Effects of Ultrasound Irradiation on the Preparation of Ethyl Cellulose Nanocapsules Containing Spirooxazine Dye

This article presents the influence of low frequency, high intensity ultrasonic irradiation on the characteristics (average size, polydispersity index) of ethyl cellulose nanocapsules encapsulating a photochromic dye. Photochromic nanocapsuleswere prepared by the emulsion-solvent evaporationmethod.The acoustic densities entering the systemwere systematically studied with respect to their abilities tomodify and reduce the average sizes and polydispersity indexes of the nanocapsules. Scanning electronmicroscope, confocal laser microscope, and dynamic light scattering were utilised to characterise the structure, shape, size, and polydispersity of ethyl cellulose photochromic nanocapsules. We were able to tailor the size of the photochromic nanocapsules simply by varying the acoustic densities entering the system. At an acoustic density of 1.5W/mL and 60 s of continuous irradiation, we were able to prepare an almost monodispersed population of the nanocapsules with an average size of 193 nm.


Introduction
Encapsulation is a very important method for improving the stabilities and protection capabilities of labile substances against degradation factors [1].So, encapsulation of photochromic dyes is an effective method for protecting dye against environmental factors such as oxygen, pH value, and light, which lead to oxidation and deterioration of the photochromic dyes [2,3].Photochromic dyes can be covalently bound to backbones [4] or immobilised by doping in polymer solids [5].
Mass coloration and coating are by far the more common industrial methods of application of photochromic colorants.Conventional methods to apply photochromic substance on fabrics include exhaustion dyeing [6].However, this method fails to provide sufficient space for photochromic molecules to accomplish the structural transformation, and consequently the photochromic effect is insignificant.In the case that photochromic dyes cannot be used directly for coloration as they do not have any affinity for the substrates the encapsulation is required [7].
Preparations of photochromic micro-and nanocapsules have been published by several authors [3,8,9].Zhou et al. [3] prepared melamine-formaldehyde microcapsules encapsulating photochromic compounds (two spirooxazine dyes and two naphtopyran dyes) with particle sizes of less than 5 m by in situ polymerisation.Han et al. [10] described the preparation of styrene photochromic nanocapsules containing diarylethenes and spirobenzopyran.In order to obtain nanosized capsules with average diameters of 50-150 nm, they adopted the miniemulsion polymerisation method.Photoresponsive devices require that either the nanostructure or the molecular structure be responsive to an external stimulus, in this case, light [11].
Spirooxazine dyes open their oxazine rings upon UV irradiation; the concentration of spirooxazine in its ringopened form increases.The ring-opened merocyanine structure has a larger conjugated molecular system and is one of several photoisomers formed upon ring-opening.After UV exposure, the "ring-open" form returns automatically to a "spiro" noncoloured structure because the "spiro" form is thermodynamically a stable one [12].A schematic illustration of the photochromic effect supported by photography of spirooxazine dye within a nanocapsule is shown in Figure 1.
The ring-opened form has partial zwitterionic character.
In general the fabrication of nanocapsules is based either on the miniemulsion principle [24,25] using the differences of interfacial tension, or on the phase separation process [26].Amongst all the available methods, the polymer emulsionsolvent evaporation method is the more common [27].The main issue is obtaining homogeneous dispersions.Mechanical stirring is often applied for improving the homogeneity of the dispersion but can hardly prevent particles from aggregation or agglomeration.In order to overcome this problem ultrasound irradiation has proved to be very efficient for eliminating agglomeration [28,29].
Ultrasound irradiation of liquids induces a phenomenon called acoustic cavitation, which means the formation, growth, and implosion of microbubbles.The extreme temperature and pressure released during adiabatic bubble collapse are normally accompanied by shear forces and high speed microjects [30,31].The powerful stirring effect of ultrasound is mainly due to the microstreaming caused by the oscillations of numerous resonant bubbles before they collapse.In this respect, intensities and durations of the ultrasound irradiation are important factors affecting the process.In general, to improve any process assisted by ultrasonic irradiation there exist a threshold limit and optimum operational parameters in the light of acoustic intensity, acoustic density, irradiation time, and so on needing to be determined.
This article presents a tailoring of the sizes of spirooxazine nanocapsules assisted by low frequency (20 kHz) ultrasound irradiation varying the acoustic densities.An ultrasound probe system was applied with two different accessories, namely, replaceable and microtip, that differentiate between probe areas.The acoustic density entering the system and the influence of the latter on spirooxazine nanocapsules' shape, average size, and polydispersity index (PDI) were systematically studied.The influence of ultrasound irradiation time on nanocapsules formation was studied as well.

