Photo-Switchable Nanomechanical Systems Comprising a

Article Cite This: ACS Biomater. Sci. Eng. 2018, 4, 184−192

Photo-Switchable Nanomechanical Systems Comprising a Nanocontainer (Montmorillonite) and Light-Driven Molecular Jack (Azobenzene-Imidazolium Ionic Liquids) as Drug Delivery Systems; Synthesis, Characterization, and in Vitro Release Studies Abdolrahim Abbaszad Rafi,†,‡ Nazila Hamidi,† Abdollah Bashir-Hashemi,§ and Mehrdad Mahkam*,† †

Department of Chemistry, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran Department of Chemical Engineering, Mid Sweden University, Sundsvall SE-85170, Sweden § Department of Chemistry, University of Alabama, Tuscaloosa, Alabama 35487, United States ‡

S Supporting Information *

ABSTRACT: In this work, photoresponsive nanomechanical systems were prepared through the intercalation of positively charged photoswitching molecular jacks (azobenzene ionic liquids, Azo-ILs) within montmorillonite (MMT) layers (MMT@Azo-ILs). The study shows that MMT@Azo-ILs are photosensitive and the synthesized molecular jacks could change the basal distances of MMT layers upon UV irradiation. These changes come from changes in the structure and geometry of Azo molecules (i.e., cis−trans isomerization) between clay layers upon UV irradiation. The prepared photoresponsive nanomechanical systems were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), energy-dispersive X-ray spectroscopy (EDX), field-emission scanning electron microscope (FE-SEM). Moreover, the in vitro release studies were performed in different conditions (upon UV irradiation and darkness) in pH 5.8 at 34 ± 1 °C, and it was found that the release rates from drug loaded MMT@Azo-ILs were higher upon UV irradiation in comparison with the release rates in darkness. According to the release studies, the prepared photoresponsive carriers might be considered as an excellent potential candidate in order to formulate smart sunscreens. KEYWORDS: photoresponsive nanomechaical system, drug delivery system, molecular jack, smart sunscreen, azobenzene ionic liquid Clays and clay materials play a significant role in the field of cosmetic and health products.15,16 Montmorillonite (MMT), one of the most frequently used clays, is one of the smectite group, composed of silica tetrahedral layers placed between alumina octahedral layers. The imperfection of the crystal lattice and the isomorphous substitution induce net negative charges on MMT layers that lead to the adsorption and introduction of alkaline earth metal cations within the MMT interlayer gallery. These imperfections and negative charges are responsible for the interactions and cation exchange reactions with organic molecules. The intercalation of organic species within the clay layers provides an appropriate convenient route to develop organic−clay hybrid materials that contain the properties of both the clay host and organic guest in a sole material.17 Furthermore, nontoxicity of MMT has been proved by hematological, biochemical and histopathological analysis in rat models. Therefore, MMT with its unique properties is a

1. INTRODUCTION In recent decades, controlled release systems (CRSs) have attracted researchers’ attention because of their applications especially in drug delivery systems (DDSs). DDSs are designed in order to overcome or reduce the weaknesses and shortcomings of conventional drug formulations (i.e., decrease the side effects of drugs and/or increase their desired effects). Nowadays, to address the drug delivery challenges, not only are typical organic and inorganic systems such as hydrogels,1 micelles,2 nanofibers,3 nanocomposites,4−6 and clays7 employed as drug carriers but novel smart nanomechanical systems are also developed.8−11 Nanomechanical systems that are prepared to encapsulate and then release drug molecules in response to an external trigger or stimulus, are recently have received considerable attentions for their potential applications in DDSs. Nanosystems suitable for this type of operations must comprise both an appropriate nanocontainer and a molecular switching motif or moving components. For instance, photoresponsive materials could release therapeutic compounds (triggered by light) at a specific time and place for phototherapy.12−14 © 2017 American Chemical Society

Received: August 28, 2017 Accepted: November 26, 2017 Published: November 27, 2017 184

DOI: 10.1021/acsbiomaterials.7b00621 ACS Biomater. Sci. Eng. 2018, 4, 184−192

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ACS Biomaterials Science & Engineering

improve and modify the side effects seems essential. As described elsewhere, encapsulation technique could be used to diminish contact between the skin and the sunscreen active ingredients.35,36 The prepared nanosystems (MMT@Azo-IL) can encapsulate the PABA (as a model drug), and then smart and on demand release would occur upon UV radiation; this would minimize the contact time between skin and sunscreen active ingredients.

frequent ingredient as both active and excipient substance in pharmaceutical products.16,18 Besides classic applications, pristine clays have been investigated as DDSs; however, they suffer from some serious drawbacks such as uncontrolled release behavior.7,19 Several approaches like intercalation of organic molecules are employed for modification and improvement of drug release behavior. Intercalation of different positively charged organic molecules (e.g., ILs) between clay layers and their possible application in DDS,20 contaminants removal,21 etc.,22 have been investigated elsewhere. Moreover, further investigations are required in order to make clays more smart and the state of art compounds. Azobenzene (Azo) based molecules have been widely used as a reversible photoresponsive motif and moving components in photoresponsive systems.12,13,23,24 The unique features of Azo systems mainly originate from the isomerization of NN bonds that readily takes place in the presence of a light source. The more stable trans isomer photoisomerizes to the less stable cis isomer and the cis isomer could subsequently convert to the trans isomer either photochemically or thermally. Since the isomers have diverse molecular and structural properties, introducing Azo molecules within a system could lead to a macroscopic change in the system upon photoisomerization (Figure 1).25−30

