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Photo-Switchable Nanomechanical Systems Comprised a Nanocontainer (Montmorillonite) and Light Driven Molecular Jack (Azobenzen-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 ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00621 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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ACS Biomaterials Science & Engineering

Photo-Switchable Nanomechanical Systems Comprised a Nanocontainer (Montmorillonite) and Light Driven Molecular Jack (AzobenzenImidazolium Ionic Liquids) as Drug Delivery Systems; Synthesis, Characterization and In-Vitro Release Studies

Abdolrahim Abbaszad Rafi a,b, Nazila Hamidi a, Abdollah Bashir-Hashemi c, Mehrdad Mahkam a* a) Department of Chemistry, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran. b) Department of Natural Sciences, Mid Sweden University, Holmgatan 10, 851 70 Sundsvall, Sweden. c) Department of Chemistry, University of Alabama, Tuscaloosa, AL 35487, USA. Corresponding author’s E-mail: [email protected]; Tel-fax: +984124327541

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: In this work, photo-responsive nanomechanical systems were prepared through the intercalation of positively charged photo-switching molecular jacks (Azobenzene Ionic Liquids, Azo-ILs) within Montmorillonite (MMT) layers (MMT@Azo-ILs). The study shows that MMT@Azo-ILs are photo-sensitive 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 photo-responsive nanomechanical systems were characterized by Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Powder Diffraction (XRD), Thermo-Gravimetric Analysis (TGA), Energy-Dispersive Xray 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 photo-responsive carriers might be considered as an excellent potential candidate in order to formulate smart sunscreens. Keywords: Photo-Responsive Nanomechaical System, Drug Delivery System, Molecular Jack, Smart Sunscreen, Azobenzene Ionic liquid, Smart Sunscreen; 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, in order to address the drug delivery challenges, not only typical organic and inorganic systems such as hydrogels Nanocomposites

4-6

, and clays

7

1

, micelles

2

, nano-fibers

3

,

are employed as drug carriers, but also novel smart

nanomechanical systems are developed

8-11

. Nanomechanical systems that are prepared to

encapsulate and then release drug molecules in response to an external trigger or stimulus, 2 ACS Paragon Plus Environment

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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 photo-therapy 12-14. 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 inter-layer 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, non-toxicity of MMT has been

proved by hematological, biochemical and histopathological analysis in rat models. Therefore, MMT with its unique properties is a 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 un-controlled release behaviour

7, 19

. Several approaches like intercalation of

organic molecules are employed for modification and improvement of drug release behaviour. 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

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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 photo-responsive motif and moving components in photo-responsive 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 photo-isomerizes to the less stable cis isomer and the cis isomer could subsequently convert to the trans isomer either photo-chemically 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 photo-isomerization (Fig. 1) 25-30.

Figure 1. Changes in molecular structure & length of Azo molecules upon photo-isomerization.

This photo-isomerization could also occur between solid micro and nano layers and would lead to changes in the distance of nanolayers 31-33. Herein, photo-responsive nanomechanical systems were prepared, a layered natural clay (i.e. MMT) was employed as nano-container for the encapsulation of drug molecules and photo-switchable organic molecules (positively charged azobenzene molecules) were used as molecular jacks. For the first time, two new Azobenzene-Imidazolium Ionic Liquids 4 ACS Paragon Plus Environment

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(Azo-ILs) with different molecular length were synthesized and intercalated within MMT layers (MMT@Azo-IL). The study shows that MMT@Azo-ILs were photo-sensitive 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 photo-responsive 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, para-aminobenzoic 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 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 3536

. 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. 2. Experimental 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. 5 ACS Paragon Plus Environment


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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 FESEM images recorded by a 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 Fig. 2. 2.3.1 4,4’-bis(hydroxy)-azobenzene or 4,4’-diazophenol (Azo-OH): The starting Azo compound (Azo-OH) was synthesized through the alkali fusion of 4Nitrophenol. 25 g 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 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


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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 re-crystallized from 50% ethanol/water (v/v) to obtain yellow/orange crystals of compound (Azo-OH). mp: 215-218 ºC. 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(4chlorobutoxy)azobenzene (Azo-OC4H8Cl) Firstly, 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 water) was added drop-wise to the mixture and stirred for 90 min. Secondly, the prepared solution was added gradually and drop-wise 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 (Fig S1); FT-IR (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, ArH);

13

C-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);



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Azo-OC4H8Cl: mp: 126 °C, and decomp.: 292 °C (Fig S1); FT-IR (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);

1

H-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);

13

C-NMR (400

MHz, CDC13): δppm 25-28 (-O-C-C-C-C-Cl), 43 (-O-C-C-C-C-Cl), 66 (-O-C-C-CC-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 (Azo-ILs); 4,4’-bis(2ethoxymethylimidazolium)azobenzene (Azo-OC2H4Im+) 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 Nmethylimidazole 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 (Fig S2); FT-IR (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-


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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 (Azo-OC4H8Im+) Azo-OC4H8Cl and N-methylimidazole were placed in a flask and all procedure was carried out the same as for Azo-OC2H4Im+. Azo-OC4H8Im+: mp:187 °C, and decomp.: 287 °C (Fig S3); FT-IR (KBr, cm-1): 3074 (aromatic -CH), 2829 (aliphatic -CH), 1498 (Ar C=C), 1594 (N=N), 1253 (C–O), 1145 (CN), 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-CH2CH2-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-CH2-CH2-CH2-CH2-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 drop-wise to the MMT mixture. The reaction was refluxed and continued at 70 °C in darkness for 3 days. The 9 ACS Paragon Plus Environment


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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 photo-isomerization kinetics Spectroscopic characterization and photo-isomerization 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 (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 the Azo solution was irradiated at 365 nm until it reached the Photo Stationary State (PSS), afterward it was irradiated by Vis-light and then the spectra were recorded. The kinetics of the photo-isomerizations were assessed by eq. 1:

= (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 photo-isomerization process. 2.6 Drug Loading 5 mg 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 10 ACS Paragon Plus Environment

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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 the following equation (Eq. 2):

Entrapment efficiency (%) =

Initial amount of drug − Supernatant free amount of drug × 100 (2) Initial amount of drug

2.7 Drug Release Studies The release behaviors of [email protected] were studied in darkness and under UV irradiation. Briefly, 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 (3) Amount of loaded drug into the nanosystem



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Data points in the release profiles (Fig. 9 and Fig. 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

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