arene Nanoassembly for Curcumin Delivery - ACS Publications

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Potential Eye Drop Based on a Calix[4]arene Nanoassembly for Curcumin Delivery: Enhanced Drug Solubility, Stability, and AntiInflammatory Effect Giuseppe Granata,† Irene Paterniti,‡ Corrada Geraci,† Francesca Cunsolo,† Emanuela Esposito,‡ Marika Cordaro,‡ Anna Rita Blanco,§ Salvatore Cuzzocrea,*,‡ and Grazia M. L. Consoli*,† †

Institute of Biomolecular Chemistry, National Research Council (C.N.R.), 95126 Catania, Italy Department of Chemical, Biological, Pharmaceutical and Environmental Science, University of Messina, 98166 Messina, Italy § SIFI SpA, 95025 Lavinaio, Catania, Italy ‡

S Supporting Information *

ABSTRACT: Curcumin is an Indian spice with a wide spectrum of biological and pharmacological activities but poor aqueous solubility, rapid degradation, and low bioavailability that affect medical benefits. To overcome these limits in ophthalmic application, curcumin was entrapped in a polycationic calix[4]arene-based nanoaggregate by a simple and reproducible method. The calix[4]arene−curcumin supramolecular assembly (Calix−Cur) appeared as a clear colloidal solution consisting in micellar nanoaggregates with size, polydispersity index, surface potential, and drug loading percentage meeting the requirements for an ocular drug delivery system. The encapsulation in the calix[4]arene nanoassembly markedly enhanced the solubility, reduced the degradation, and improved the anti-inflammatory effects of curcumin compared to free curcumin in both in vitro and in vivo experiments. Calix−Cur did not compromise the viability of J774A.1 macrophages and suppressed pro-inflammatory marker expression in J774A.1 macrophages subjected to LPS-induced oxidative stress. Histological and immunohistochemical analyses showed that Calix−Cur reduced signs of inflammation in a rat model of LPS-induced uveitis when topically administrated in the eyes. Overall, the results supported the calix[4]arene nanoassembly as a promising nanocarrier for delivering curcumin to anterior ocular tissues. KEYWORDS: drug delivery, calix[4]arene, curcumin, inflammation, uveitis

1. INTRODUCTION

In an attempt to enhance curcumin bioavailability and therapeutic efficacy, a variety of nanostructured delivery systems has been developed.10,11 Among them, an orally administrated curcumin−phosphatidylcholine formulation (Meriva) showed to improve at least 10 times the bioavailability of curcumin in humans12 and formulated as a food supplement (Norflo tablets) showed a positive role in the adjunctive therapy of anterior uveitis.13 Eye drop is the most convenient and patient compliant route of drug administration, especially for the treatment of anterior segment diseases, but the topical administration of a drug must deal with anatomy and physiology of the eye. Corneal barrier, tear formation, blinking, and flow of the drug through nasolacrimal duct are factors limiting the bioavailability of topically administrated drugs. The encapsulation in nano-

Curcumin is a natural polyphenolic compound with multiple molecular targets and therapeutic properties including antioxidant,1 anti-inflammatory,2 antimicrobial,3 and anticancer4 activities. Many studies have shown that curcumin can modulate the expression and activation of many cellular regulatory proteins such as chemokines, interleukins, hematopoietic growth factors, and transcription factors involved in cellular inflammatory responses.5,6 The anti-inflammatory activity makes curcumin beneficial also in eye inflammatory and degenerative diseases, as chronic anterior uveitis, dry eye, maculopathy, glaucoma, and diabetic retinopathy.7 Lal et al. showed that the efficacy of curcumin on chronic anterior uveitis is comparable to corticosteroid therapy but with fewer side effects.8 Despite significant medicinal efficacy and biosafety profile, low water solubility, chemical instability, poor bioavailability, rapid metabolism, and interaction with plasma proteins have limited the clinical success of curcumin.9 © XXXX American Chemical Society

Received: November 23, 2016 Revised: February 15, 2017 Accepted: March 31, 2017

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DOI: 10.1021/acs.molpharmaceut.6b01066 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

calix[4]arene35 (4.1 g, 3.2 mmol) dissolved in THF (60 mL), a solution of N,N-dimethylethanolamine (1.5 mL, 14.9 mmol) in THF (15 mL) was added. The reaction mixture was refluxed for 24 h. After cooling, the suspension was centrifuged at 2933 × g for 5 min. The precipitate was washed with THF (40 mL) and after with acetonitrile (4 × 20 mL) by repeated centrifugation (2933 × g, 5 min) and removal of the solvent. The white precipitate was recovered and dried under vacuum to give a white powder (4.2 g, 80% yield). Compound 1 (MW 1648.2 for C96H168Cl4N4O8) was characterized by NMR spectroscopy, and the spectral data were consistent with those reported in the literature.31 For the synthesis of calix[4]arene derivative 2, a solution of N,N-dimethylethanolamine (47 μL, 0.47 mmol) in THF (0.5 mL) was added to a stirring solution of 25,26,27,28-tetrakis(6bromohexyloxy) calix[4]arene36 (107 mg, 99 μmol) in THF (7 mL). The reaction mixture was refluxed for 24 h. After cooling, the suspension was centrifuged at 2933 × g, 5 min. The precipitate was washed with THF and with acetone by repeated centrifugation (2933 × g, 5 min) and removal of the solvent. The white precipitate was recovered and dried under vacuum to give a white powder (104 mg, 80% yield). 1H NMR (MeOD): δ 1.52 (br m, 16H, CH2), 1.88 (br t, 8H, CH2), 2.00 (t, 8H, J = 6.8 Hz, CH2), 3.15 and 4.45 (AX system, 4H each, J = 13.2 Hz, CH2), 3.22 (s, 24H, CH3), 3.45−3.58 (overlapped, 16H, CH2N), 3.93 (t, 8H, J = 6.8 Hz, OCH2), 4.02 (br t, 8H, CH2OH), 6.54 (t, 4H, J = 7.0 Hz, ArH), 6.60 (d, 8H, J = 7.0 Hz, ArH). 13C NMR (MeOD): δ 22.5, 25.5, 26.1, 29.8, 30.5, 34.8, 50.4, 50.8, 65.2, 73.3 (t), 121.6, 127.4, 127.8 (d), 134.1, 156.2 (s). MW 1313.2 for C64H104Br4N4O4. 1 H NMR (400.13 MHz) and 13C NMR (100.62 MHz) spectra were acquired on a Bruker Avance 400 spectrometer. Chemical shifts (δ) are expressed in parts per million (ppm) and reported relative to the residual proton solvent peak; coupling constant (J) values are given in Hz. 2.3. Preparation of Calix−Cur Nanoassembly. Calix[4]arene 1 (20.8 mg) was dissolved, by manual shaking at rt, in 20 mL of 10 mM PBS (pH 7.4) to give a colloidal solution. Curcumin excess was then added (1:4 molar ratio); the mixture was sonicated for 15 min and shaken at 300 rpm, 25 °C for 3 days. Sonication was performed in a Ultrasonic cleaner 600TH, frequency 45 kHz, power 1200 W, 20−27 °C. After centrifugation at 2933 × g, 30 min, the supernatant was recovered and filtered through a 0.2 μm GHP filter (Acrodisc) to give a clear red-orange colloidal solution. 2.4. Physicochemical Characterization of Calix−Cur. All the characterization was performed on a Calix−Cur sample containing 1 mg/mL calix[4]arene 1 and 0.1 mg/mL curcumin. Size, polydispersity index, and zeta potential of Calix−Cur assemblies were determined by dynamic light scattering (DLS) on a ZetaSizer NanoZS90 Malvern Instrument (UK), equipped with a 633 nm laser, at the scattering angle of 90° and 25 °C temperature. Each measurement was performed three times. The morphology of Calix−Cur was analyzed under a transmission electron microscope (TEM, JEOL, Japan) using an accelerating voltage of 200 kV, at room temperature. The unstained specimens were prepared by placing a drop of Calix− Cur on copper TEM grids coated with a thin amorphous carbon film. The grids were dried in air, and the dried specimens were examined. UV−vis and fluorescence spectra were recorded on an Agilent Technologies 8453 UV−vis spectrophotometer and Horiba-Jobin-Yvon Fluoromax-3 fluorescence spectrometer