2. EXPERIMENTAL SECTION 2.1. Materials. 4-Nitrophenol, N-methylimidazole, para-aminobenzoic acid (PABA) 1,2-dichloroethane, 1,4-dichlorobutane, and silver nitrate (AgNO3) were obtained from Merck. Montmorillonite (MMT) was bought from Fulka. All Solvents were purchased from Acros. All other chemicals were chemical grade and used as received without further purification. 2.2. Apparatus Measurements. The FT-IR spectra were recorded using a “Bruker Vector 22” FT-IR spectrometer. Powder samples were put into the KBr pellet and spectra were recorded at room temperature. The 1H NMR spectra were recorded on a Bruker 400 AC spectrometer using tetramethylsilane (TMS) as the internal reference. Powder X-ray diffraction patterns of the samples were recorded on a Bruker AXS model D8 Advance diffractometer using CuKα radiation, with the Bragg angle ranging from 2−70°. The thermogravimetric analysis (TGA) of the samples was measured using Mettler Toledo instrument under N2 with a heating rate of 10 °C min−1. Morphology and surface characteristics of the samples were studied by FE-SEM images recorded by scanning electron microscopy (TESCAN MIRA3). The UV light source was “Philips Hg lamp (8 W), Actinic BL TL TL 8W/10 1FM/10X25CC, made in Poland”; the irradiation power was about 1.1 mW/cm2. The visible light source was an LED lamp (Philips, LED PLC 8.5W 840 2P G24d-3); the irradiation power was about 12 mW/cm2. The release amounts were determined and measured on a Philips PU 8620 ultraviolet spectrophotometer at the absorption maximum (λ max) of the drug. 2.3. Synthesis of Azo Compounds. The schematic illustration for the synthesis of Azo compounds are shown in Figure 2. 2.3.1. 4,4′-Bis(hydroxy)-azobenzene or 4,4′-Diazophenol (AzoOH). The starting Azo compound (Azo-OH) was synthesized through the alkali fusion of 4-nitrophenol. Twenty-five grams of KOH (380 mmol), 5 g of 4-nitrophenol (36 mmol), and water (8 mL) were poured into a flask, then heated to 120 °C using heating mantle and

Figure 1. Changes in molecular structure and length of Azo molecules upon photoisomerization.

This photoisomerization could also occur between solid micro and nano layers and would lead to changes in the distance of nanolayers.31−33 Herein, photoresponsive nanomechanical systems were prepared, a layered natural clay (i.e., MMT) was employed as nanocontainer for the encapsulation of drug molecules and photoswitchable organic molecules (positively charged azobenzene molecules) were used as molecular jacks. For the first time, two new azobenzene-imidazolium ionic liquids (Azo-ILs) with different molecular length were synthesized and intercalated within MMT layers (MMT@Azo-IL). The study shows that MMT@Azo-ILs were photosensitive and the basal distances of MMT layers changed upon UV irradiations, resulting in changes in the drug loading and release rates. The results suggest that the prepared nanomechanical systems can be considered as photoresponsive drug carriers and/or as an excellent potential candidate to produce smart sunscreens. Sunscreens have some known and unknown undesired side effects that can restrict their usages; for example, paraaminobenzoic acid (PABA) was one of the most widely used sunscreen ingredients; however, its usage has been limited due to adverse dermatological effects.34 On the other hand, dermatologists strongly advise people against exposure to solar radiations unless they wear sunscreens. Regarding all these issues, preparing some new sunscreens or systems that can

Figure 2. Synthesis of Azo-ILs. Reagents and conditions: (a) KOH, 200 °C; (b) NaOH (aq), RCl2 (C4H8Cl2 and C2H4Cl2), 70−80 °C, reflux, 72 h; (c) N-methylimidazol, reflux, 72 h, 80 °C. 185


