cyclic molecules, Pluronic P123 as an axle polymer, and N-Trt groups as acid-cleavable stopper molecules was synthesized as previously described.19,49 HEE-modified nonlabile PRX (nl-HEE-PRX) composed of HEE-modified βCDs as cyclic molecules, Pluronic P123 as an axle polymer, and C-Trt groups as acid-stable stopper molecules was synthesized using 3,3,3-triphenylpropionic acid (Supporting Information). The modification of HEE-PRX and nl-HEE-PRX with HiLyte Fluor 488 (HF488) (Anaspec, Fremont, CA, USA) was performed as described in the Supporting Information. 2Hydroxypropyl β-cyclodextrin (HP-β-CD) (average molecular weight of 1,478, with 5.9 HP groups per β-CD) and retinol were obtained from Sigma-Aldrich (Milwaukee, WI, USA). NRetinylidene-N-retinylethanolamine (A2E) was obtained from Gene and Cell Technologies (Vallejo, CA, USA). pH-Dependent Dissociation of HEE-PRX by Size Exclusion Chromatography (SEC). HEE-PRX or nl-HEEPRX were dissolved in buffer solutions at different pH (pH 4.0 to 5.0:10 mM CH3COOH/CH3COONa, 150 mM NaCl; pH 5.5 to 9.0:10 mM NaH2 PO4 /Na 2HPO4, 150 mM NaCl) at a concentration of 5.0 mg/mL and the solutions were incubated for 24 h at 37 °C. An aliquot of each solution (500 μL) was then collected and combined with 50 mM NaHCO3/Na2CO3 buffer at pH 9.0 (1 mL) to neutralize the solutions. The solutions were then freeze-dried. The resulting powder was dissolved in dimethyl sulfoxide (DMSO) containing 10 mM LiBr (500 μL). SEC was performed on an HLC-8120 system (Tosoh, Tokyo, Japan) equipped with a combination of TSKgel AW-4000 and AW-2500 columns (150 mm × 6 mm I.D.) (Tosoh). Samples were eluted with DMSO containing 10 mM LiBr at a flow rate of 0.15 mL/min at 65 °C. Cell Culture and the Loading of A2E. ARPE-19 cells, a human retinal pigment epithelium cell line, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 units/mL penicillin (Wako Pure Chemical Industries), and 100 μg/mL streptomycin (Wako Pure Chemical Industries) in 5% CO2 at 37 °C. To load A2E in ARPE-19 cells, the cells were treated with A2E (10 μM) for 24 h at 37 °C.50 Cellular Uptake Analysis by Flow Cytometry and Confocal Laser Scanning Microscopy (CLSM). ARPE-19 cells were plated in 24-well plates at a density of 1 × 105 cells/well and incubated overnight. Cells were cultured in the treatment medium containing HF488-HEE-PRX or HF488-nl-HEE-PRX (0.5 mM β-CD) for 24 h. Cells were then washed with PBS and harvested by 0.25% trypsin-EDTA treatment. Cells were then collected by centrifugation, washed three times with PBS containing 0.1% bovine serum albumin (BSA), and passed through a 35-μm cell strainer (Corning, Corning, NY, USA). The fluorescence intensity of the cells was measured on a NovoCyte 2000 flow cytometer (ACEA Biosciences, San Diego, CA, USA). The HF488-HEE-PRX was excited with a 488 nm laser and detected using a 530 ± 30 nm bandpass filter. A total of 10 000 cells were counted for each sample and the mean fluorescence intensity of the cell population was determined using Novo Express software (ACEA Biosciences). B