structured delivery systems is proving to be a promising strategy for the topical administration of a drug in the eye,14 including curcumin.15−20 Cationic nanocarriers are particularly attractive as drug delivery systems in ophthalmology. They by a large contact surface and electrostatic interactions with the negative charged ocular surface, can enhance adhesion, spreading, and residence time of a drug on the ocular surface and improve the penetration across cornea and sclera.21 Calix[n]arenes are polyphenolic macrocycles that have gained great appeal in supramolecular chemistry. Due to their synthetic versatility and host−guest properties, calix[n]arenes have found a notable diversity of applications ranging from material science to biomedical field.22 The opportune functionalization of the calixarene skeleton has provided low cytotoxic and immunogenic derivatives active as drugs23,24 but also amphiphilic structures self-assembling in well-defined supramolecular architectures suitable as nanocarriers for drug delivery.25−27 To the best of our knowledge, differently from other molecular scaffolds such as polymers28 and cyclodextrins,29,30 never has a calix[n]arene platform been investigated for applications in ophthalmology. The aim of this study was to demonstrate the potential of an amphiphilic calix[4]arene derivative (1) self-assembling in welldefined micellar structures,31−34 as a novel nanocarrier for delivering curcumin in the eye. To this end, we investigated the capability of the calix[4]arene 1 nanoassembly to enhance solubility and chemical stability of curcumin and evaluated the anti-inflammatory effects of the nanoentrapped curcumin compared to curcumin and calix[4]arene 1 singularly administrated, in in vitro and in vivo experiments. In particular, the effects on pro-inflammatory markers as IkB-α, NF-κB p65, COX2, iNOS, and nitrite levels were tested in J774A.1 macrophages subjected to LPS-induced oxidative stress, whereas recovery of inflammation signs, reduction of nucleophile infiltration, ICAM-1, nitrotyrosine, and protein and VEGF levels were analyzed in a model of rat LPS-induced uveitis.

2. MATERIALS AND METHODS 2.1. Materials. Curcumin and all the other chemicals were purchased from Sigma-Aldrich (Milan, Italy) and used without purification. Solvents were of analytical or high performance liquid chromatography (HPLC) grade. Specific primary antibody anti-iNOS (1:500) was purchased form BD Trasduction (CA, USA), anti-COX2 (1:500) from Cayman Chemical (MI, USA), anti-IκBα (1:500), anti-ICAM-1 (1:500), antinitrotyrosine, and anti-VEGF (1:500) from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-NFκB p65 (1:500) from Cell Signaling (Frankfurt, Germany). Bicinchoninic acid protein assay kit was purchased form Pierce (Rockford, IL, USA). Biotin and avidin were purchased from Vector Laboratories (Burlingame, CA, USA). Peroxidaseconjugated bovine anti mouse IgG secondary antibody and peroxidase-conjugated goat anti rabbit IgG (1:2000) were purchased from Jackson Immunoresearch (West Grove, PA). Antibody β-actin (1:500) was purchased from Santa Cruz Biotechnology. ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Salmonella typhimurium was purchased from Sigma-Aldrich (St Louis, MO, USA). 2.2. Syntheses and Characterization of Calix[4]arene Derivatives 1 and 2. The synthesis of calix[4]arene derivative 1 was performed by adapting the procedure reported in ref 31. Briefly, to a stirring solution of tetrachloromethyl-O-dodecyl B


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from Harlan Nossan, Italy. All the animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 2.6. Cytotoxicity. J774A.1 cells (3 × 104 cells) were plated in a volume of 150 μL in 96-well plates and treated with increasing concentrations of calix[4]arene 1, Calix−Cur, and curcumin, for 24 h. Curcumin was applied as a 1% DMSO solution. J774A.1 cell line cultures were divided into the following groups: (a) control group, cells cultured with normal medium; (b) control + vehicle, cells with 1% DMSO; (c) control + curcumin, cells with curcumin solubilized in DMSO at different concentrations 0.05, 0.0025, and 0.00125 mg/mL for 24 h; (d) control + calix[4]arene, cells with calix[4]arene at different concentrations 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015 mg/mL for 24 h; (e) control + Calix−Cur, cells with Calix− Cur at different concentrations, referring to the concentration of the calix[4]arene in Calix−Cur, 0.5, 0.25, 0.125, 0.06, 0.03, and 0.015 mg/mL for 24 h. At 24 h viability of cell cultures was assessed by using a mitochondria-dependent dye for live cells (tetrazolium dye; MTT). Cultures were incubated at 37 °C with MTT (0.2 mg/mL) for 1 h. Culture medium was removed by aspiration, and the cells were lysed with DMSO. The extent of reduction of MTT to formazan within cells was quantified by the measurement of optical density at 550 nm (OD550) with the microplate reader.38 Moreover, J774A.1 cells were also pretreated for 2 h with the increasing concentrations of curcumin, calix[4]arene 1, and Calix−Cur and stimulated with LPS (10 μg/mL). At 24 h cell viability was assessed by MTT assay as described before. 2.7. Protein Concentrations: Western Blot Analysis. J774A.1 cells cultures were treated with a lysis buffer containing 20 mM Tris-HCl at pH 7.5, 10 mM NaF, 150 μL NaCl, 1% Nonident P-40, and protease inhibitors. After 1 h, cell lysates were obtained by centrifugation at 15294 × g, for 15 min at 4 °C. Protein concentrations were estimated by the Bio-Rad protein assay using bovine serum albumin as standard. Briefly, samples were heated to 100 °C for 5 min, and equal amounts of protein were separated on 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Specific primary antibody antiiNOS, anti-COX2, anti-IκBα, and anti-NFκB p65 were mixed in 1 × PBS, 5% w/v nonfat dried milk, and 0.1% Tween-20 (PMT) and incubated at 4 °C overnight. After, membranes were incubated with peroxidase-conjugated bovine anti mouse IgG secondary antibody or peroxidase-conjugated goat anti rabbit IgG (1:2000) for 1 h at room temperature. To ascertain that blots were loaded with equal amounts of protein lysates, they were also incubated in the presence of the antibody β-actin (1:500). Signals were detected with enhanced chemiluminescence detection system reagent according to manufacturer’s instructions. The relative expression of the protein bands was quantified by densitometry with Gel Logic 2200 PRO software and standardized to β-actin levels. Images of blot signals (8 bit/ 600 dpi resolution) were imported to analysis software (Image Quant TL, v2003). A preparation of commercially available molecular weight markers (proteins from 10 to 250 kDa) was used to define molecular weight and as reference concentrations. 2.8. Measurement of Nitrite Levels. Total nitrite levels, as an indicator of nitric oxide (NO) synthesis, were measured in the supernatant. Briefly, the nitrate in the medium was reduced to nitrite by incubation with nitrate reductase (670 mU/mL) and β-nicotinamide adenine dinucleotide 3-phosphate (160 mM) at room temperature for 3 h. The total nitrite