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Azo-OC4H8Im+: mp:187 °C, and decomp.: 287 °C (Figure S3); FTIR (KBr, cm−1): 3074 (aromatic −CH), 2829 (aliphatic −CH), 1498 (Ar CC), 1594 (NN), 1253 (C−O), 1145 (C−N), 850 (para substituted aromatic rings); 1H NMR (400 MHz, DMSO-d6): δppm 1.7 (4H, quintet, −O−CH2−CH2−CH2−CH2−Im), 1.9 (4H, quintet, −O−CH2−CH2-CH2−CH2−Im), 3.8 (6H, s, Im ring, −N−CH3), 4.1 (4H, t, −O−CH2−CH2−CH2−CH2−Im), 4.2 (4H, t, −O−CH2− CH2−CH2−CH2−Im), 7.1 (4H, d, Ar−H), 7.8 (8H, m, Im ring, −N− CH−CH−N− and Ar−H), 9.2 (2H, s, Im ring, −N−CH−N−). 13 C NMR (400 MHz, DMSO-d6): δppm 35.7 (Im ring, -N−CH3), 25.2 (−O−CH2−CH2−CH2−CH2−Im), 26.2 (−O−CH2−CH2− CH2 −CH2 −Im), 48.4 (−O−CH2−CH2−CH2−CH2−Im), 67.2 (−O−CH 2 −CH 2 −CH 2 −CH 2 −Im), 114 (Ar ring, −C*−C− O−),122−123 (Im ring, −N−C−C−N−), 124 (Ar ring, −N−C− C−), 136 (Ar ring, −N−C−), 146 (Ar ring, −C−O−), 160 (Im ring, −N−CH−N−). 2.4. Preparation of MMT@Azo-IL (Intercalation of Azo-ILs between MMT Layers). The MMT@Azo-IL hybrid systems were prepared by the conventional ion-exchange reaction. Briefly, MMT (2 g) was dispersed in a water/ethanol mixture (water 100 mL + ethanol 60 mL) via magnetically stirring for 1 h, and then in order to obtain a bubble-free, well-dispersed mixture, it was ultrasonicated for 30 min. The Azo-IL (Azo-OC2H4Im+ or Azo-OC4H8Im+) was dissolved in 60 mL of ethanol and added dropwise to the MMT mixture. The reaction was refluxed and continued at 70 °C in darkness for 3 days. The precipitates were centrifuged and washed several times with water and ethanol until no Azo-ILs leached out and negative AgNO3 test was observed. The yellow/orange precipitates dried under vacuum for 3 days and kept in darkness. 2.5. Spectroscopic Characterization and Photoisomerization Kinetics. Spectroscopic characterization and photoisomerization studies of the synthesized Azo compounds were performed by UV/ vis absorption spectroscopy. To do so, 3 mL of a low concentrated Azo compound solution in ethanol (1 × 10−4 mol/L) was irradiated at 365 nm (in different durations) and then the UV/vis spectra of the solution was recorded at the wavelength of 250−500 nm (trans to cis isomerization). To study the cis to trans isomerization, first we irradiated the Azo solution at 365 nm until it reached the photo stationary state (PSS), and afterward it was irradiated by Vis light and then the spectra were recorded. The kinetics of the photoisomerizations were assessed by eq 1

left to stand for 60 min. When the temperature slowly rose to 190− 200 °C, the mixture vigorously started to give a brown viscous bubbling liquid. When the bubbling finished, the mixture was cooled down to room temperature and dissolved in water. The obtained red solution was acidified via concentrated HCl (pH 3) and then extracted with diethyl ether. The combined organic phases were dried over Mg2SO4 and filtered, then diethyl ether was evaporated using a rotary evaporator. For further purification, the residues were recrystallized from 50% ethanol/water (v/v) to obtain yellow/orange crystals of compound (Azo−OH). mp: 215−218 °C. FT-IR (KBr, cm−1): 3457 (−OH), 3199 (C−H, benzene aromatic ring), 1506 (ring skeletal vibrations), 1590 (NN), 1250 (C−O), 844 (para-substituted aromatic rings).37 2.3.2. 4,4′-Bis(2-chloroethoxy)azobenzene (Azo-OC2H4Cl) and 4,4′-Bis(4-chlorobutoxy)azobenzene (Azo-OC4H8Cl). First, 0.7 g of Azo-OH (3.2 mmol) was dispersed in 4 mL of distilled water for 35 min, then a solution of NaOH (about 7 mmol in 4 mL of water) was added dropwise to the mixture and stirred for 90 min. Second, the prepared solution was added gradually and dropwise to an excess amount of 1,2-dichloroethane (or 1,4-Dichlorobutane) and a catalytic amount of KI solution in ethanol and the reaction was refluxed at 70− 80 °C for 3 days. Finally, the crude products were isolated and purified via silica gel chromatography [n-hexane: dichloromethane: methanol 20:20:1 (v), for Azo-OC2H4Cl; and n-hexane: dichloromethane 1:2 (v), for Azo-OC4H8Cl]. Azo-OC2H4Cl. mp: 153 °C, and decomp.: 300 °C (Figure S1); FTIR (KBr, cm−1): 3080 (aromatic −CH), 2930 (aliphatic −CH), 1509 (ring skeletal vibrations), 1593 (NN), 1258 and 1034 (C−O), 843 (para substituted aromatic rings), 654 (C−Cl); 1H NMR (400 MHz, CDC13): δppm 3.8 (4H, t, −CH2−Cl), 4.3 (4H, t, Ar−O−CH2−), 7 (4H, d, Ar−H), 7.8 (4H, d, Ar−H); 13C NMR (400 MHz, CDC13): δppm 40 (−O−C−C−Cl), 67 (−O−C−C−Cl), 113 (Ar ring, −C−C− O−), 123 (Ar ring, N−C−C−), 146 (Ar ring, C−N), 159 (Ar ring, C−O). Azo-OC4H8Cl. mp: 126 °C, and decomp.: 292 °C (Figure S1); FTIR (KBr, cm−1): 3069 (aromatic −CH), 2948 (aliphatic −CH), 1581 (Ar CC), 1601 (NN), 1244 (C−O), 843 (para substituted aromatic rings), 729 (C−Cl); 1H NMR (400 MHz, CDC13): δppm 2, 1.5 (m, 8H, −O−C−CH2−CH2−C−Cl), 3.6 (t, 4H, −O−C−C−C− CH2−Cl), 4 (t, 4H, −O−CH2−C−C−C−Cl), 6.9 (d, 4H, Ar−H), 7.8 (d, 4H, Ar−H); 13C NMR (400 MHz, CDC13): δppm 25−28 (−O− C−C−C−C−Cl), 43 (−O−C−C−C−C−Cl), 66 (−O−C−C−C− C−Cl), 113 (Ar ring, -C−C−O−), 123 (Ar ring, N−C−C−), 146 (Ar ring, N−C−), 159 (Ar ring, C−O); 2.3.3. Synthesis of Azobenzene-Imidazolium Ionic Liquids (AzoILs); 4,4′-Bis(2-ethoxymethylimidazolium)azobenzene (AzoOC2H4Im+). N-Methylimidazole and synthesized Azo-OC2H4Cl were placed in a flask equipped with a condenser and the reaction was refluxed at 80 °C for 3 days (Azo-OC2H4Cl to N-methylimidazole ratio was 1:2.5 mmol). The progress of the reaction was monitored by thin layer chromatography (TLC). For extraction and purification, toluene was added gradually to the reaction solution in order to precipitate the Azo-OC2H4Im+. The obtained orange solids were washed again with toluene and then dried under vacuum overnight. Azo-OC2H4Im+. mp: 208 °C, and decomp.: 279 °C (Figure S2); FTIR (KBr, cm−1): 3153 (aromatic −CH), 2877 (aliphatic −CH), 1505 (Ar CC), 1593 (NN), 1258 and 1053 (C−O), 858 (para substituted aromatic rings), 1111 (C−N); 1H NMR (400 MHz, DMSO-d6): δppm 3.8 (6H, s, Im ring, -N−CH3), 4.5−4.6 (8H, m, −O−CH2−CH2−Im), 7.5 (2H, d, Im ring, −CH−), 7.8 (2H, d, Im ring, −CH−), 7.1−8.2 (8H, m, Ar−H), 9.3 (2H, s, Im, -N−CH-N-); 13 C NMR (400 MHz, DMSO-d6): δppm 35.8 (Im, −N−CH3), 48 (−O−CH2−CH2−Im), 66 (−O−CH2−CH2−Im), 115 (Ar ring, −C− C−O−), 122−123 (Im ring, −N−C−C−N−), 125 (Ar ring, −N−C− C−), 146 (Ar ring, −N−C−), 159 (Ar ring, −C−O−), 162 (Im ring, −N−CH−N−). 2.3.4. 4,4′-Bis(2-butoxymethylimidazolium)azobenzene (AzoOC4H8Im+). Azo-OC4H8Cl and N-methylimidazole were placed in a flask and all procedure was carried out the same as for Azo-OC2H4Im+.