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undissolved retinol, and the filtrates (250 μL) were diluted with acetonitrile (250 μL). The solubility of retinol was measured on a high performance liquid chromatography (HPLC) system consisting of an AS-2057i Plus autosampler (Jasco, Tokyo, Japan), a DG-2080-53 degasser (Jasco), a PU-2080i Plus pump (Jasco), a CO-965 column oven (Jasco), an RI-2031 Plus refractive index detector (Jasco), an MD-2018 Plus photodiode array detector (Jasco), and a combination of a Cosmosil 5C18AR-II packed column (250 mm × 4.6 mm I.D.) (Nakalai Tesque, Kyoto, Japan) and a Cosmosil 5C18-AR-II guard column (10 mm × 4.6 mm I.D.) (Nakalai Tesque). Solutions (50 μL) were injected into the HPLC system and samples were eluted with a mixture of methanol and acetonitrile (the volume ratio of methanol:acetonitrile was 20:80) at a flow rate of 1.0 mL/min at 40 °C. Absorption intensities at 322 nm were used for the quantification of retinol. The apparent stability constant (K1:1) of the complexes was calculated using the equation; K1:1 = slope/ [intercept(1−slope)],51 where the slopes of retinol complexes were determined from the phase-solubility diagram and the intercept was the intrinsic solubility of retinol at 37 °C. Attenuation of A2E Toxicity. ARPE-19 cells were plated in 96-well plates at a density of 1 × 104 cells/well and incubated overnight. The medium was exchanged to treatment medium containing HEE-PRX, nl-HEE-PRX, or HP-β-CD (1 mM β-CD) and incubated for 24 h. After the cells were washed with fresh medium, they were treated with A2E (30 μM) for 5 h. Finally, Cell Counting Kit-8 reagent (Dojindo Laboratories) (10 μL/ well) was added to the medium and cells were incubated for 2 h at 37 °C. Then, the absorbance at 450 nm was measured using a Multiskan FC plate reader (Thermo Fisher Scientific). Cellular viability was calculated relative to untreated cells. Phototoxicity of A2E-Loaded ARPE-19 Cells. ARPE-19 cells were plated in 24-well plates at a density of 5 × 104 cells/well and incubated overnight. After the medium was replaced with fresh medium, the cells were treated with A2E (10 μM) for 24 h at 37 °C. The medium was then exchanged for treatment medium containing HEE-PRX, nl-HEE-PRX, or HP-β-CD (1 mM β-CD) and cells were incubated for an additional 24 h. After incubation, cells were exposed to blue light (465.5 nm, 10,000 lx) using an LED illumination system (MLEK-A230W1LR and MDBL-CB100, Moritex, Saitama, Japan) for 10 min at room temperature and further incubated for 24 h at 37 °C. Finally, cell viability was assessed as described above. Statistical Analysis. Statistical analyses were performed using OriginPro 8 (OriginLab, Northampton, MA, USA). The statistical differences between the means of individual groups were determined by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test. In the case of Figure 3B, the statistical difference was determined by twosided Student’s t test. A P value less than 0.05 was considered statistically significant.