Preparation of the Photochromic Nanocapsules
Organic Phase.70 mg of ethyl cellulose powder was dissolved in 4 mL dichloromethane in 50 mL vessel using magnetic stirring at room temperature (22 ∘ C).Different amounts of spirooxazine dye were added to the ethyl cellulose solution (5 mg, 10 mg, 20 mg, 30 mg, and 40 mg).The final concentrations of the spirooxazine dye in solution were 1.25, 2.5, 5.0, 7.7, and 10 mg/mL.Organic phase was continuously stirred at 600 rpm (electric overhead stirrer IKA C-MAG HS 7) at room temperature (22 ∘ C) until complete dissolution.
Water Phase.1% polyvinyl alcohol (PVA) water solution was prepared.The PVA powder was slowly added to water to avoid formation of lumps.Water phase was continuously stirred at 600 rpm (electric overhead stirrer IKA C-MAG HS 7) at room temperature (22 ∘ C) until the PVA is fully solubilized (at least 1 h).
The size of nanocapsules, synthesised by oil-water emulsion process, can be affected by numerous parameters.One important step was to find a suitable emulsifier and adjust its concentration.In previous study [32] PVA was found to be a very effective surfactant in a similar system for emulsifying organic droplets in an aqueous phase [8].According to previous study 1% w/v PVA was applied, and, upon ultrasound irradiation, nanocapsules were formulated.The increase of PVA concentration did not affect the size of the particles significantly [8].Spirooxazine nanoparticles have been synthesised with 1% w/v PVA.
The photochromic nanocapsules were synthesised according to the method described in the previous work [8] with some modifications.Photochromic nanocapsules were prepared by an oil-in-water emulsion using the solvent evaporation method.
The oil-in-water emulsion was formulated when 10 mL of water phase was added drop wise into prepared organic phase in 50 mL vessel.The two phases were treated with mechanical stirring process (Procedure 1) or with ultrasound irradiation (Procedure 2).
Procedure 1.The two phases were stirred at a constant speed of 800 rpm (electric overhead stirrer IKA C-MAG HS 7) at room temperature for 10 minutes in closed 50 mL vessels.The dispersion was then concentrated by continuous open-top stirring at room (22 ∘ C) for 2 hours.Upon complete removal of the dichloromethane from the dispersion by the evaporation during mechanical mixing, the formed capsules were stabilized.To remove PVA, the capsules were centrifuged by an Eppendorf Centrifuge MiniSpin with 11,000 rpm for 30 min and supernatant was removed and redisposed within the distilled water for further analysis.
Procedure 2. The two phases were treated with ultrasound irradiation for different periods of time in continuous mode.
Different acoustic densities, determined via calorimetric measurements, and ultrasonic irradiation durations (30-180 s) were applied.Irradiation was performed using 20 kHz low frequency ultrasound equipment from Sonics & Materials.Two different ultrasound probe systems were used; probe with replaceable tip and with tip area of 1.3 cm 2 , and tapered microtip with tip area of 0.07 cm 2 .
All experiments were performed in an ice-water bath (0-3 ∘ C) to keep the reaction mixture at constant temperature during ultrasound irradiation.The dispersion was then concentrated by continuous open-top stirring at room (22 ∘ C) for 2 hours.Upon complete removal of the dichloromethane from the dispersion by the evaporation during mechanical mixing, the formed capsules were stabilized.To remove PVA, the capsules were centrifuged by an Eppendorf Centrifuge MiniSpin with 11,000 rpm for 30 min and supernatant was removed and redispersed within the distilled water for further analysis.
All experiments were run in triplicate.