(λexc 420 nm, slit 5), respectively. To record the UV−vis and fluorescence spectra of Calix−Cur, an aliquot of the colloidal solution was diluted with 10 mM PBS to a curcumin concentration of 15 × 10−6 and 13 × 10−6 M, respectively. To calculate the loading capacity %, the amount of curcumin in Calix−Cur was determined by using a Dionex HPLC system (P680 pump, ASI-100 autosampler, UVD170U detector, TCC100 temperature-controlled column compartment), and Phenomenex Luna 5 μm C18 reverse-phase column (250 × 4.6 mm). Eluents A, CH3CN; B, 0.25% AcOH; gradient, A from 40% to 76%, 18 min, flow 1 mL/min, T = 48 °C, λ = 425 nm. Calix−Cur (50 μL) was diluted with 550 μL of CH3CN, and 50 μL of sample was injected. The amount of calix[4]arene 1 was calculated by UV−vis spectroscopy. In brief, 10 μL of the colloidal solution were diluted with 2 mL of methanol and the amount of calix[4]arene 1 was measured by the absorbance at 210 nm, corrected from the small curcumin absorbance and compared to calibration curve. Loading capacity % (LC%) was calculated applying the following formula: LC(%) =

mass of the loaded drug × 100 mass of the drug‐loaded nanoparticle

The phase-solubility study was performed by the method reported by Higuchi and Connors.37 Curcumin (2.5 mg) was added to 10 mM PBS (500 μL, pH 7.4) containing increasing concentrations of calix[4]arene 1 (0.01−1 mM). The suspensions were sonicated and shaken at 25 °C, at 300 rpm for 2 days, then centrifuged at 2933 × g, 30 min. Opportune amounts of sample (400−25 μL) were diluted with PBS to a total volume of 400 μL, then 400 μL of acetonitrile was added. After stirring, curcumin in the samples was quantified by UV− vis spectroscopy (λ = 425 nm) in relation with the corresponding calibration plot. The phase-solubility diagram was constructed by plotting the concentrations of solubilized curcumin against the calix[4]arene 1 concentrations. To evaluate the stability of curcumin (0.1 mg/mL) in Calix− Cur, the same amount of curcumin was dissolved in 30% ethanol/PBS mixture. The amount of free curcumin and entrapped curcumin was monitored at 37 °C, at different time intervals by HPLC in triplicate. Viscosity of the Calix−Cur colloidal solution (1.35 mg/mL calix[4]arene 1 and 0.135 mg/mL curcumin) used for in vivo experiments was determined to be 3.0 mPa·s by using a Haake RS-600 rheometer, thermocryostat DC30K10 set at 25 °C, cone C60/1 Ti, plate MP60, at a shear rate of 150 s−1. To evaluate the stability of Calix−Cur to the freeze-drying process, the colloidal solution (1 mg of calix[4]arene 1 entrapping 0.1 mg/mL curcumin) was diluted with water (1:3 v/v) and subjected to freeze-drying by using a Lyoquest85, Telstar (MI, Italy). The lyophilized was rehydrated, by addition of 1 mL of pure water, and analyzed by DLS and HPLC to evaluate size, PDI, and curcumin amount. The same analyses were performed on a lyophilized sample stored at 25 °C and rehydrated after 3 months. 2.5. Cells and Animals. Monocyte/macrophage (J774A.1) cell-line were purchased from ATTC. J774A.1 cell-line was grown in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 5% CO2/95% air. Male Lewis rats (160−180 g) were obtained C


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deparaffinized with xylene, stained with May−Grünwald− Giemsa, and studied by light microscopy. 2.12. Localization of ICAM-1, Nitrotyrosine, and VEGF by Immunohistochemistry. After 16 or 72 h from the injection of LPS, iris−ciliary bodies were collected and fixed in 10% (w/v) PBS-buffered formaldehyde, and 7 μm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the sections in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Tissue sections were incubated overnight with purified antimouse ICAM-1 (1:500 in PBS, w/v), antinitrotyrosine antibody (1:500 in PBS, w/v), or anti-VEGF antibody (1:500 in PBS, w/v). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was determined with a biotin-conjugated goat antirabbit IgG and avidin−biotin peroxidase complex. The counterstain was developed with diaminobenzidine (brown color) and nuclear fast red (red background). Positive staining (brown color) was found in the sections, indicating that the immunoreactions were positive. To verify the binding specificity for ICAM-1 and nitrotyrosine, some sections were also incubated with primary antibody only (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections. The immunohistochemical pictures were collected by Zeiss microscope using Axio Vision software. For graphic representation of densitometric analysis, we measured the intensity of positive staining (brown staining) by computerassisted color image analysis (Leica QWin V3, UK). The percentage area of immunoreactivity (determined by the number of positive pixels) was expressed as percent of total tissue area (red staining). Replicates for each experimental condition and histochemical staining were obtained from each mouse in each experimental group. 2.13. Statistical Analysis. All values are expressed as mean ± standard error of the mean (SEM) of N observations. The results were analyzed by one-way ANOVA followed by a Kruskall−Wallis (Dunns test) posthoc test for multiple comparisons. P < 0.0001 was considered as significant. The figures from histological and immunohistochemical experiments are representative of at least three experiments (five slides for each eye from different animals) performed on different days.

concentration was then measured using the Griess reaction by adding 100 μL of Griess reagent, a 1:1 mixture of 0.1% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride in H2O and 1% (w/v) sulfanilamide in 5% (v/v) concentrated H3PO4, to a 100 μL sample. OD550 was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Tecan, Männedorf, Switzerland). Nitrite concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrite prepared in H2O. 2.9. Endotoxin Induced Uveitis (EIU) in Rats. EIU was induced by footpad injection of 200 μg of lipopolysaccharide (LPS) from Salmonella typhimurium that had been diluted in 0.2 mL of PBS, pH 7.4. Different groups of rats were created for the experimental design: (a) Sham group, rats that were not injected by LPS; (b) LPS group, rats received an injection of LPS (200 μg) in the footpad; (c) Curcumin dissolved in 1% DMSO + LPS group; (d) calix[4]arene + LPS group; (e) Calix−Cur + LPS group, rats received a pretreatment of curcumin, calix[4]arene 1, or Calix−Cur topically applied to both eyes, for 3 days and 1 h before LPS injection and 7 h after. The dosage was one drop per day of solutions containing calix[4]arene 1.35 mg/mL and/or curcumin 0.135 mg/mL. Sixteen hours after uveitis induction, the rats were killed and aqueous humor and iris−ciliary body of right and left eyes were collected. Other groups of rats were treated as abovementioned, but the treatment was continued (one drop per day) up to 72 h. 2.10. Clinical Grading of Uveitis and Protein Concentration. After the induction of uveitis at 16 or 72 h, the eyes were examined by slit lamp by an observer unaware of the treatment. Uveitis in each eye was graded according to a previously reported scoring system:39 (0) no inflammatory reaction; (1) discrete dilatation of the iris and conjunctival vessels; (2) moderate dilatation of the iris and conjunctival vessels; (3) intense iridal hyperemia with flare in anterior chamber; and (4) same clinical signs as grade 3 plus the presence of fibrinous exudate in the pupillary area with intense flare in the anterior chamber. No signs of uveitis were observed in the animals at the beginning of each experiment. Immediately after the slit lamp examination, the rats were killed and the aqueous humor was collected from both eyes by an anterior chamber puncture (15−20 μL per rat) using a 30gauge needle under a surgical microscope. The aqueous humor from each eye of five rats from the same group was then pooled and diluted 10 times with PBS (pH 7.4). The total protein concentration in the aqueous humor samples was measured with a bicinchoninic acid protein assay kit. The levels of TNF-α in the aqueous humor were assessed with a commercially available ELISA kit. Intra- and interassay coefficients of variations were lower than 10%. The aqueous humor samples were in all cases stored in ice water until tested on the day of sample collection. 2.11. Histological Examination. A separate set of rats (four rats for each groups) treated in the same way as described earlier were used for the histological study. For microscopic histological evaluation, rat eyes were collected at 16 or 72 h after LPS induction and fixed in a solution containing 0.1% glutaraldehyde (25%) and 4% paraformaldehyde for 24 h at room temperature, dehydrated by graded ethanol, and embedded in paraffin (Paraplast; Sherwood Medical, Mahwah, NJ, USA). Tissue sections (7 mm), cut near the optic nerve (obtained from eight eyes of four rats in each group), were