ln

A 0 −A pss A t −A pss

= kt (1)

Where A0, At, and Apss are the absorbance of the Azo compounds at time 0, t and at the photo stationary stage, respectively; k is the rate constant of the photoisomerization process. 2.6. Drug Loading. Five milligrams of PABA, as a model drug and one of the effective ingredients of sunscreens, was dissolved in an ethanol−water mixture (5 mL, 1:1), then 50 mg of [email protected] was added to PABA solution. The mixture was ultrasonicated for 30 min and stirred at room temperature for 8 h (under UV irradiation). The precipitants (drug-loaded carriers) collected via centrifugation, washed with deionized water/ethanol, dried under vacuum, and kept in darkness. The supernatant was kept in order to calculate the loading content. The difference between initial concentration and final (supernatant) concentration of PABA was construed to be the sum of loaded PABA. By comparing the absorbance of PABA in the supernatant with its calibration curve (obtained at the same conditions, y = 0.0376x − 0.0378; R2 = 0.997), drug entrapment efficiency was assessed via eq 2:

entrapment efficiency(%) initial amount of drug − supernatant free amount of drug = initial amount of drug × 100

(2)

2.7. Drug Release Studies. The release behaviors of MMT@ Azo.ILs were studied in darkness and under UV irradiation. Briefly, 186


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ACS Biomaterials Science & Engineering drug-loaded nanocarriers (10 mg) were placed into dialysis bags and the bags were subsequently placed into the release medium (phosphate buffer solution, pH 5.8) at 34 ± 1 °C. At appropriate time intervals (60 min), 3 mL of the solution was withdrawn and the drug amount in the release medium was measured spectrophotometrically (by using a standard calibration curve obtained under the same conditions, y = 0.0479x-0.0078; R2 = 0.998). The withdrawn solution was replaced with the same volume of fresh buffer solution to keep the volume of the release medium constant. The release contents were calculated by eq 3. release content (%) =

total amount of drug in release medium 100 amount of loaded drug into the nanosystem

(3) Data points in the release profiles (Figure 9 and Figure S10) are for 3 independent experiments as “mean ± the standard error of the mean”. Moreover, student’s t test was employed for calculation of the statistical significance between different groups at each time point (p < 0.05).