Characterisations of Ultrasound Systems.
The transducer's efficiency for electrical to sound conversion was generally limited due to the fact that the real power dissipating into the liquid was determined by calorimetry.Acoustic power entering the system was defined using calorimetric measurements [33].
During the calorimetric measurements the cooling jacket contained air instead of water in order to minimize heat losses.For the selected amplitudes' measurements, the temperature () was recorded against time () at 10 s intervals using a thermocouple placed within the reactor.From the  versus  data, the temperature rise (/) was estimated by fitting the data to a polynomial curve.The dissipated power was determined according to the equation [34] where   is the heat capacity of the water,  is the mass of water, and (/) represents the initial slope of the temperature rise versus time.

Scanning Electron Microscope.
A scanning electron microscope (SEM) was used for observing the encapsulated spirooxazine dye within ethyl cellulose nanocapsules.The samples were examined using a Philips XL-30 environmental scanning electron microscope at 25 kV.
After synthesis the photochromic nanocapsules were centrifuged by an Eppendorf Centrifuge MiniSpin with 11,000 rpm for 30 min and redispersed in distilled water for the scanning electron microscope measurements.

Measurements of Photochromic Nanocapsules Size and
Polydispersity Index (PDI).Photochromic nanocapsules size and polydispersity index (PDI) were determined by the Zetasizer Nano ZS5 (Malvern Instruments, Ltd., UK) equipped with DLS technology.Triplicate measurements were carried out using He-Ne laser at wavelength of 633 nm and at scattering angle of 173 ∘ at 20 ∘ C. Intensity weighted average hydrodynamic diameter was computed from intensity autocorrelation data using the cumulative method with DTS (nano) software (SOP) provided with the instrument.The -average diameter was reported as the mean droplet diameter of the emulsion.The PDI was a dimensionless measurement of the width of size distribution calculated from the cumulate analysis and ranged from 0 to 1.0.A small PDI value indicates an almost monodispersed population, whilst a large PDI indicates a very broad particle-size distribution.The calculations are defined in the ISO standard document 13321 : 1996 E.

Confocal Laser Microscope.
The confocal and multiphoton microscope system Leica TCS SP5 MP is suitable for deep imaging.The system has no limitation regarding IR laser configurations.Photochromic dyes in nanocapsules were detected using an argon laser at 488 nm.