3. RESULTS 3.1. Physicochemical Characterization of Calix−Cur. Calix−Cur was assembled by using a simple and reproducible phase solubility method, consisting of shaking an excess of curcumin in a colloidal solution of calix[4]arene derivative 1 (Figure 1), which we previously reported spontaneously selfassemble in well-defined and stable micellar nanoaggregates in mimicking physiological conditions (10 mM PBS, pH 7.4).32,33 Size and morphology of Calix−Cur were derived from DLS measurements and TEM images (Figure 2). The nanoaggregates showed a mean hydrodynamic diameter of 82 nm (Z average) with polydispersity index of 0.30, surface zeta potential of +24.3 mV, spherical shape, and micellar structure. The formation of micellar aggregates was in good D


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(Figure 3a,b) and the shift of the emission maximum from 610 to 540 nm, typical of free curcumin, when the Calix−Cur was disrupted by acetonitrile addition (Figure 3b), evidenced that the enhanced solubility of curcumin is associated to the entrapment in the calix[4]arene nanoassembly. This was corroborated by the absence of significant variation of the surface zeta potential of the calix[4]arene nanoaggregate empty and loaded with curcumin, which ruled out significant interactions of curcumin with the surface of the calix[4]arene nanoassembly. Since the observed red-shifts are indicative of curcumin in anionic form, to better understand the role played by the electrostatic interactions between anionic curcumin and cationic calix[4]arene 1 in curcumin solubilization, we purposely synthesized cationic calix[4]arene derivative 2, bearing four choline groups tethered at the calix[4]arene lower rim (Figure 1). When curcumin was dissolved in the presence of calix[4]arene 2, which, differently from calix[4]arene 1, did not assemble into well-defined nanoaggregates in water solution, no significant solubilization of curcumin was observed. This result clearly demonstrated that the entrapment of hydrophobic curcumin in the hydrophobic environment created by the calix[4]arene nanoaggregate is crucial for the solubilization process. The amount of calix[4]arene 1 and curcumin in Calix−Cur, measured by UV−vis spectrophotometry and HPLC analyses, provided a drug loading capacity of 9% (0.1 mg/mL curcumin per 1 mg/mL calix[4]arene 1). Since the solubility of curcumin was reported to be 11 ng/mL,42 the entrapment in the calix[4]arene nanoaggregate improved the curcumin solubility more than 9000 times. A phase solubility study by using Higuchi and Connors method37 showed that the amount of solubilized curcumin exhibited a linear relationship with the calix[4]arene concentration (Figure 4a). The encapsulation of curcumin in the calix[4]arene nanoaggregate protected curcumin from rapid degradation reported to be 90% at 25 μM concentration in phosphate buffer, 37 °C, 10 min incubation.43 To compare samples at the same concentration corresponding to curcumin loaded in 1 mg/mL of calix[4]arene, 0.1 mg/mL of curcumin was solubilized in PBS containing 30% ethanol. Despite the presence of ethanol, known to reduce degradation of curcumin in PBS medium,44

Figure 1. Structures of calix[4]arene derivatives 1 and 2.

Figure 2. (a) Intensity weighted hydrodynamic diameter distribution and (b) TEM image of Calix−Cur (1−0.1 mg/mL, 10 mM PBS, pH 7.4).

agreement with the cone-shaped geometry and high curvature of the calix[4]arene 1 structure.40,41 The calix[4]arene nanoassembly enhanced the curcumin solubility in PBS medium, as evidenced by the increased fluorescence intensity of Calix−Cur compared to free curcumin (Figure 3b). Red-shift of the absorption (λ 450 nm) and emission (λ 610 nm) maxima of curcumin in Calix−Cur spectra

Figure 3. (a) UV−vis spectrum of Calix−Cur in 10 mM PBS, pH 7.4, sample diluted to curcumin concentration 15 × 10−6 M. (b) Fluorescence spectra (λexc 420 nm, 10 mM PBS, pH 7.4) of curcumin (dashed line) and Calix−Cur diluted to curcumin concentration 13 × 10−6 M (filled line) and after 1:1 dilution with acetonitrile (dotted line). E


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Figure 4. (a) Linear plot (R2 = 0.997) of phase solubility of curcumin related to calix[4]arene 1 concentration, 25 °C. (b) Stability of curcumin (0.1 mg/mL) in 30% ethanol/PBS and in Calix−Cur (1−0.1 mg/mL) at 37 °C and different incubation time.

Figure 5. Cell viability study of curcumin, calix[4]arene 1, and Calix−Cur at different concentrations performed on J774A.1 cells (a) and LPSstimulated J774A.1 cells (b).

Figure 6. Analysis of IκB-α (a,a1) and NF-κB p65 factor (b,b1) levels in control cells and in LPS stimulated J774A.1 cells treated with curcumin, calix[4]arene 1, or Calix−Cur. The densitometric expression of protein bands was normalized to β-actin level. Western blot analyses are representative of three different gels obtained by dividing the number of samples for each experimental group on different days.

F


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Figure 7. Levels of iNOS (a,a1) and COX2 (b,b1) and NO2− in LPS stimulated J774A.1 cells treated with curcumin, calix[4]arene 1, or Calix−Cur. The densitometric expression of protein bands was normalized to β-actin level. Western blot analyses are representative of three different gels obtained by dividing the number of samples for each experimental group on different days.