For CLSM analysis, ARPE-19 cells were plated in a 35 mm glass bottom dish (diameter of glass area: 12 mm) (Iwaki, Tokyo, Japan) at a density of 1 × 104 cells/dish and incubated overnight. Cells were cultured in treatment medium containing HF488HEE-PRX or HF488-nl-HEE-PRX (0.5 mM β-CD) for 24 h. Cells were then stained with LysoTracker Deep Red (Thermo Fisher Scientific, Waltham, Massachusetts, USA) (100 nM) for 15 min, followed by staining with Hoechst 33342 (Dojindo Laboratories, Kumamoto, Japan) (1 μg/mL) for 10 min at 37 °C. CLSM observations were performed using a FluoView FV10i (Olympus, Tokyo, Japan) equipped with a 60× water-immersion objective lens (N/A 1.2). The excitation/emission wavelengths for Hoechst 33342 were 405 nm/455 nm, HF488-labeled PRXs were 473 nm/520 nm, and LysoTracker Deep Red were 635 nm/ 668 nm. To observe A2E accumulated in the cells, the excitation and emission wavelengths of A2E were set at 430 and 600 nm, respectively. Quantification of Intracellular Triphenylmethanol by GC-MS. ARPE-19 cells were plated in 6-well plates at a density of 5 × 105 cells/well and incubated overnight. Cells were cultured in treatment medium containing HEE-PRX, nl-HEE-PRX, or HPβ-CD (1 mM β-CD) for 24 h. After incubation, cells were harvested with trypsin-EDTA treatment and washed twice with PBS. The cells were mixed with cholesterol-d7 (10 μL, 1 mg/mL in pyridine) (Avanti Polar Lipids, Alabaster, AL, USA) as an internal standard and homogenized in PBS (500 μL). Homogenized samples were extracted with a mixture of chloroform and methanol (chloroform:methanol = 1:2; 1.9 mL), followed by chloroform (1.25 mL). The organic phase was combined and evaporated to dryness via nitrogen flow on an MGS-2200 instrument (Eyela, Tokyo, Japan) at 40 °C. Dried residues were dissolved in dehydrated pyridine (150 μL) and derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA, Sigma-Aldrich) (50 μL) for 30 min at 60 °C. The concentration of triphenylmethanol (Trt−OH) liberated from the acid-labile HEE-PRX was quantified by GC-MS measurement on a GCMS-QP2020 instrument (Shimadzu, Tokyo, Japan) equipped with an AOC-20i auto injector (Shimadzu) and an MXT-1 cross-linked poly(dimethylsiloxane) capillary column (30 m × 0.25 mm I.D., 0.25 μm phase thickness) (Restek, Bellefonte, PA, USA). Ultrahigh-purity helium (>99.999%) was used as a carrier gas at a column head pressure of 50 kPa and a column flow rate of 0.75 mL/min. The ionization energy was relative to the tuning and the ion source temperature was 230 °C. Sample solutions (1 μL) were injected in splitless mode. The oven temperature was initially held at 100 °C for 1 min (0 to 1 min), increased to 300 °C at a rate of 20 °C/min (1 to 21 min), increased further to 330 °C at a rate of 10 °C/min (21 to 24 min), and finally held at 330 °C for 46 min (24 to 60 min). For quantitative analysis, measurements were performed with a selected-ion monitoring (SIM) mode. The ions used for the determination ions were m/z = 77, 105, and 260 and the quantification was m/z = 183. Inclusion Complex Formation with Retinol. HEE-PRX, nl-HEE-PRX, and HP-β-CD were dissolved in buffer solutions at different pH values (pH 5.0:10 mM CH3COOH/CH3COONa, 150 mM NaCl; pH 7.4:10 mM NaH2PO4/Na2HPO4, 150 mM NaCl) and the solutions were incubated at 37 °C for 24 h (the concentrations of β-CD were 2.5, 5, 10, 15, and 20 mM). Approximately 2 mg of retinol (Sigma-Aldrich) was then combined with the solutions and the suspensions were shaken for 24 h at 37 °C. Suspensions were passed through a 0.22-μm polyvinylidene difluoride (PVDF) membrane filter to eliminate

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RESULTS AND DISCUSSION Characterization of Acid-Labile and Nonlabile HEEPRX. The acid-labile HEE-PRX, composed of HEE groupsmodified β-CDs (HEE-β-CDs) as a cyclic molecule, Pluronic P123 as an axle polymer, and acid-cleavable N-Trt groups as bulky stopper molecules, was used to investigate the effect of intracellular release of β-CDs from the HEE-PRX on the accumulation of the bisretinoids, A2E.19,49 Pluronic P123 (PEGb-PPG-b-PEG triblock copolymer) was employed as an axle of HEE-PRX, because β-CDs form an inclusion complex with PPG segment.52,53 Because pluronic copolymers are water-soluble, C


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Figure 1. Schematic illustration of HEE group-modified acid-labile polyrotaxane (HEE-PRX) and nonlabile polyrotaxane (nl-HEE-PRX) and intracellular dissociation of HEE-PRX in response to endosomal/lysosomal acidification.

chemical composition and molecular weight of HEE-PRX and nlHEE-PRX were almost comparable. Because we have previously clarified that the number of HEE groups on HEE-PRX has little effect on cellular uptake efficiency, intracellular distributions, and the dissociation kinetics of HEE-PRX,19 we synthesized only one composition of HEE-PRX and nl-HEE-PRX for this study. pH-dependent cleavage of N-Trt groups in HEE-PRX and the pH-tolerability of C-Trt groups in the nl-HEE-PRX were assessed by incubating these PRXs in various pH conditions and monitoring the dissociation of PRX structure by SEC (Figure 2A,B). Under a neutral to alkaline pH range (7.4 to 9), peaks corresponding to HEE-PRX and nl-HEE-PRX were observed after 24 h, indicating that both PRXs are stable under these pH conditions. Consistent with our previous results, dissociation of HEE-PRX was observed with decreasing the pH values, as confirmed by the appearance of peaks corresponding to dethreaded HEE-β-CDs.19 By comparing the peak area ratio of dethreaded HEE-β-CD to HEE-PRX, it was revealed that HEEPRX was completely dissociated after incubation for 24 h below pH 5.0 (Figure 2C). In contrast, dissociation of nl-HEE-PRX was not observed after incubation for 24 h at the pH range of 4.0 to 9.0, suggesting that the C-Trt groups in nl-HEE-PRX are stable, even at acidic pH. Accordingly, C-Trt groups can serve as acidstable stopper molecules for β-CD-threaded PRXs (Figure 2B,C). In further investigations, liberated triphenylmethyl groups were quantified by HPLC after incubation for 24 h. When the terminal N-Trt groups of HEE-PRX are cleaved in aqueous solutions, triphenylmethyl cations are released and immediately converted into triphenylmethanol (Trt−OH) through reaction with water molecules.55,57 The release of Trt−OH was observed