Effect of Ultrasonic Irradiation on the Characteristics of the Ethyl Cellulose Spirooxazine
Nanocapsules.Preparation of ethyl cellulose nanocapsules encapsulating 10 mg of spirooxazine dye was carried out using the oil-in-water emulsion evaporation process.After the addition of the water phase to the organic phase, the two phases were treated, firstly with the mechanical stirring process (Procedure 1), and, secondly, with ultrasonic irradiation in continuous mode for 60 s (Procedure 2).Ultrasonic irradiation was performed with low frequency probe type ultrasound (20 kHz) using two different accessories, namely, the replaceable tip and the microtip.The systematic study of the influence of acoustic density on nanocapsules average size formations and PDI values was performed by applying different acoustic densities for the replaceable tip (Table 1) and for the microtip (Table 2), which differentiate in the active vibrating area.
Compared to mechanical stirring, low frequency high intensity ultrasound offers a better alternative technique during the preparation of more uniform sized capsules     Nevertheless, the results presented in Tables 1 and 2 indicated that the acoustic density had an important influence on the spirooxazine nanocapsules' average sizes and their PDI values regardless of the used concentration of dye.In the case of the replaceable tip a decrease in nanocapsules average size was observed with increasing acoustic density reaching the minimum average size of 193 nm and PDI value of 0.067 at acoustic density of 1.5 W/mL.After that the subsequent increase of acoustic density resulted repeatedly in gradual increases of the average sizes of those nanocapsules reaching the average size of 404 nm at maximum acoustic density of 8.2 W/mL.This observation could be explained by too high vibrational amplitudes leading to decoupling between the vibrating plate and the liquid near the radiating surface resulting in a cloud of bubbles, which diminish the penetration of the sound into the liquid [35].
The tapered microtip had in comparison to the replaceable tip an almost 20-fold smaller vibrating area.The same trend can be observed regarding the maximum plateau of acoustic density necessary for the minimum possible average size acquisition of nanocapsules with uniform size distribution as evident from the PDI measurements.In the case of the tapered microtip, an acoustic density of 1.07 W/mL seems to be the optimum ultrasound parameter for the production of nanocapsules with average sizes of 216 nm and with PDI of 0.091.
The particle sizes observed by SEM were in good agreement with the dynamic light scattering investigations.Figure 2 presents SEM images of ethyl cellulose nanocapsules containing spirooxazine dye prepared with the support of classical mechanical stirring (a) and with the use of ultrasound irradiation at acoustic density of 1.5 W/mL and sonication time of 60 s (b).
We proved that with nanoencapsulation of spirooxazine dye the photochromic effect still remained.Photo-induced reversible structural transformation of incorporated spirooxazine dye still responded to the light.Upon UV irradiation the color of the nanocapsules with spirooxazine dye turned blue (Figure 1) and then bleached within a few minutes.According to naked eye observation there were no significant differences in the photochromic effect when the sizes of the nanocapsules decreased to average size of about 193 nm.

Effect of Spirooxazine Dye Mass.
In this set of experiments, the effects of five different masses (5 mg, 10 mg, 20 mg, 30 mg, and 40 mg) of spirooxazine dye in dichloromethane (4 mL) solution on the size distributions of photochromic nanoparticles were investigated.All experiments were performed with a replaceable tip at optimum acoustic density of 1.5 W/mL and sonication time of 60 s.The smallest, averagesized nanocapsules of 193 nm were obtained when 10 mg of the dye was used.Increase in the spirooxazine dye mass from 10 to 40 mg slightly increased the sizes of the nanocapsules, namely, up to 240 nm at 40 mg of the dye.Nevertheless, low values of the polydispersity indexes in all cases indicated an almost monodispersed population of nanocapsules prepared under the above stated experimental conditions.We can conclude that the mass of the spirooxazine dye did not significantly affect the average size of the photochromic nanocapsules (Figure 3).In parallel during the same experimental conditions nanocapsules in the absence of dye were prepared.The average size of the nanocapsules without the spirooxazine dyes was 206 nm with PDI 0.074.

Effect of the Sonication Time.
The influence of the ultrasonic irradiation duration on the spirooxazine nanocapsules average size was also studied.Experiments were performed with replaceable tip at 1.5 W/mL of acoustic density with a spirooxazine dye mass of 30 mg, whilst we concluded that the masses of the spirooxazine dye did not significantly influence  the average size of the photochromic nanocapsules.Sonication was applied for 30, 60, 120, and 180 s after the addition of the water phase to the organic phase.From the results presented in Figure 4 it is evident that an optimum sonication time exists when producing nanocapsules of average size below 200 nm.Any increase in irradiation times resulted in gradual increases in the average size.The reason for this phenomenon probably lies in the small working volume in which the ultrasound waves after a certain time period start to interfere and cause the so-called cushioning effect.The nanocapsules diameter and polydispersity index sharply decreased as the ultrasonic irradiation time increased up to 30 s.The smallest average size of nanocapsules was obtained at the sonication time of 60 s.With prolonged sonication up to 180 s we were unable to produce nanocapsules below 200 nm.Sample in Figure 4, at time 0 s, presented photochromic capsules prepared via classical mechanical stirring (Procedure 1), rather than via ultrasonic irradiation.