κB activation subsequently down-regulates COX2 and iNOS expression.47 By comparing the effect of Calix−Cur, calix[4]arene 1, and curcumin on IκB-α level, NF-κB p65 nuclear translocation, COX2 and iNOS expression, and nitrosative stress in LPSstimulated J774A.1 cells, we observed that the Calix−Cur elicited an enhanced anti-inflammatory activity. In particular, Western blot analysis showed a basal expression of IκB-α in the cytoplasmic fraction of control cells and significant decrease of IκB-α levels (Figure 6a and a1) and increase of the nuclear translocation of NF-κB p65 after LPS stimulation (Figure 6b and b1). Pretreatment with Calix−Cur significantly inhibited the LPS-induced degradation of IκB-α, maintaining its expression at levels comparable to value of the control group (Figure 6a,a1), and reduced the nuclear translocation of NF-κB p65 (Figure 6b,b1). No considerable effect was instead observed after the treatment with curcumin or calix[4]arene singularly (Figure 6a,a1; b,b1). In agreement with the reduction of the nuclear translocation of NF-κB p65, pretreatment with Calix−Cur significantly reduced the expression of both iNOS (Figure 7a,a1) and COX2 (Figure 7b,b1). Also calix[4]arene 1 administered alone reduced the levels of iNOS and COX2 expression, whereas no significant effect was observed with curcumin administered alone (Figure 7a,a1; b,b1). A different behavior was observed on the levels of nitrite released into the culture medium after LPS-stimulated nitrosative stress. The untreated control group released low levels of NO2−, instead LPS stimulation significantly enhanced NO2− production. The pretreatment with either Calix−Cur or

free curcumin at 37 °C degraded faster than nanoencapsulated curcumin. In the presence of calix[4]arene more than 95% and 72% of curcumin was kept after 24 h and 5 days incubation, respectively (Figure 4b). By data analysis (see SI) half-life of 22 and 162 h, were estimated for free and nanoentrapped curcumin, respectively. The capability of the cationic calix[4]arene nanoassembly to preserve curcumin from degradation is ascribable to the stabilization of the readily degradable curcumin anions as reported for other cationic micelles.45,46 3.2. Cell Viability. The cytotoxicity of calix[4]arene 1, Calix−Cur, or curcumin (range 0.5−0.015 mg/mL) was tested in vitro on J774A.1 cell lines by MTT assay. The highest concentration of all the three compounds induced a mortality of 60% in J774A.1 cell line (Figure 5a). The cytotoxicity of curcumin, Calix−Cur, or calix[4]arene 1 was also tested on J774A.1 cells insulated by lipopolysaccharide (LPS, 10 μg/mL, 24 h) (Figure 5b). The LPS stimulation significantly reduced the cell viability compared to the control group; noteworthy pretreatment with curcumin, calix[4]arene 1 and Calix−Cur at the lowest concentrations for 2 h reduced cell death compared to the LPS-stimulated group (Figure 5b). 3.3. In Vitro Anti-Inflammatory Effects. Curcumin is a pleiotropic agent for which over 100 different molecular targets have been identified. Anti-inflammatory effect is one of the best-explored actions of curcumin.6 Curcumin is thought to suppress the nuclear activation of factor-kappa B (NF-κB) and proinflammatory gene expression by blocking phosphorylation of inhibitory factor I-kappa B kinase (I-κB). Suppression of NFG


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Figure 8. Clinical inflammation score (a) and proteins levels (b) detected in aqueous humor in LPS injected animals and in animals treated with calix[4]arene 1, curcumin, or Calix−Cur. P < 0.0001.

Figure 9. Histological analysis of iris-ciliary body tissues from sham-treated rats (a,a′) and LPS injected animals before (b,b′) and after treatment with calix[4]arene 1 (c,c′), curcumin (d,d′), or Calix−Cur (e,e′). Images are illustrative of at least three independent experiments. Values are mean ± SEM of 10 animals for each group. P < 0.0001.

calix[4]arene 1 alone significantly reduced the levels of NO2− to those of the control group (Figure 7c). 3.4. In Vivo Anti-Inflammatory Effects on LPS-Induced Uveitis. To explore the potential of Calix−Cur in an in vivo model of LPS-induced uveitis, curcumin, calix[4]arene 1, and Calix−Cur were topically administrated in the eyes of rats. The eyes of the prepost treated rats were analyzed 16 h after the LPS induction, corresponding to the expected peak of disease, to evaluate clinical inflammation score and protein levels in aqueous humor (Figure 8), grade of inflammation by histological analysis (Figure 9), and localization of ICAM-1 (Figure 10), nitrotyrosine (Figure 11), and VEGF (Figure 12) by immunohistochemical analyses of tissues from iris-ciliary body. The same results were obtained when the treatment was continued for 72 h after LPS induction (data not shown). The treatment with curcumin significantly reduced clinical inflammation score and protein levels increased in aqueous humor of LPS injected rats. However, interestedly, a more

pronounced trend of protection was observed in the rats treated with Calix−Cur and no significant anti-inflammatory effect was observed for calix[4]arene 1 alone (Figure 8a,b). Histological analysis of iris-ciliary body tissues from LPS injected animals showed severe signs of uveitis with reduced cytoplasm, visible nucleus and an important infiltration of neutrophils (Figure 9b,b′; histological score f). Curcumin (Figure 9d,d′) and more effectively Calix−Cur (Figure 9e,e′; histological score f), significantly reduced the uveitis signs, degree of inflammation and neutrophil infiltration, upon to characteristics similar to the sham-treated rats (Figure 9a,a′). Sign of recovery, which did not reach statistical significance, was instead observed in the rats treated with calix[4]arene 1 alone (Figure 9c,c′; histological score f). During inflammatory conditions, adhesion molecules such as ICAM-1 are expressed on endothelium48 and are activated by different cytokines such as TNF-α. In the present study we performed immunohistochemical staining for ICAM-1 that H


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Figure 10. Immunohistochemical staining for ICAM-1 in iris-ciliary body sections from sham-treated rats (a), rats injected with LPS (b), and from animals treated with calix[4]arene 1 (c), curcumin (d), or Calix−Cur (e). Images are illustrative of at least three independent experiments. (f) Densitometric analysis. Values are mean ± SEM of 10 animals for each group. P < 0.001.

Figure 11. Immunohistochemical staining for nitrotyrosine from sham rats (a), rats injected with LPS (b), and from animals treated with calix[4]arene 1 (c), curcumin (d), or Calix−Cur (e). Images are illustrative of at least three independent experiments. (f) Densitometric analysis. Values are mean ± SEM of 10 animals for each group. P < 0.001.

the uvea (Figure 11b, see densitometric analysis f) compared to the sham treated rats (Figure 11a). The analysis showed that the treatment with curcumin or Calix−Cur reduced positive staining for nitrotyrosine (Figure 11d,e; densitometric analysis f), no reduction was instead observed in rats treated with calix[4]arene 1 alone (Figure 11c). Also the vascular endothelial growth factor (VEGF), through the promotion of angiogenesis and vascular permeability, plays an important role in the inflammatory process. The expression of VEGF is intimately linked to that of major cytokines in the inflammatory cascade. We observed that the expression of VEGF significantly increased in rats with LPS-induced uveitis (Figure 12b; densitometric analysis f) and was significantly reduced in the group treated with curcumin or Calix−Cur

perfectly correlated with the histological results and the intensity of cell infiltration. The positive staining for ICAM-1 in iris-ciliary body sections from rats injected with LPS (Figure 10b; densitometric analysis f) compared to the sham treated animals (Figure 10a) significantly reduced in the sections from rats treated with curcumin (Figure 10d) or Calix−Cur (Figure 10e; densitometric analysis f). Also in this case no protection was observed in the group of rats treated with calix[4]arene 1 alone (Figure 10c; densitometric analysis f). It is known that in LPS-induced uveitis both inflammation and oxidative stress contribute to its pathogenesis.49 For this reason, we also performed immunohistochemistry staining for nitrotyrosine considered as an indicator of nitrosative stress. LPS injection increased the positive staining for nitrotyrosine in I


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Figure 12. Immunohistochemical staining for VEGF from sham rats (a), rats injected with LPS (b), and from animals treated with calix[4]arene 1 (c), curcumin (d), or Calix−Cur (e). Images are illustrative of at least three independent experiments. (f) Densitometric analysis. Values are mean ± SEM of 10 animals for each group. P < 0.001.

loading capacity to give a clear colloidal solution (Figure 13) suitable for ocular applications. The curcumin-loaded calix[4]-

(Figure 12d,e; densitometric analysis f), whereas no protective effect was observed in the group of rats treated with calix[4]arene alone (Figure 12c; densitometric analysis f).