3. RESULTS AND DISCUSSIONS 3.1. Synthesis and Characterization of Azo.ILs (Molecular Jacks). As illustrated in Figure 2, Azo.ILs synthesis includes three steps. The first step is the synthesis of Azo−OH through the potash fusion of 4-nitrophenol as starting material. In the second step, nucleophilic substitution reaction between halides and Azo−OH (in the presence of a base and a catalytic amount of KI) lead to Azo-ORCl. In the third step, the AzoORIm+ was synthesized through the second nucleophilic substitution reaction between N-methylimidazole and AzoORCl. 3.1.1. FT-IR and 1H NMR and 13C NMR Characterization. The FT-IR spectrum of Azo-OH, Azo-OC2H4Cl, AzoOC4H8Cl, Azo-OC2H4Im+, and Azo-OC4H8Im+ are shown in Figure S4. The characteristic peaks of NN, C−O, and 4,4′disubstituted benzene rings can be detected in all spectra (details for each compound can be found in the Experimental Section). Moreover, it can be noticed that after attaching R-Cl group on Azo-OH, not only is the broadband related to −OH bond weakened (i.e., the band at 3457 cm−1 in Figure S4a almost disappeared in Figure S4b, d) but other peaks related to aliphatic CH vibrations (at 2930 cm−1 for Azo-OC2H4Cl and at 2948 cm−1 for Azo-OC4H8Cl) and R-Cl vibrations (at 654 cm−1 for Azo-OC2H4Cl and 729 cm−1 for Azo-OC4H8Cl) also arise. In Figure S4c, e, the strengthened peaks of aromatic CH vibrations (at 3153 cm−1 for Azo-OC2H4Cl and 3074 cm−1 for Azo-OC4H8Cl), the elimination of peaks related to R-Cl, and sharp peaks assigned to C−N bonds (at 1111 cm−1 for AzoOC2H4Cl and 1145 cm−1 for Azo-OC4H8Cl) confirm that AzoOC2H4Im+ and Azo-OC4H8Im+ are synthesized. The assigned 1H NMR and 13C NMR spectra of Azo-ILs and Azo-ORCl and are depicted in Figures S5 and S6, respectively. 3.1.2. Photoisomerization of Azo-ILs. The photosensitivity and photoisomerization behaviors of prepared Azo-ILs were studied by UV/vis absorption spectroscopy (Figure 3). The UV−vis spectra of Azo compounds show two absorption bands: the strong one around 350 nm and the weak one around 445 nm. These bands are regular for Azo compounds and can be assigned to the π−π* and n−π* electronic transitions.38,39 Upon UV irradiation at 365 nm, all synthesized Azo compounds show significant trans to cis photoisomerization that lead to a noticeable continuous decline in the intensity of the π−π* transition bands around 350 nm, and on the other

Figure 3. UV−vis spectral changes in dependence of time for the AzoILs solutions (1 × 10−4 mol/Lit) in ethanol: trans to cis photoisomerization of (a) Azo-OC2H4Im+, and (b) Azo-OC4H8Im+ upon irradiation at 365 nm for different durations; following the visible-light irradiation (dashed spectra). The kinetics of the photoisomerization are shown in the insets. The rate constants were found to be 9.4 × 10−3 s−1 (Azo-OC2H4Im+) and 12.6 × 10−3 s−1 (Azo-OC4H8Im+).

hand slight increases in n−π* electron transition bands around 445 nm are seen (Figure 3). Moreover, the Azo solutions that had been irradiated with UV light, were irradiated with visible light. This resulted in the increase in intensity recovery of the absorption band (π−π* transition) because of the cis to trans back-isomerization of Azo compounds (dashed spectra). These changes in UV/vis absorption spectra confirm that photoisomerizations happen and the structures of Azo compounds are changed upon UV irradiation. The kinetics of the photoisomerizations are depicted in the insets and the isomerization rate constants were comparable and similar to other azobenzene molecules reported in literatures.38 Photoisomerization behaviors of AzoOC2H4Cl and Azo-OC4H8Cl are depicted in Figure S7. 3.2. Preparation of Photoresponsive Nanomechanical Systems. As it can be seen in Figure 4, the photoresponsive nanomechanical systems MMT@Azo-ILs consist of two main parts: the nanocontainer part that has the duty to contain and protect the drug molecules (MMT, an inexpensive layered clay, was used for this aim). The second and most important part of these nanosystems are photoswitchable molecules that act as 187


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Figure 4. Schematic illustration for intercalation of Azo-ILs between MMT layers (not to scale), and molecular dimensions of trans-Azo-OC2H4Im+.

Figure 5. FE-SEM images of (a) pristine MMT, (b) MMT@Azo-OC2H4Im+, (c) MMT@Azo-OC4H8Im+.

molecular jacks and could convert light energy to mechanical energy. These molecular jacks would transfer microscopic changes in the molecular level of the chromophore to the macroscopic changes of the whole systems. Herein, two Azo-IL molecules with different molecular length were synthesized and played this role. Generally, photoisomerization alters the molecular dimensions of Azo molecules, consequently, the distance between the 4 and 4′ carbons of the aromatic rings is decreased by 3.5 Å (from 9.0 in trans isomer to 5.5 Å in cis isomer). The trans isomers are almost flat and planar whereas the cis isomers have angular unplanar geometries (one of the aromatic rings rotates in order to avoid steric repulsions owing to facing of the π clouds of aromatic rings). Briefly, photoisomerization converts the long planar trans isomer to the short unplanar cis isomer and vice versa.30 To prepare these nanomechanical systems, we must intercalate the molecular jacks between the MMT layers. The driving force of intercalation process is electrostatic forces between negatively charged MMT layers and positively charged

