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Nanohydrogel formation within halloysite lumen for triggered and sustained release Giuseppe Cavallaro, Giuseppe Lazzara, Stefana Milioto, Filippo Parisi, Vladimir G. Evtugyn, Elvira Rozhina, and Rawil F. Fakhrullin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19361 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018
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ACS Applied Materials & Interfaces
Nanohydrogel formation within halloysite lumen for triggered and sustained release
Giuseppe Cavallaro,a Giuseppe Lazzara,a,* Stefana Milioto,a Filippo Parisi,a Vladimir Evtugyn,b Elvira Rozhina,b Rawil Fakhrullinb,* a
Dipartimento di Fisica e Chimica, Università degli Studi di Palermo, Viale delle Scienze,
pad. 17, 90128 Palermo, Italy.
[email protected] b
Institute of fundamental biology and medicine, Kazan federal university, Kreml uramı 18,
Kazan, Republic of Tatarstan 420008, Russian Federation.
[email protected] Abstract An easy strategy to obtain nanohydrogel within the halloysite nanotubes (HNTs) lumen was investigated. Inorganic reverse micelles based on HNTs and hexadecyltrimethylammonium bromides were dispersed in chloroform and the hydrophilic cavity was used as nano-reactor to confine the gel formation based on alginate cross-linked by calcium ions. Spectroscopy and electron microscopy experiments proved the confinement of the polymer into the HNTs lumen and the formation of calcium mediated networks. Biological tests proved the biocompatibility of the hybrid hydrogel. The nanogel in HNTs was suitable for drug loading and sustained release with the opportunity of triggered burst release by chemical stimuli. Here we propose a new strategy based on inorganic reverse micelles for nanohydrogel formation that are suitable for industrial and biological applications as well as for selective and triggered adsorption and/or release.
Keywords: halloysite, inorganic reverse micelles, nanohydrogel, sustained release, alginate.
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Introduction Nanotechnology based delivery systems offer interesting alternative ways of administration to improve patient compliance and to target delivery at the proper site. Novel micro/nanohydrogel based formulations for delivering molecular therapeutics have attracted scientific interests.1 Inverse microemulsion is a well established method to obtain the confinement of water for controlled nanoparticle synthesis2,3 or micro/nanohydrogel preparation in a variety of sizes.4 Reducing the size of a gel matrixes improves their response time in comparison with the bulk
counterparts
to
external
physico-chemical
stimuli.
Literature
reports
nanohydrogels, nanogels or microgels are promising in sustained drug delivery.
5–9
that One
major limitation of nanohydrogels for clinical applications is the structural instability for the precise control of the release of drug during the treatment protocol. To overcome these limitations, montmorillonite nanocomposites hydrogel were proposed as they possess interesting anti-fatigue and delivery properties.10 Among clays, halloysite nanotubes (HNTs) are a very promising because of versatile properties, such as hollow tubular morphology, tunable surface chemistry, and biocompatibility.11 microorganisms
12,13
Tests
demonstrated
the
low
toxicity
of
halloysite
towards
14
and nematodes. These features make HNTs suitable for development
of hybrid nanomaterials for wastewater remediation,15-18 smart coating,19,20 catalysis,21,22 nanocomposite packaging23-26 and pharmaceutical applications.27-30 HNTs is obtained from natural sources with polydisperse size, the length is in the micrometers range while the external and internal diameters range between 50-80 nm and 10-15 nm, respectively.31,32 Although the chemical composition is dependent on the deposit,33 halloysite has a unit cell formed by Al2Si2O5(OH)4·2H2O, where the Al is disposed in a gibbsite octahedral sheet (AlOH) at the inner surface and siloxane (Si−O−Si) groups are exposed at the external surface. HNTs dispersed in water in a wide pH interval (from 3 to 8) show a charge separation and therefore a positively charged lumen and a negative charge at the outer surface which dominates the net nanotubes charge value.31 Such a peculiarity, not only influences the liquid crystalline and assembling behavior of aqueous halloysite dispersions,34,35 but allows for the selective modification of HNTs surfaces by ion exchange.36-38 The selective adsorption of sodium alkanoates and SDS onto HNTs lumen generates tubular inorganic micelles with efficient solubilization ability towards hydrophobic molecules.16,39 Dioctyl sulfosuccinate sodium salt are effective in stabilizing oil-in-water Pickering emulsions.40 2
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Covalent modification of HNTs lumen to enhance the loading capacity towards hydrophobic derivatives was achieved with octadecylphosphonic acid.41 Here we prepared inorganic reverse micelles inspired by halloysite surface chemistry and our recent findings.42 The HNTs with hydrophobic external surface was dispersed in chloroform to encapsulate aqueous sodium alginate and calcium chloride into their lumen. By mixing the two dispersions one expects the Ca-Alginate hydrogel formation confined into the HNTs lumen. Doxycycline, a broad-spectrum antibiotic of the tetracycline class, was selected as model drug for loading and release from the confined hydrogel. The possibility to trigger the drug release from the hydrogel was exploited by adding ethylenediaminetetraacetic acid that is a Ca2+ chelant and therefore it can control the hydrogel rupturing. This approach represents an alternative to end-capped nanotubes for sustained release.43,44