4. DISCUSSION The realization of nanocarriers able to enhance solubility and chemical stability of hydrophobic drugs and improve eye surface permanence and penetrability is a topic of interest in ophthalmology where rapid elimination and low penetration are factors limiting the therapeutic efficacy of many drugs administrated as eye drop. Generally, only 1−5% of an applied drug is absorbed in the eye. To give a contribution in the search of innovative and more effective nanocarriers for topical ocular drug delivery, in this work we focused on the potential of the micellar nanoaggregate of the calix[4]arene derivative 1 as a nanocarrier for administrating curcumin to the eye. The amphiphilic calix[4]arene derivative 1, bearing lipid-like dodecyl chains, was selected to take advantage of the choline groups, eligible for ocular applications: (i) choline is an alkoxyamine well tolerated by the eye; (ii) it can cross the cornea via low-affinity facilitated diffusion, and choline transporters are present in the iris;50,51 (iii) choline, covalently tethered to the calix[4]arene wide rim, forms quaternary ammonium salts, which by ensuring a positively charged surface, favor the formation of stable nanoaggregates by electrostatic repulsive forces but also ocular adhesion, spreading, and corneal penetration by electrostatic interactions with the negatively charged ocular surface and mucus.21 Moreover, the presence of quaternary ammonia could confer to the calix[4]arene derivative 1 antibacterial properties useful to achieve a preservative free formulation. As a support, the antibacterial activity of polycationic calix[4]arene derivatives has been demonstrated as well as their low cytotoxicity on eukaryotic cells.52 Interestedly, the low cytotoxicity showed by nanoformulated benzalkonium or cetalkonium chlorides on corneal cells has revaluated the quaternary ammonium salts for ophthalmic formulations.21 The micellar nanoaggregate of calix[4]arene 1, by a simple and reproducible protocol, entrapped curcumin with good drug

Figure 13. Photo showing the clearness of Calix−Cur formulation compared with distilled deionized water.

arene micelles showed uniform nanosize and stability for months at room temperature (see Table S1 in SI). The surface potential of +24.3 mV, known to stabilize cationic nanoaggregates by electrostatic repulsive forces,21 explained the notable stability of Calix−Cur, which was also resistant to the lyophilization process performed in the absence of cryoprotectants (see Table S1 in SI). Hydrophobic interactions are crucial for the tremendous enhancement of curcumin solubilization (by 9000-fold), whereas the stabilization of the curcumin anions (by 7.5-fold) by charge-to-charge interactions with the cationic choline groups is involved in the protection of curcumin from rapid degradation in PBS medium. This is an important result since solubility and rapid degradation are factors limiting the clinical success of curcumin and a research challenge yet. Curcumin is a drug of interest in ophthalmology as it has shown beneficial effects on diverse ocular diseases associated with inflammatory phenomena. The entrapment of curcumin in the calix[4]arene nanoassembly enhanced the effects of curcumin on inflammation and oxidative biomarkers. In J



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particular, Calix−Cur reduced IκB-α degradation, NF-κB p65 nuclear translocation, COX2 and iNOS expression, and nitrosative stress on LPS-stimulated J774A.1 macrophages better than free curcumin. The same trend was observed in vivo in rats with LPSinduced uveitis. Anterior uveitis is a condition characterized by inflammation of the uveal tract of the eye, including the iris, and if untreated can result in blurred vision and permanent damage. Although the exact cause of anterior uveitis is not certain, actually the pharmacological treatment aim at decreasing inflammation. The prepost treatment with Calix−Cur topically administrated in the eye reduced clinical inflammation score and protein levels in aqueous humor, and in iris-ciliary body tissues recovered signs of uveitis such as reduced cytoplasm, visible nucleus, and important infiltration of neutrophils better than free curcumin. Calix−Cur also reduced ICAM-1, activated by pro-inflammatory cytokines, and VEGF expression, downregulated by curcumin via the suppression of NF-kB, better than curcumin and calix[4]arene 1 singularly administrated. The higher activity of curcumin entrapped in the amphiphilic calix[4]arene nanoassembly compared to free curcumin can be ascribed to the capability of the calix[4]arene nanoaggregate to protect curcumin from degradation but also to a calix[4]arenemediated cellular uptake. This latter assumption is supported by the known capability of choline and polycationic calix[4]arene derivatives to cross the cellular membranes.53,54 Only in the case of the nitrosative stress in in vitro experiments, calix[4]arene 1 showed a beneficial effect when administrated alone. This behavior, which could agree with the antiantioxidant properties described for choline derivatives55,56 and for an analogous cationic amphiphilic calix[4]arene derivative bearing long alkyl chains,57 needs a more in-depth study. Noteworthy, calix[4]arene 1 nanocarrier can be a curcumin solubilizing agent preferable to DMSO solvent, reported to be toxic also at unexpected low concentrations and therefore not suitable to clinical applications.58

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Grazia M. L. Consoli: 0000-0003-4189-930X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge project PON R&C 02_00355_2964193 (MIUR, Rome) for financial assistance and Prof. E. Caponetti, Prof. D. F. Chillura Martino and Dr. G. Nasillo for TEM image, which was provided by Centro Grandi Apparecchiature-Uni NetLab−Università di Palermo funded by P.O.R. Sicilia 2000−2006, Misura 3.15.