nontoxic, and sufficiently biocompatible as a component for biomaterials and their biodistributions are clarified so far,54 pluronic was selected as an axle polymer for HEE-PRX instead of PPG. To better understand the contributions of the intracellular dissociation of HEE-PRX, nonlabile HEE-PRX (nl-HEE-PRX) was synthesized with a similar chemical structure to HEE-PRX (Figure 1, Supporting Information Figures S1−S3). In the molecular design of nl-HEE-PRX, a C-Trt group was employed as an acid-stable stopper molecule. In the case of triphenylmethyl ethers (O-Trt), triphenylmethyl sulfides (S-Trt), and N-Trt, the protonation of ethers, sulfides, and amines adjacent to the triphenylmethyl group results in α-elimination of a triphenylmethyl cation.55,56 On the contrary, it is considered that the protonation of an aliphatic carbon adjacent to the triphenylmethyl group is difficult under acidic conditions and C-Trt groups may avoid α-elimination of a triphenylmethyl cation and provide stable linkage at any pH condition. The characterization of HEE-PRX and nl-HEE-PRX is summarized in Table 1. The Table 1. Characterization of HEE-PRX and nl-HEE-PRX sample code

number of threading β-CDsa

number of HEE groups on PRXa

Mn,NMRb

Mw/Mnc

HEEPRX nl-HEEPRX

11.2

62.5 (5.6)

29,000

1.09

10.3

45.8 (4.4)

25,800

1.15

a

Determined by 1H NMR in DMSO-d6. The values in parentheses denote the average number of HEE groups per threaded β-CD. b Calculated based on the chemical composition of HEE-PRX and nlHEE-PRX determined by 1H NMR. cDetermined by SEC in DMSO containing 10 mM LiBr at 60 °C. D


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The intracellular distribution of the PRXs was assessed by CLSM after treatment for 24 h. To clarify the site of localization, endosomes and lysosomes were stained with LysoTracker Deep Red dye, which specifically stains intracellular acidic compartments. The fluorescence signals of both HF488-HEE-PRX and HF488-nl-HEE-PRX were colocalized with the signals of the LysoTracker dye (Figure 3C). Additionally, Pearson’s correlation coefficients of the localization of HEE-PRX and nl-HEEPRX to endosomes/lysosomes were comparable (Figure 3D). These results indicate that both PRXs are localized in endosomes and lysosomes and intracellular distributions of both PRXs are comparable. Because A2E accumulates in endosomes and lysosomes in AMD,50,58 the intracellular localization of the PRXs is favorable for interaction with A2E. Intracellular Cleavage of N-Trt Groups of Acid-Labile HEE-PRX. To ascertain the intracellular cleavage of the N-Trt end groups of HEE-PRX, we performed qualitative and quantitative analyses of intracellularly liberated N-Trt end groups by GC-MS. First, we investigated whether Trt−OH could be detected by GC-MS in the lysates of treated cells. Figure 4A shows the total ion chromatogram (TIC) and mass spectrum pattern of Trt−OH coupled with a trimethylsilyl group (TrtOTMS). The Trt-OTMS was observed at 10.68 min and specific ions for Trt-OTMS were m/z = 77, 105, 154, 183, and 260. Next, the lysates of ARPE-19 cells treated with HEE-PRX and nl-HEEPRX were measured on selected ion mode (SIM) to detect intracellularly liberated Trt−OH (Figure 4B). In the chromatograms of untreated ARPE-19 cells and nl-HEE-PRX-treated cells, no peaks were observed at 10.68 min for each mass (m/z = 77, 105, 183, and 260). In contrast, the peaks were observed at 10.68 min in all mass chromatograms of HEE-PRX-treated cells, strongly suggesting the liberation of Trt−OH from HEE-PRX in the intracellular environment. Figure 4C shows the time-course of the SIM chromatograms (m/z = 183) of cells treated with HEE-PRX or nl-HEE-PRX. The chromatograms of HEE-PRX-treated cells showed time-dependent increases in the peak intensities of Trt-OTMS. The intracellular amount of liberated Trt−OH increased proportionally with time during treatment of the cells with HEE-PRX (Figure 4D). This result suggests that the dissociation of HEEPRX occurs immediately after cellular internalization, to release threaded HEE-β-CDs, in a time-dependent manner. In contrast, the generation of Trt−OH was not confirmed for nl-HEE-PRX, indicating that nl-HEE-PRX is stable, even under cellular environments. Additionally, when lysosomal acidification was blocked with specific pharmacological inhibition of vacuolar-type H+-ATPase (bafilomycin A1), the amount of liberated Trt−OH was significantly reduced (Supporting Information Figure S5). This result supports our hypothesis that the N-Trt groups in HEE-PRX are liberated by reaction with lysosomal protons. Because the intracellular release of threaded HEE-β-CDs from HEE-PRX occurred by the cleavage of terminal N-Trt groups (Figure 2), these results strongly suggest that the threaded HEEβ-CDs are released from HEE-PRX in response to the intracellular acidic environment. When treatment was stopped by exchanging the culture medium, the amount of intracellular Trt−OH decreased after incubation for an additional 24 h (Figure 4E). Because the triphenylmethyl groups are known to be metabolized and excreted in feces and urine in rats,59 these results suggest that the liberated Trt−OH is metabolized and excreted from cells, although the detailed mechanisms are unclear at this time.