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Experimental Materials Halloysite nanotubes (HNTs), hexadecyltrimethylammonium bromide, sodium alginate, calcium chloride (CaCl2), ethylenediaminetetraacetic acid and 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) are from Sigma. Doxicicline chlorhydrate is from Alfa Aesar. All the products were used without further purification. Water from reverse osmosis was used. HNTs outer surface modification HNTs was modified at the outer surface by ion-exchange with cationic surfactant by means of a procedure already reported in the literature.42 Briefly, aqueous surfactant solutions were prepared by dissolving 4 g of hexadecyltrimethylammonium bromide in 500 cm3 of water. Then, 8 g of HNTs were added and the obtained dispersion was stirred for 48 h. The functionalized material was recovered by centrifugation at 700 rpm for 40 min. Excess of surfactant was removed washing with water until the presence of bromide was not detected in the washing water by AgNO3 addition. The obtained powder (f-HNT) was dried at 80 °C for seven days. Encapsulation of Sodium Alginate and CaCl2 solutions into modified HNTs A dispersion of the f-HNTs composite in chloroform was prepared by dissolving 0.2 g of the modified nanotubes into 60 cm3 of solvent. Then, the dispersion was mixed with a aqueous solution of sodium alginate (2 wt% in water) or CaCl2 (0.1M) at 25 °C. The volume ratio between the chloroform dispersion and the aqueous solution was 3:1. The biphasic system was stirred overnight. Finally, the chloroform dispersions were recovered with a microsyringe and mixed. After 12 hours stirring, the nanopowder (Ca-alg@f-HNT) was separated by centrifugation. Synthesis of Sodium Alginate labeled with fluorescent probe According to the literature,45 alginate was fluorescently labelled using DTAF (5-(4,6dichlorotriazinyl) aminofluorescein. To this purpose, alginate was solubilized in 50 mM sodium bicarbonate. The alginate concentration was fixed at 10 mg ml-1, while the pH was adjusted to 9.0 with 1.0 M NaOH. The polymer solution was mixed overnight with a solution of DTAF (concentration of 10 mg mL-1 in DMSO) at room temperature. The ratio between alginate and DTAF solutions was 1:0.4 v/v. Finally, the reaction mixture was 4
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dialysed in 10kDa cut-off dialysis tubing against PBS until no residual DTAF could be detected in the dialysate by UV absorbance at 490 nm. Loading and release of doxycycline from the Ca-Alginate confined within the HNTs lumen The procedure for encapsulation of sodium alginate and CaCl2 solutions into f-HNTs was repeated but doxycycline was added to the alginate solution (ca. 0.35 g doxycycline/g fHNTs). After Ca2+ coordination, the powder was separated by centrifugation. The release of doxycycline was monitored placing the powder into a dialysis membrane (Medicell International Ltd MWCO 12-14000 with a diameter of 21.5 mm) and 100 cm3 of Phosphate buffer (0.05 M, pH 7.4). The flask was maintained under stirring at 37 °C. Aliquots of 2 cm3 have been withdrawn and analyzed by UV-VIS spectra. Methods The thermogravimetric (TG) analyses were performed by using a Q5000 IR apparatus (TA Instruments) under nitrogen flow of 25 cm3 min-1 for the sample and 10 cm3 min-1 for the balance. The explored temperature interval ranged between 25 and 900 °C at a heating rate of 20 °C min-1. Dynamic Light Scattering (DLS) and ζ-potential measurements were carried out by means of a Zetasizer NANO-ZS (Malvern Instruments) at 25.0 ± 0.1 °C. The fieldtime autocorrelation functions were analyzed by ILT. The wavelength of 632.8 nm the scattering angle of 173° were used. DLS experiments were conducted in chloroform dispersions with variable concentration (see Supporting Information). FT-IR spectra were determined at room temperature using a Frontier FT-IR spectrometer (Perkin-Elmer) in KBr. The spectral resolution was 2 cm-1. The steady-state fluorescence spectra for alginate labeled with fluorescein, were registered with a Fluoromax 4 (Jobin-Yvon) spectrofluorometer (right angle geometry, 1 cm x 1 cm quartz cell) at 25.0±0.1 °C. The excitation wavelength was of 470 nm and the emission spectra were recorded from 500 nm to 680 nm. The widths of slits were set at 1.5 and 1.5 nm for excitation and emission, respectively. UV-VIS spectra of Doxycycline chlorhydrate were recorded by a Specord S600 Analytik Jena. Doxicicline chlorhydrate in water presents a peak at 362 nm with a extinction coefficient of 23.6 ± 0.3 cm2 mg-1. For transmission electron microscopy imaging a Hitachi HT7700 Exalens transmission electron microscope was used. A droplet of the suspension (10 µL) was placed on a carbon lacey 3mm copper grid, then dried at room temperature. TEM imaging was 5
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performed at 100kV accelerating voltage. EDX analysis was carried out in STEM mode using Oxford Instruments X-Max ™ 80T detector. The surface morphology of the prepared materials was studied by using a microscope ESEM FEI QUANTA 200F. Before each SEM experiment, the surface of the sample was coated with gold in argon by means of an Edwards Sputter Coater S150A to avoid charging under electron beam. The measurements were carried out in high vacuum mode (