Azo-ILs. After intercalation, the photoisomerization of Azo-ILs could change the basal distances of MMT layers (interlayer expansion), which would consequently alter the drug loading contents and drug release rates (more details will be presented in sections 3.2.3 and 3.2.4). 3.2.1. Morphology and SEM Images. The SEM images of parent MMT and MMT@Azo-IL hybrid nanosystems and their sample colors are shown in Figure 5. The SEM images of MMT and MMT@Azo-IL hybrid nanosystems are similar and no noticeable changes can be detected after intercalation, suggesting that the intercalations have not changed the morphology. The higher-magnification reveals the constituent spherical nanoparticles. 3.2.2. Compositions of Hybrid Nanosystems (TGA&EDX Analysis). Thermal gravimetric analysis was employed to evaluate the amounts of organic parts in the prepared hybrid nanosystems. Figure 6 shows TGA thermograms of parent MMT, MMT@Azo-OC4H8Im+ and MMT@Azo-OC2H4Im+ in the temperature range of 50−900 °C. Figure 6a shows a 5 wt % 188


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Figure 6. TGA curves for (a) MMT, (b) MMT@Azo-OC2H4Im+, (c) MMT@Azo-OC4H8Im+.

weight loss for parent MMT (mainly for water loss). TGA curves of MMT@Azo-OC2H4Im+ (Figure 6b) and MMT@ Azo-OC4H8Im+ (Figure 6c) show a total weight loss of about 28 and 29 wt %, respectively, that correspond to the intercalated organic molecules (Azo-ILs) between MMT layers. According to these weight loss amounts, the percentages of organic molecules intercalated between MMT layers are assessed to be about 23 and 24 wt % for MMT@AzoOC2H4Im+ and MMT@Azo-OC4H8Im+, respectively. The EDX spectrum of parent MMT (Figure S9) shows the bands related to the elements that are in the structure of MMT (Si, O, Al, Mg, ...). Figure 7 depicts the EDX spectra of MMT@ Azo-ILs, a moderate band related to the carbon atom and a small band related to the nitrogen atom can be observed in the spectra of MMT@Azo-OC2H4Im+ (Figure 7a) and MMT@ Azo-OC4H8Im+ (Figure 7b), that come from the intercalated Azo-ILs molecules. In addition, the quantitative results reveal that the sum of carbon and nitrogen percentages (i.e., 25 wt % for MMT@Azo-OC2H4Im+; and 20.2 wt % for MMT@AzoOC4H8Im+), which correspond to the approximate amounts of organic molecules, are in good agreement with the results obtained from TGA analysis (23 wt % for MMT@AzoOC2H4Im+ and 24 wt % for MMT@Azo-OC4H8Im+). 3.2.3. XRD Analysis. The XRD measurements provide a powerful tool to study the changes in the clay microenvironments and importantly, the basal distances of the MMT layers are obtained from the position of the d001 peak. The XRD patterns of parent MMT and MMT@Azo-IL hybrid nanosystems are illustrated in Figure 8. The characteristic d001 peak of parent MMT appeared at 2θ = 9.12 (spectrum a)40 and after intercalation of Azo-ILs, the d001 peak shifted to lower angles (about 2θ = 6.0 for both MMT@Azo-OC2H4Im+ and MMT@ Azo-OC4H8Im+). According to the Bragg’s law, the peak shifting from higher diffraction angle to lower diffraction angle is due to increase in the d-spacing and it indicates that Azo-ILs have successfully intercalated between the MMT layers. By subtracting the thickness of the silicate layer of MMT (9.67 Å) from the observed basal spacings of MMT@Azo-OC2H4Im+ (14.72 Å) and MMT@Azo-OC4H8Im+ (14.56 Å), the interlayer galleries were assessed to be 5.05 and 4.9 Å for MMT@Azo-OC2H4Im+ and MMT@Azo-OC4H8Im+, respectively.40−42 Upon UV radiation, the intercalated Azo-ILs photoisomerize to cis forms and as a result, the d001 peaks shifted to 2θ = 5.77 (basal distance = 15.29; interlayer space = 5.62 Å) and 2θ =

Figure 7. EDX spectra of (a) MMT@Azo-OC2H4Im+, (b) MMT@ Azo-OC4H8Im+ (with quantitative data).

5.67 (basal distance = 15.55; interlayer space = 5.88 Å) for MMT@Azo-OC2H4Im+ and MMT@Azo-OC4H8Im+, respectively. Comparing of interlayer spaces indicates that the interlayer gallery between MMT layers is increased from 5.05 to 5.62 Å for MMT@Azo-OC2H4Im+ and from 4.9 to 5.8 Å for MMT@Azo-OC4H8Im+. The results and data obtained from XRD analysis can also be used to estimate how the Azo-ILs lie between the MMT layers. We first expected that the Azo-ILs should have been placed vertically within the MMT layers, in this case, trans Azo-ILs (with higher molecular length) should have increased the basal distances more than cis-Azo-ILs (with shorter molecular length), but the reverse events were observed and in the trans state the basal distances were lower than that of cis state. Replacing Azo-OC2H4Im+ with a longer molecule (AzoOC4H8Im+) does not increase the basal space, suggesting that changes in molecular length do not alter the basal distances of MMT layers. It can be said that trans-Azo-ILs are flat planar molecules and when these planar molecules are horizontally placed between the MMT layers, the lower expansion for basal distances are expected. Only in this circumstance, the changes in molecular length could not significantly affect the interlayer expansions. In addition, the dimensions of trans-Azo-ILs 189


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Table 2. Drug Loading Contents for MMT@Azo-ILs in Darkness and upon UV Irradiation MMT@ AzoOC2H4Im+ loading time (h) drug loading %

MMT@ AzoOC4H8Im+

24

24

78.6

89.1

MMT@AzoOC2H4Im+ (UVIrad.)