Figure 2. (A,B) SEC charts of HEE-PRX (A) and nl-HEE-PRX (B) after incubation for 24 h at various pHs. (C) The SEC peak area ratios for released β-CD from HEE-PRX (squares) and nl-HEE-PRX (circles) after incubation for 24 h at various pHs. (D) The rate of liberated triphenylmethanol (Trt−OH) from HEE-PRX (squares) and nl-HEEPRX (circles) after incubation for 24 h at various pHs. Liberated Trt− OH was quantified by HPLC. Data were expressed as the mean ± SD (n = 3).

for HEE-PRX after incubation for 24 h below pH 7.0 (Figure S4). The pH dependency of the amount of liberated Trt−OH was almost consistent with the dissociation profiles of HEE-PRX (Figure 2C,D). This result indicates that the liberation of N-Trt stopper molecules from HEE-PRX triggers dissociation of the PRX structure and the release of HEE-β-CDs. On the contrary, the release of Trt−OH and other Trt species was not observed in nl-HEE-PRX, again confirming that the C-Trt groups are stable at acidic pH conditions (Figures S4 and 2D). Intracellular Localization of Acid-Labile and Nonlabile HEE-PRX. To gain fundamental knowledge for in vitro investigations, cellular internalization efficiency and intracellular distribution of HEE-PRX and nl-HEE-PRX in ARPE-19 cells were compared. Cellular uptake of the PRXs was investigated with flow cytometry using fluorescent molecule (HF488)-labeled HEE-PRX and nl-HEE-PRX. The fluorescence intensity of the treated cells increased with time for both HF488-HEE-PRX and HF488-nl-HEE-PRX (Figure 3A). However, the cells treated with HF488-HEE-PRX showed higher intensities than cells treated with HF488-nl-HEE-PRX (Figure 3B). It is possible that slight differences in the chemical compositions of HEE-PRX and nl-HEE-PRX, such as the number of threading β-CDs and the hydrophobicity of bulky stopper molecules, affect their interaction with the cellular membrane and cellular internalization efficiency. E


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Figure 3. (A) Flow cytometric histograms of ARPE-19 cells treated with HF488-HEE-PRX or HF488-nl-HEE-PRX for various times (concentration of threaded β-CD: 0.5 mM). (B) Time course of fluorescence intensity of ARPE-19 cells treated with HF488-HEE-PRX or HF488-nl-HEE-PRX for various times (concentration of threaded β-CD: 0.5 mM). Data are expressed as the mean ± SD (n = 3). (C) CLSM images of ARPE-19 cells treated with HF488-HEE-PRX or HF488-nl-HEE-PRX (green) for 24 h (concentration of threaded β-CD: 0.5 mM). The nuclei and endosomes/lysosomes were stained with Hoechst 33342 (blue) and LysoTracker Deep Red (Red), respectively (scale bars: 10 μm). (D) Pearson’s correlation coefficient for the localization of HF488-HEE-PRX or HF488-nl-HEE-PRX to endosomes/lysosomes (LysoTracker Deep Red staining). Data are expressed as the mean ± standard error of 20 cells.