Spirooxazine Nanocapsules Stabilities over Time.
As mentioned earlier, spirooxazine nanocapsules were prepared by an oil-in-water emulsion using the solvent evaporation method, by adding the water phase to the organic phase.For physical stability measurements of 30 mg of spirooxazine dye were used; the irradiation time was 60 seconds with acoustic densities of 0.71 W/mL and 1.5 W/mL.Both experiments in Table 3 were run in triplicate.The spirooxazine nanocapsules prepared according to the Procedure 2 were stored in distilled water at room temperature for 6 months.The results showed that the average size of spirooxazine nanocapsules did not change; moreover, under these storage conditions we did not observe any aggregation (Table 3).The photochromic effect during this period still remained.As shown in Figure 5 the spirooxazine nanocapsules were spherical.Based on confocal laser microscope images we supposed that the significant part of nanocapsules interior corresponds to spirooxazine dye molecules.

Conclusions
The influence of ultrasound irradiation on the ethyl cellulose spirooxazine nanocapsules formation and average size distributions was systematically studied by varying the acoustic densities and process times.We were able to prepare spherically uniform sized nanocapsules with diameter sizes below 200 nm simply by optimising experimental conditions related to the acoustic densities and irradiation times.At 1.5 W/mL and sonication time of 60 s we obtained the smallest average size of spirooxazine nanocapsules, that is, 193 nm and PDI 0.067, prepared with 10 mg of dye.In all cases the spirooxazine nanocapsules did not agglomerate and they conserved the photochromic effect over time.Preparation of the uniform sized nanocapsules is important for future applications.Our future applications will focus on studies related to the photochromism as well as the incorporation of spirooxazine nanocapsules into silica matrices for sensing application.

Figure 1 :
Figure 1: Schematic illustration of the photochromic effect of spirooxazine dye within a nanocapsule from "spiro" to "ring-open" form upon UV irradiation.
values of almost 1, indicating a very broad distribution of the particle sizes.In contrast ultrasonic irradiation was essential for the production of uniform sized nanocapsules below 1 m.

Figure 3 :
Figure 3: Average sizes (nm) and PDIs of spirooxazine nanocapsules in the presence of different spirooxazine concentrations.

3. 5 .
Confocal Laser Microscopic Analysis.Spirooxazine nanocapsules, spirooxazine dye, and empty nanocapsules were characterised by the confocal laser scanning microscopy (CLSM), in order to confirm that the interior of nanocapsules is fulfilled with spirooxazine dye molecules.Comparably, the visualisation of spirooxazine nanocapsules was possible also using normal light microscope (Figures5(a), 6(a), and 7(a)).Spirooxazine nanocapsules, spirooxazine dye, and empty nanocapsules were excited with an argon laser beam at 488 nm and 10x magnification.CLSM images of spirooxazine nanocapsules are shown in Figure 5(b) and CLSM images of spirooxazine dye and empty nanocapsules are shown in Figures 6(b) and 7(b).CLSM image (Figure 5(b)) of spirooxazine nanocapsules illustrated localization of spirooxazine dye within the ethyl cellulose nanocapsules.CLSM image of spirooxazine dye and CLSM image of empty nanocapsules confirm this.

Figure
Figure (a) Visualisation of empty nanocapsules by light microscopy image.(b) Confocal (CLSM) images of empty nanocapsules.

Table 1 :
). Effects of acoustic density with replaceable tip on the average sizes and PDI values of ethyl cellulose nanocapsules containing spirooxazine dye.
*All experiments were run in triplicate.

Table 2 :
Effects of acoustic density using tapered microtip on the average sizes and PDI values of ethyl cellulose nanocapsules containing spirooxazine dye.
*All experiments were run in triplicate.within the nanoscale range.Classical mechanical stirring was insufficient in the preparation of those photochromic capsules with average diameter sizes less than 1 m and with small values of PDI.Samples number 1 in Table1presented photochromic capsules prepared with classical mechanical stirring according to Procedure 1.The sizes of capsules synthesised without ultrasonic irradiation were above 1 m with PDI