MMT@AzoOC4H8Im+ (UV-Irad.)

8

8

99.3

99.8

and upon UV irradiation as a function of time at pH 5.8 (pH of skin). The control system (i.e., drug-loaded MMT, with no intercalated Azo-ILs) shows no noticeable differences between release profiles in darkness and under UV irradiation (Figure S10). Unlike the control system, the prepared MMT@Azo-ILs are photoresponsive and the drug release rates obviously depend on UV irradiation (Figure 9). The amounts of released

Figure 8. Powder X-ray diffraction patterns of (a) pristine MMT, (b) MMT@Azo-OC2H4Im+, (c) MMT@Azo-OC4H8Im+, (d) MMT@ Azo-OC2H4Im+ (UV irradiated), (e) MMT@Azo-OC4H8Im+ (UV irradiated).

(Figure 4) exactly fit the interlayer space (5 Å) if the molecule lies horizontally between the layers. On the other hand, cis-Azo-ILs are nonplanar molecules. Upon UV radiation, Planar trans Azo molecules photoisomerize to nonplanar cis molecules and this deformation leads to an increase in basal distance of MMT layers. Finally, comparing of basal distance expansions for Azo-OC2H4Im+ and AzoOC4H8Im+ in their cis form indicates that the molecular length affects the basal distance expansions (i.e., the basal distance expansion of cis-Azo-OC4H8Im+ is higher than cis-AzoOC2H4Im+). All data extracted from XRD spectra are summarized in Table 1. Table 1. Data Extracted from d001 Peak Positions

MMT d001 peak position (2θ) basal distance (Å) interlayer gallery (Å)

MMT@ MMT@ AzoAzo+ OC2H4Im OC4H8Im+

MMT@ AzoOC2H4Im+ (UV-Irad.)

MMT@ AzoOC4H8Im+ (UV-Irad.)

9.12

6.00

6.06

5.77

5.67

9.67

14.72

14.56

15.29

15.55

5.05

4.9

5.62

5.88

Figure 9. In vitro release profiles of PABA from (a) MMT@AzoOC2H4Im+ and (b) MMT@Azo-OC4H8Im+ in darkness and upon UV radiation at 34 ± 1 °C; * The mean difference is significant at the p < 0.05 level.

PABA from MMT@Azo-ILs in darkness are about 30% for MMT@Azo-OC2H4Im+ and 33% for MMT@Azo-OC4H8Im+; and the released amounts upon UV irradiation are about 71% for MMT@Azo-OC 2 H 4 Im + and 80% for MMT@AzoOC4H8Im+. These differences in release rates could be ascribed to the sensitivity of Azo-ILs molecules to UV radiation. As it was mentioned before, upon UV radiation, the interlayer galleries between MMT layers are expanded, therefore the room for drug encapsulation is enlarged and the drug penetrates into MMT interlayer spaces more easily. All these cases lead to an increase in drug loading content in a short time (8 h). In the case of loading in darkness, the restricted drug penetration and small room for drug encapsulation result in a low drug loading percentages even in a long loading time (24 h). The similar explanation can be used for release behaviors, upon UV

3.2.4. Drug Loading and Release Studies. Drug loading was carried out in two different conditions (in darkness for 24 h, and upon UV irradiation for 8 h). The loading percentages are diverse and it can be seen that loading amounts upon UV irradiation are higher than that of darkness, although in dark conditions the given time for the loading process was longer (Table 2). The in vitro drug release curves are drawn by plotting the cumulative release of PABA from MMT@AZ-ILs in darkness 190


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powder diffraction; TGA, thermogravimetric analysis; EDX, energy-dispersive X-ray spectroscopy; FE-SEM, field-emission scanning electron microscope; CRSs, controlled release systems; DDSs, drug delivery systems; Azo, Azobenzene; Azo-ILs, azobenzene-imidazolium ionic liquids; Im, imidazolium ring; PABA, para-aminobenzoic acid; TMS, tetramethylsilane; PSS, photostationary state; TLC, thin layer chromatography; Azo-OH, 4,4′-bis(hydroxy)-azobenzene or 4,4′-diazophenol; Azo-OC2H4Cl, 4,4′-bis(2-chloroethoxy)azobenzene; Azo-OC 4H8Cl, 4,4′-bis(4-chlorobutoxy)azobenzene; AzoOC2H4Im+, 4,4′-bis(2-ethoxymethylimidazolium)azobenzene; Azo-OC 4 H 8 Im + , 4,4′-bis(2-butoxymethylimidazolium)azobenzene; MMT@Azo-IL, intercalation of Azo-ILs between MMT layers (the prepared nanomechanical systems)

radiation, the encapsulated drugs can leach out from expanded MMT layers more easily, in comparison to dark conditions (Figure 10).