complexation. In general, β-CD can encapsulate various retinoids, such as retinol and retinoic acid, at 1:1 stoichiometry.48,60 The stability constants for the complex of retinol at pH 7.4 and 5.0 were determined as shown in Table 2. The stability constant for the HEE-PRX was 34.6-fold high at pH 5.0 compared to pH 7.4, again confirming that the inclusion of the retinoid can be controlled by pH for the acid-labile HEE-PRX. Although HEE-PRX and nl-HEE-PRX slightly solubilize retinol at pH 7.4, this may be attributed to a nonspecific interaction with a PRX chain or an axle polymer. Removal of Intracellularly Deposited A2E. A2E is a major byproduct of the visual cycle and it progressively accumulates forms lipofuscin with aging in RPE cells.39,40 To assess the ability of HEE-PRX to reduce the accumulation of the bisretinoid, A2E, in lysosomes of RPE cells, A2E was loaded into ARPE-19 cells according to the previously published methods.50 When cells were treated with 10 μM of A2E for 24 h, bright autofluorescence derived from punctate A2E was observed in the intracellular space by CLSM.50 Most of the A2E-positive puncta were localized to lysosomes (LysoTracker stain), indicating A2E is stored in lysosomes (Figure 6A). The A2E-loaded ARPE-19 cells were then treated with HEEPRX, nl-HEE-PRX, and HP-β-CD at various concentrations for 24 h. Intracellular A2E was then observed (Figure 6B) and the number of A2E-positive puncta in the treated cells was determined (Figure 6C). The treatment with HP-β-CD significantly reduced the number of A2E-positive puncta in ARPE-19 cells, especially at a high concentration (1 mM). In

Inclusion Complexation with Retinol. The inclusion of a retinoid moiety with β-CD derivatives plays a pivotal role in the excretion of intracellular A2E.48 The interlocked structure of PRX prevents the inclusion of additional molecules, because the β-CDs cavity is occupied with a polymer chain. However, the release of the threaded HEE-β-CDs via the dissociation of the PRX may recover the ability to form an inclusion complex. This means that the ability of the HEE-PRX to form an inclusion complex is dependent on the pH. To verify whether dethreaded HEE-β-CDs from the HEE-PRX in lysosomes can form an inclusion complex with retinoids, the inclusion complexation of retinol, which has a retinoid backbone structure similar to A2E (Figure 5A), was investigated in terms of the solubility of retinol. Herein, in order to compare the HEE-PRX with commercially available and clinically approved β-CD derivatives, HP-β-CD was used as a control. In the case of HP-β-CD, retinol solubility increased with concentration, regardless of pH value (Figure 5B, 5C). This suggests that HP-β-CD forms an inclusion complex with retinol and this ability is not affected with pH. In the case of the PRXs, both acid-labile and nonlabile HEE-PRXs did not solubilize retinol at pH 7.4 (Figure 5B). Because both PRXs retain their interlocked structure at pH 7.4, it is difficult to solubilize retinol through inclusion complexation. In contrast, the solubility of retinol proportionally increased in the presence of acid-labile HEE-PRX at pH 5.0, whereas the nonlabile HEEPRX was not affect the solubility of retinol at pH 5.0 (Figure 5C). These results suggest that the acid-induced release of threaded HEE-β-CDs occurs to solubilize retinol through inclusion F


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Figure 4. (A) Total ion chromatogram (TIC) of Tt−OH. The inset figure shows the mass spectrometric profile of Trt−OH. (B) Selected ion mode (SIM) profiles for treated ARPE-19 cells detected at fragment mass for Trt−OH (m/z = 77, 108, 183, and 260). ARPE-19 cells treated with the HEEPRX or nl-HEE-PRX (concentration: 1 mM β-CD) for 24 h. (C) Time-course of the SIM profile (m/z = 183) of ARPE-19 cells treated with HEE-PRX or nl-HEE-PRX (concentration: 1 mM β-CD). (D) Time-course of the intracellular amount of Trt−OH in ARPE-19 cells treated with HEE-PRX or nlHEE-PRX (concentration: 1 mM β-CD). The amount of Trt−OH was normalized to the number of cells (n = 3). (E) Time-course of the intracellular amount of Trt−OH in ARPE-19 cells treated with HEE-PRX or nl-HEE-PRX (concentration: 1 mM β-CD) for 24 h, followed by incubation without PRXs for an additional 72 h. Data are expressed as the mean ± SD (n = 3).