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REFERENCES

(1) Alrawaiq, N. S.; Abdullah, A. A review of antioxidant polyphenol curcumin and its role in detoxification. Int. J. Pharm. Technol. Res. 2014, 6, 280−289. (2) He, Y.; Yue, Y.; Zheng, X.; Zhang, K.; Chen, S.; Du, Z. Curcumin, inflammation, and chronic diseases: How are they linked? Molecules 2015, 20, 9183−9213. (3) Moghadamtousi, S. Z.; Kadir, H. A.; Hassandarvish, P.; Tajik, H.; Abubakar, S.; Zandi, K. A review on antibacterial, antiviral, and antifungal activity of curcumin. BioMed Res. Int. 2014, 2014, 12. (4) Vallianou, N. G.; Evangelopoulos, A.; Schizas, N.; Kazazis, C. Potential anticancer properties and mechanisms of action of curcumin. Anticancer Res. 2015, 35, 645−651. (5) Yadav, V. S.; Mishra, K. P.; Singh, D. P.; Mehrotra, S.; Singh, V. K. Immunomodulatory effects of curcumin. Immunopharmacol. Immunotoxicol. 2005, 27, 485−497. (6) Jurenka, J. S. Anti-inflammatory properties of curcumin, a major constituent of curcuma longa: A review of preclinical and clinical research. Alt. Med. Rev. 2009, 14, 141−153. (7) Pescosolido, N.; Giannotti, R.; Plateroti, A. M.; Pascarella, A.; Nebbioso, M. Curcumin: therapeutical potential in ophthalmology. Planta Med. 2014, 80, 249−254. (8) Lal, B.; Kapoor, A. K.; Asthana, O. P.; Agrawal, P. K.; Prasad, R.; Kumar, P.; Srimal, R. C. Efficacy of curcumin in the management of chronic anterior uveitis. Phytother. Res. 1999, 13, 318−322. (9) Anand, P.; Kunnumakkara, A. B.; Newman, R. A.; Aggarwal, B. B. Bioavailability of curcumin: problems and promises. Mol. Pharmaceutics 2007, 4, 807−818. (10) Sun, M.; Su, X.; Ding, B.; He, X.; Liu, X.; Yu, A.; Lou, H.; Zhai, G. Advances in nanotechnology-based delivery systems for curcumin. Nanomedicine 2012, 7, 1085−1100. (11) Ghalandarlaki, N.; Alizadeh, A. M.; Ashkani-Esfahani, S. Nanotechnology-applied curcumin for different diseases therapy. BioMed Res. Int. 2014, 2014, 23. (12) Marczylo, T. H.; Verschoyle, R. D.; Cooke, D. N.; Morazzoni, P.; Steward, W. P.; Gescher, A. J. Comparison of systemic availability of curcumin with that of curcumin formulated with phosphatidylcholine. Cancer Chemother. Pharmacol. 2007, 60, 171−177. (13) Allegri, P.; Mastromarino, A.; Neri, P. Management of chronic anterior uveitis relapses: efficacy of oral phospholipidic curcumin treatment. Long-term follow-up. Clin. Ophthalmol. 2010, 4, 1201− 1206. (14) Pignatello, R.; Puglisi, G. Nanotechnology in ophthalmic drug delivery: A survey of recent developments and patenting activity. Recent Pat. Nanomed. 2011, 1, 42−54. (15) Anjana, D.; Anitha Nair, K.; Somashekara, N.; Venkata, M.; Sripathy, R.; Yelucheri, R.; Parmar, H.; Upadhyay, R.; Rama Verma, S.; Ramchand, C. N. Development of curcumin based ophthalmic formulation. Am. J. Infect. Dis. 2012, 8, 41−49.

5. CONCLUSIONS For the first time a calix[4]arene-based nanoassembly was investigated as a potential vehicle for ophthalmic drug delivery. The calix[4]arene nanoaggregate solubilized curcumin, prevented its degradation, and enhanced the anti-inflammatory effects of curcumin in vitro and in vivo models of ocular inflammation. Easy and reproducible preparation, clearness, nanosize, low polydispersity index, stability, and biocompatibility are requisites that make the Calix−Cur supramolecular assembly a promising nanotechnological formulation for ophthalmic administration of curcumin. Noteworthy, the here described polycationic calix[4]arene nanoassembly could be a versatile nanocarrier suitable for delivering not only curcumin but also other hydrophobic drugs in the eye. Our findings advise the calix[n]arene macrocycles as a new class of molecular scaffolds for ophthalmic applications.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b01066. NMR data, fitting curves of curcumin decay, stability monitored over time, and Western blot of Cox-2, iNOS, NF-κB, B Actin (PDF) K


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sensitizers by calix[4]arene nanoassemblies. RSC Adv. 2016, 6, 105573−105577. (35) Eggers, P. K.; Becker, T.; Melvin, M. K.; Boulos, R. A.; James, E.; Morellini, N.; Harvey, A. R.; Dunlop, S. A.; Fitzgerald, M.; Stubbse, K. A.; Colin, L.; Raston, C. L. Composite fluorescent vesicles based on ionic and cationic amphiphilic calix[4]arenes. RSC Adv. 2012, 2, 6250−6257. (36) Safa, K. D.; Oskoei, Y. M. Synthesis of new calix[4]arenes bearing silylether groups. Arkivoc 2010, 1−10. (37) Connors, K. A. Binding Constants; Wiley: New York, 1987; pp 261−281. (38) Abe, K.; Matsuki, N. Measurement of cellular 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT. Neurosci. Res. 2000, 4, 325−329. (39) Behar-Cohen, F. F.; Savoldelli, M.; Parel, J. M.; Goureau, O.; Thillaye-Goldenberg, B.; Courtois, Y.; Pouliquen, Y.; de Kozak, Y. Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis. Invest. Ophthalmol. Vis. Sci. 1998, 6, 897−904. (40) Consoli, G. M. L.; Granata, G.; Lo Nigro, R.; Malandrino, G.; Geraci, C. Spontaneous self-assembly of water-soluble nucleotide− calixarene conjugates in small micelles coalescing to microspheres. Langmuir 2008, 24, 6194−6200. (41) Houmadi, S.; Coquiere; Legrand, L.; Faure, M. C.; Goldmann, M.; Reinaud, O.; Remita, S. Architecture-controlled “SMART” calix[6]Arene self-assemblies in aqueous solution. Langmuir 2007, 23, 4849−4855. (42) Dutta, A. K.; Ikiki, E. Novel drug delivery systems to improve bioavailability of curcumin. J. Bioequivalence Bioavailability 2013, 6, 1− 9. (43) Oetari, S.; Sudibyo, M.; Commandeur, J. N.; Samhoedi, R.; Vermeulen, N. P. Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat liver. Biochem. Pharmacol. 1996, 51, 39−45. (44) Zhu, M.; Li, S. The stability of curcumin and drug-loading property of starch microspheres for it. Adv. Biomed. Eng. 2012, 9, 44− 48. (45) Leung, M.; Colangelo, H.; Kee, T. W. Encapsulation of curcumin in cationic micelles suppresses alkaline hydrolysis. Langmuir 2008, 24, 5672−5675. (46) Wang, Z.; Leung, M. H. M.; Kee, T. W.; English, D. S. The role of charge in the surfactant-assisted stabilization of the natural product curcumin. Langmuir 2010, 26, 5520−5526. (47) Surh, Y.-J.; Chun, K. S.; Cha, H. H.; Han, S. S.; Keum, Y.-S.; Park, K.-K.; Lee, S. S. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: downregulation of COX-2 and iNOS through suppression of NF-κB activation. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2001, 480, 243− 268. (48) Roebuck, K. A.; Finnegan, A. Regulation of intercellular adhesion molecule-1 (CD54) gene expression. J. Leukoc. Biol. 1999, 6, 876−888. (49) Hollyfield, J. G.; Bonilha, V. L.; Rayborn, M. E.; Yang, X.; Shadrach, K. G.; Lu, L.; Ufret, R. L.; Salomon, R. G.; Perez, V. L. Oxidative damage-induced inflammation initiates age-related macular degeneration. Nat. Med. 2008, 2, 194−198. (50) Marchi, M.; Hoffman, D. W.; Mussini, I.; Giacobini, E. Development and aging of cholinergic synapses III. Choline uptake in the developing iris of the chick. Dev. Neurosci. 1980, 3, 185−198. (51) Mannermaa, E.; Vellonen, K.-S.; Urtti, A. Drug transport in corneal epithelium and blood−retina barrier: Emerging role of transporters in ocular pharmacokinetics. Adv. Drug Delivery Rev. 2006, 58, 1136−1163. (52) Grare, M.; Mourer, M.; Fontanay, S.; Regnouf-de-Vains, J.-B.; Finance, C.; Duval, R. E. In vitro activity of paraguanidinoethylcalix[4]arene against susceptible and antibiotic-resistant Gram-negative and Gram-positive bacteria. J. Antimicrob. Chemother. 2007, 60, 575−581.