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(1) Abbaszad Rafi, A.; Mahkam, M. Preparation of magnetic pHsensitive microcapsules with an alginate base as colon specific drug delivery systems through an entirely green route. RSC Adv. 2015, 5 (6), 4628−4638. (2) Biswas, S.; Kumari, P.; Lakhani, P. M.; Ghosh, B. Recent advances in polymeric micelles for anti-cancer drug delivery. Eur. J. Pharm. Sci. 2016, 83, 184−202. (3) Thakkar, S.; Misra, M. Electrospun polymeric nanofibers: New horizons in drug delivery. Eur. J. Pharm. Sci. 2017, 107, 148−167. (4) Abbaszad Rafi, A.; Fakheri, F.; Mahkam, M. Synthesis and preparation of new pH-sensitivenanocomposite and nanocapsule based on “MCM-41/poly methacrylic acid” as drug carriers. Polym. Bull. 2016, 73 (10), 2649−2659. (5) Mahkam, M.; Abbaszad Rafi, A.; Mohammadzadeh Gheshlaghi, L. Preparation of novel pH-sensitive nanocomposites based on ionicliquid modified montmorillonite for colon specific drug delivery system. Polym. Compos. 2016, 37 (1), 182−187. (6) Rafi, A. A.; Jeddi, M. K.; Bashir-Hashemi, A.; Mahkam, M. Synthesis of Multiwalled Carbon Nanotubes−Poly (Methacrylic Acid) Nanohybrid Systems: Characterization, Thermal Properties, and In Vitro Release Studies of Naproxen as a Model Drug. Polym.-Plast. Technol. Eng. 2017, 56 (16), 1723−1729. (7) Viseras, C.; Cerezo, P.; Sanchez, R.; Salcedo, I.; Aguzzi, C. Current challenges in clay minerals for drug delivery. Appl. Clay Sci. 2010, 48 (3), 291−295. (8) Coti, K. K.; Belowich, M. E.; Liong, M.; Ambrogio, M. W.; Lau, Y. A.; Khatib, H. A.; Zink, J. I.; Khashab, N. M.; Stoddart, J. F. Mechanised nanoparticles for drug delivery. Nanoscale 2009, 1 (1), 16−39. (9) Abbaszad Rafi, A.; Mahkam, M.; Davaran, S.; Hamishehkar, H. A Smart pH-responsive Nano-Carrier as a Drug Delivery System: A hybrid system comprised of mesoporous nanosilica MCM-41 (as a nano-container) & a pH-sensitive polymer (as smart reversible gatekeepers): Preparation, characterization and in vitro release studies of an anti-cancer drug. Eur. J. Pharm. Sci. 2016, 93, 64−73. (10) Abendroth, J. M.; Bushuyev, O. S.; Weiss, P. S.; Barrett, C. J. Controlling Motion at the Nanoscale: Rise of the Molecular Machines. ACS Nano 2015, 9 (8), 7746−7768. (11) Zheng, Y. B.; Kiraly, B.; Huang, T. J. Molecular machines drive smart drug delivery. Nanomedicine 2010, 5 (9), 1309−1312. (12) Lu, J.; Choi, E.; Tamanoi, F.; Zink, J. I. Light-Activated Nanoimpeller-Controlled Drug Release in Cancer Cells. Small 2008, 4 (4), 421−426. (13) Fomina, N.; Sankaranarayanan, J.; Almutairi, A. Photochemical mechanisms of light-triggered release from nanocarriers. Adv. Drug Delivery Rev. 2012, 64 (11), 1005−1020. (14) Benova, E.; Zelenak, V.; Halamova, D.; Almasi, M.; Petrul’ova, V.; Psotka, M.; Zelenakova, A.; Backor, M.; Hornebecq, V. A drug delivery system based on switchable photo-controlled p-coumaric acid derivatives anchored on mesoporous silica. J. Mater. Chem. B 2017, 5 (4), 817−825.

Figure 10. Molecular jacks switch to the cis and trans states that lead to changes in basal distances and room for drug encapsulation (triggered by UV radiation).

4. CONCLUSIONS Photoresponsive nanomechanical systems have been prepared through simple intercalation of synthesized Azo-ILs between MMT layers. The study shows that the prepared nanomechanical systems (MMT@Azo-ILs) are photosensitive and the basal distances of MMT layers are changed upon UV irradiations. Azo-ILs (molecular jacks) could change the basal distances, which consequently would alter the drug loading and release rates. Upon UV irradiation, drug loading and release rates were higher, and therefore the prepared photosensitive nanosystems can be considered as photoresponsive drug carriers.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00621. DSC analysis, FTIR, 1H NMR, and 13C NMR for Azo compounds; UV/vis spectra and TGA analysis for AzoORCl; EDX analysis and release behavior of pristine MMT (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Telfax: +984124327541. ORCID

Abdolrahim Abbaszad Rafi: 0000-0001-8717-3198 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study has been supported by the office of the research vice chancellor of “Azarbaijan Shahid Madani University”. ABBREVIATIONS Azo-ILs, azobenzene ionic liquids; MMT, montmorillonite; FTIR, Fourier transform infrared spectroscopy; XRD, X-ray 191


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Photo-Switchable Nanomechanical Systems Comprising a

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