Figure 5. (A) Chemical structure of A2E and retinol. (B, C) Solubility of retinol in the presence of various concentrations of HEE-PRX (green squares), nl-HEE-PRX (blue circles), or HP-β-CD (orange triangles) in phosphate buffer at pH 7.4 (B) and acetate buffer at pH 5.0 (C). The solubility of retinol was determined after incubation for 24 h at 37 °C. Data are expressed as the mean ± SD (n = 3).

cellular dissociation of HEE-PRX and the subsequent release of threaded HEE-β-CDs might contribute to the removal of A2E from the cells. We have previously reported that HEE-PRX can also reduce the lysosomal cholesterol accumulation in NPC disease at a much lower concentration than HP-β-CD.17,19 Therefore, it is considered that the function of acid-labile HEEPRX in reducing excess metabolites in lysosomes is effective for both cholesterol and bisretinoids. Attenuating Toxic Effects of A2E. Intracellular accumulation of A2E induces cytotoxic and phototoxic effects in RPE cells.39,40,42,58 To verify whether the removal of A2E by the HEEPRX attenuates the toxic effects of A2E, ARPE-19 cell viability was investigated. Cells were pretreated with HEE-PRX for 24 h,

Table 2. Stability Constants (K1:1) for the Complexes of Retinol at Different pH Conditions at 37°C sample code HEE-PRX nl-HEEPRX HP-β-CD

K1:1 at pH 7.4 (M−1)

K1:1 at pH 5.0 (M−1)

fold increase pH 5.0/pH 7.4

2.13 × 103 4.40 × 103

7.38 × 104 3.20 × 103

34.6 0.72

5.57 × 104

5.69 × 104

1.02

contrast, HEE-PRX treatment reduced the number of A2Epositive puncta to an approximately 10-fold lower concentration than HP-β-CD. Because nonlabile HEE-PRX did not alter the number of A2E-positive puncta in ARPE-19 cells, the intraG


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Figure 6. (A) CLSM images of untreated and A2E-loaded ARPE-19 cells. CLSM observations of the cells were performed after treatment with A2E (10 μM) for 24 h. The excitation and emission wavelengths for A2E (shown as green) were 430 and 600 nm, respectively. Endosomes/lysosomes were stained with LysoTracker Deep Red (Red) (scale bars: 20 μm). (B) CLSM images of A2E-loaded ARPE-19 cells after treatment with HEE-PRX, nlHEE-PRX, or HP-β-CD at various β-CD concentrations for 24 h (scale bars: 50 μm). (C) Number of A2E-positive puncta in A2E (10 μM)-loaded ARPE-19 cells after treatment with HEE-PRX, nl-HEE-PRX, or HP-β-CD at various β-CD concentrations for 24 h. Data are expressed as the mean ± standard error of 55 cells (*p < 0.05).

Figure 7. (A) Experimental protocol for investigating the attenuation of A2E toxicity in ARPE-19 cells. (B) The viability of ARPE-19 cells pretreated with HEE-PRX, nl-HEE-PRX, or HP-β-CD (concentrations: 1 mM β-CD) for 24h, followed by treatment with A2E at a toxic concentration (30 μM) for 5 h (n = 6). (C) Experimental protocol for investigating the attenuation of the A2E phototoxicity in ARPE-19 cells. (D) The viability of ARPE-19 cells pretreated with A2E (10 μM) for 24 h, followed by treatment with HEE-PRX, nl-HEE-PRX, or HP-β-CD (concentrations: 1 mM β-CD) for 24 h and irradiated with blue light for 10 min (n = 3). Data are expressed as the mean ± SD (*p < 0.05).

followed by A2E treatment (Figure 7A). In this experiment, A2E treatment was carried out at a toxic concentration (30 μM) (Supporting Information Figure S6). Note that the pretreatment with HEE-PRX, nl-HEE-PRX, and HP-β-CD (concentraiton: 1 mM β-CD) did not decrease cell viability (Figure 7B), indicating

that the PRXs and HP-β-CD are nontoxic under these experimental conditions. When cells were pretreated with nlHEE-PRX or HP-β-CD, the cell viability decreased to approximately 20% after post-treatment with A2E at a toxic concentration. The viability of cells pretreated with HP-β-CD H