(16) Duan, Y.; Cai, X.; Du, H.; Zhai, G. Novel in situ gel systems based on P123/TPGS mixed micelles and gellan gum for ophthalmic delivery of curcumin. Colloids Surf., B 2015, 128, 322−330. (17) Li, J.; Liub, D.; Tana, G.; Zhinan Zhao, Z.; Yanga, X.; Pana, W. A comparative study on the efficiency of chitosan-N-acetylcysteine, chitosan oligosaccharides or carboxymethyl chitosan surface modified nanostructured lipid carrier for ophthalmic delivery of curcumin. Carbohydr. Polym. 2016, 146, 435−444. (18) Liu, R.; Sun, L.; Fang, S.; Wang, S.; Chen, J.; Xiao, X. Thermosensitive in situ nanogel as ophthalmic delivery system of curcumin: development, characterization, in vitro permeation and in vivo pharmacokinetic studies. J. Pharm. Dev. Technol. 2016, 21, 576− 582. (19) Liu, D.; Li, J.; Pan, H.; He, F.; Liu, Z.; Wu, Q.; Bai, C.; Yu, S.; Yang, X. Potential advantages of a novel chitosan-N-acetylcysteine surface modified nanostructured lipid carrier on the performance of ophthalmic delivery of curcumin. Sci. Rep. 2016, 6, 28796. (20) Zhang, W.-L-I.; Wu, Z.-H.; Du, L.-N.; Wang, Q.; Yu, J.; Jin, Y.G. Curcumin-loaded chitosan liposomes for the treatment of rabbit corneal alkali burns. J. Int. Pharm. Res. 2016, 43, 705−710. (21) Lallemand, F.; Daull, P.; Benita, S.; Buggage, R.; Garrigue, J.-S. Successfully improving ocular drug delivery using the cationic nanoemulsion, Novasorb. J. Drug Delivery 2012, 2012, 16. (22) Neri, P.; Sessler, J. L.; Wang, M.-X. Calixarenes and Beyond; Springer: Berlin-Heidelberg, 2016. (23) Yousaf, A.; Abd Hamid, S.; Bunnori, N. M.; Ishola, A. A. Applications of calixarenes in cancer chemotherapy: facts and perspectives. Drug Des., Dev. Ther. 2015, 9, 2831−2838. (24) Geraci, C.; Consoli, G. M. L.; Granata, G.; Galante, E.; Pappalardo, M.; Spadaro, A. First self-adjuvant multicomponent potential vaccine candidates by tethering of four or eight muc1 antigenic immunodominant PDTRP units on a calixarene platform: Synthesis and biological evaluation. Bioconjugate Chem. 2013, 24, 1710−1720. (25) Zhou, Y.; Li, H.; Yang, Y.-W. Controlled drug delivery systems based on calixarenes. Chin. Chem. Lett. 2015, 26, 825−828. (26) Consoli, G. M. L.; Granata, G.; Geraci, C. Design, synthesis, and drug solubilising property of the first folate−calix[4]arene conjugate. Org. Biomol. Chem. 2011, 9, 6491−6495. (27) Consoli, G. M. L.; Granata, G.; Fragassi, G.; Grossi, M.; Sallese, M.; Geraci, C. Design and synthesis of a multivalent fluorescent folate−calix[4]arene conjugate: cancer cell penetration and intracellular localization. Org. Biomol. Chem. 2015, 13, 3298−3307. (28) Prosperi-Porta, G.; Kedzior, S.; Muirhead, B.; Sheardown, H. Phenylboronic-acid-based polymeric micelles for mucoadhesive anterior segment ocular drug delivery. Biomacromolecules 2016, 17, 1449−1457. (29) Loftsson, T.; Stefánsson, E. Cyclodextrins in ocular drug delivery: theoretical basis with dexamethasone as a sample drug. J. Drug Delivery Sci. Technol. 2007, 17, 3−9. (30) Badr-Eldin, S. M.; Elkheshen, S. A.; Ghorab, M. M. Inclusion complexes of tadalafil with natural and chemically modified betacyclodextrins. I: preparation and in-vitro evaluation. Eur. J. Pharm. Biopharm. 2008, 70, 819−827. (31) Rodik, R. V.; Anthony, A. S.; Kalchenko, V. I.; Melya, Y.; Klymchenko, A. S. Cationic amphiphilic calixarenes to compact DNA into small nanoparticles for gene delivery. New J. Chem. 2015, 39, 1654−1664. (32) Blanco, R.; Bondì, M. L.; Cavallaro, G.; Consoli, G. M. L.; et al. Nanostructured formulations for the delivery of silibinin and other active ingredients for treating ocular diseases. Patent WO/2016/ 055976. (33) Di Bari, I.; Picciotto, R.; Granata, G.; Blanco, A. R.; Consoli, G. M. L.; Sortino, S. A bactericidal calix[4]arene-based nanoconstruct with amplified NO photorelease. Org. Biomol. Chem. 2016, 14, 8047− 8052. (34) Di Bari, I.; Fraix, A.; Picciotto, R.; Blanco, A. R.; Petralia, S.; Conoci, S.; Granata, G.; Consoli, G. M. L.; Sortino, S. Supramolecular activation of the photodynamic properties of porphyrinoid photoL


Article

Molecular Pharmaceutics (53) Lalor, R.; Baillie-Johnson, H.; Redshaw, C.; Matthews, S. E.; Mueller, A. Cellular uptake of a fluorescent calix[4]arene derivative. J. Am. Chem. Soc. 2008, 130, 2892−2893. (54) Mueller, A.; Lalor, R.; Cardaba, C. M.; Matthews, S. E. Stable and sensitive probes for lysosomes: Cell-penetrating fluorescent calix[4]arenes accumulate in acidic vesicles. Cytometry, Part A 2011, 79A, 126−136. (55) Rowley, T. J.; McKinstry, A.; Greenidge, E.; Smith, W.; Flood, P. Antinociceptive and anti-inflammatory effects of choline in a mouse model of postoperative pain. Br. J. Anaesth. 2010, 105, 201−217. (56) Treede, I.; Braun, A.; Sparla, R.; Kühnel, M.; Giese, T.; Turner, J. R.; Anes, E.; Kulaksiz, H.; Füllekrug, J.; Stremmel, W.; Griffiths, G.; Ehehalt, R. Anti-inflammatory effects of phosphatidylcholine. J. Biol. Chem. 2007, 282, 27155−27164. (57) James, E.; Eggers, P. K.; Harvey, A. R.; Dunlop, S. A.; Fitzgerald, M.; Stubbs, K.; Raston, C. L. Antioxidant phospholipid calix[4]arene mimics as micellular delivery systems. Org. Biomol. Chem. 2013, 11, 6108−6112. (58) Galvao, J.; Davis, B.; Tilley, M.; Normando, E.; Duchen, M. R.; Cordeiro, M. F. Unexpected low-dose toxicity of the universal solvent DMSO. FASEB J. 2017, 28, 1317−1330.

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