Article

Molecular Pharmaceutics was slightly lower than nl-HEE-PRX-treated cells, presumably because of the destabilization of the plasma membrane through the removal of cholesterol by HP-β-CD. In contrast, the toxic effect of A2E was attenuated when cells were pretreated with HEE-PRX. HEE-β-CDs released from HEE-PRX are thought to remain in cells and interact with A2E to eliminate it from cellular environment. In further investigations, the phototoxic effect of A2E was evaluated after treatment with the PRXs (Figure 7C). Since retinoid-induced phototoxicity occurs especially under irradiation with blue light, cells were irradiated using blue light (λ = 465.5 nm) at 10 000 lx for 10 min.42 Without A2E treatment (10 μM) or light irradiation, cell viability was not significantly reduced (Figure 7D). When cells were treated with A2E and irradiated with blue light, cell viability was significantly reduced. Treatment with nl-HEE-PRX or HP-β-CD did not alter cell viability, presumably because there was an insufficient amount of A2E removed. On the contrary, treatment with HEE-PRX attenuated the phototoxicity of A2E, probably due to effective removal of A2E from the cells. Accordingly, these data suggest that the HEE-PRX-mediated reduction of intracellular A2E attenuates the toxic and phototoxic effects of A2E. For the excretion of A2E, it is considered that the ability to form an inclusion complex of A2E with dethreaded HEE-β-CDs from HEE-PRX plays an important role. Although the inclusion complexation of retinol with dethreaded HEE-β-CDs were confirmed (Figure 5C), this ability may be altered by the number of HEE groups. For further potentiating the ability to excrete A2E from RPE cells, the effect of molecular composition of HEEPRX should be investigated in future studies.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Atsushi Tamura: 0000-0003-0235-7364 Nobuhiko Yui: 0000-0001-5212-1371 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Grant-in-Aid for Young Scientists (A) from Japan Society for the Promotion of Science (JSPS) (JSPS KAKENHI Grant Number JP16H05910 to A.T.); Azuma Medical & Dental Research Grant (to A.T.); and Cooperative project among medicine, dentistry, and engineering for medical innovation “Construction of creative scientific research of the viable material via integration of biology and engineering” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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REFERENCES

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CONCLUSION In this study, the effects of acid-labile HEE-PRX treatment on the accumulation of the bisretinoid, A2E, in the lysosomes of ARPE19 cells were investigated in comparison to nl-HEE-PRX and HP-β-CD treatment. GC-MS measurements revealed that the NTrt groups of HEE-PRX are cleaved in intracellular environments, strongly indicating that HEE-PRX is dissociated in response to lysosomal acidification and threaded β-CDs in the HEE-PRX are released. This intracellular dissociation of HEEPRX contributed to reducing the A2E level in lysosomes of ARPE-19 cells, whereas A2E was still stored in lysosomes after the treatment with nonlabile HEE-PRX. Note that HEE-PRX reduced lysosomal A2E content at approximately a 10-fold lower concentration than HP-β-CD. HEE-PRX treatment attenuated the toxicity and phototoxicity of A2E through the removal of A2E, whereas nl-HEE-PRX and HP-β-CD failed to reduce A2E toxicity and phototoxicity. Accordingly, we conclude that acidlabile HEE-PRX is a promising candidate for the treatment of macular degeneration through the effective removal of toxic bisretinoids. For further potentiating the removal efficiency of A2E, the chemical modification of HEE-PRX with functional molecules, such as ligand molecules to RPE cells, is one of the essential strategies. Further studies including the molecular design of PRX are needed for the treatment of AMD by acidlabile HEE-PRX.

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Complete experimental methods, characterization of polyrotaxanes, synthesis of β-CD/pluronic P123-based polyrotaxane bearing C-Trt end groups, synthesis of HiLyte Fluor 488-labeled HEE-PRX and nl-HEE-PRX, and pH-dependent cleavage of N-Trt end groups in HEEPRX by HPLC measurements (PDF)

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00859. I


Article

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Acid-Induced Intracellular Dissociation of Î²-Cyclodextrin-Threaded

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