Facile preparation of artemisia argyi oil-loaded antibacterial microcapsules by hydroxyapatite-stabilized Pickering emulsion templating

Colloids and Surfaces B: Biointerfaces 112 (2013) 96–102

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Facile preparation of artemisia argyi oil-loaded antibacterial microcapsules by hydroxyapatite-stabilized Pickering emulsion templating Yang Hu, Yu Yang, Yin Ning, Chaoyang Wang ∗ , Zhen Tong Research Institute of Materials Science, South China University of Technology, Guangzhou 510640, China

a r t i c l e

i n f o

Article history: Received 5 June 2013 Received in revised form 30 July 2013 Accepted 2 August 2013 Available online xxx Keywords: Artemisia argyi oil Hydroxyapatite nanoparticle Antibacterial microcapsule Pickering emulsion Controlled release

a b s t r a c t Artemisia argyi oil (AAO)-loaded antibacterial microcapsules with hydroxyapatite (HAp)/poly(melamine formaldehyde) (PMF) hybrid shells were facilely prepared by oil-in-water (O/W) Pickering emulsion templating. AAO-in-water emulsions were stabilized using HAp nanoparticles as the particulate emulsifier. The hybrid shells were fabricated by in situ polymerization of melamine formaldehyde pre-polymer (pre-MF) at the interface of the O/W Pickering emulsions. The prepared microcapsules were characterized in terms of size, morphology, component and thermal stability using scanning electronic microscope (SEM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), respectively. Moreover, both in vitro release and antimicrobial activity of the microcapsules were also evaluated. The results showed that the AAO-loaded microcapsules with HAp/PMF shells had a spherical shape and a rough surface. The microcapsules maintained excellent performances in the thermal stability, controlled release activity, antimicrobial effect and long-term antimicrobial activity. The release curves of AAO from the microcapsules could be well described by Higuchi kinetic model. The microcapsules may find applications as antibacterial agents in the areas of textiles, leather, rubber and coatings. In situ polymerization based on Pickering emulsion droplets opens up a new route to prepare a variety of hybrid microcapsules with a core-shell structure. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Microcapsules have received considerable attention in both academic and industrial areas, because they can protect functional materials against contacting with the external environment and control the release behaviors of encapsulated materials [1]. In recent years, an encapsulation method has been developed on the basis of Pickering emulsions [2]. Pickering emulsions are solid particle-stabilized emulsions, where solid particles are adsorbed at an oil–water interface [3–5]. Compared with the conventional emulsions, Pickering emulsions show an excellent emulsion droplet stabilization [6–8], owning to the nearly irreversible adsorption of solid particles at the oil–water interface. Thus, Pickering emulsions are stable enough to protect the core materials in the microencapsulation procedure. Recently, there has been increasing interest in using the Pickering emulsion templates to form drugloaded microcapsules.

∗ Corresponding author. Tel.: +86 20 22236269. E-mail address: [email protected] (C. Wang). 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.08.002

There are a number of solid particles acted as particulate emulsifiers, including silica [9], titania [10,11], clays [12,13], microgels [14], metals [15,16], polymer latexes [17], flavonoids [18], and hydroxyapatite (HAp) [19]. As particulate emulsifiers, the surface property of solid particles is very important, such as charge, shape, wettability and interaction between particles [20]. Thus, the studies on the surface property and surface modification of particles [21–24] are quite significant for the preparation of the Pickering emulsions. Among the above-mentioned particles, HAp has attracted increasing attention to be used as Pickering emulsifiers, due to its biocompatibility, low cost and easy preparation. Fujii and co-workers [25,26] used HAp nanoparticles as the particulate emulsifiers to prepare oil-in-water (O/W) Pickering emulsions in the absence of any molecular surfactants. Methyl myristate was employed as oil phase to prepare porous material by sintering; dichloromethane solution of poly(l-lactideco-␧-caprolactone) was used as oil phase to fabricate injectable scaffold by solvent evaporation method. However, they did not used HAp nanoparticles to prepare drug-loaded microcapsules. Artemisia argyi oil (AAO), the main medicinal component of artemisia argyi, is an important raw material used in cosmetics, pharmaceuticals and perfume products, due to its broad-spectrum antibacterial, antifungal, anti-inflammatory,

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Fig. 1. Schematic illustration of the preparation of AAO-loaded microcapsules with HAp/PMF shells based on HAp-stabilized O/W Pickering emulsion templates.

anti-histamatic, antioxidant effects and aromatic flavor [27]. AAO is composed of ethers, alcohols, ketones, monoterpenes and sesquiterpenes, etc. [27,28]. It is relatively unstable in the presence of air, light and heat, which greatly limit its application. Hence, the microencapsulation seems to be a good choice to solve this problem, which can enhance the stability, prolong the efficiency, and expand the potentials of AAO applications. In this study, we reported a simple and effective method to fabricate AAO-loaded antibacterial microcapsules with HAp/poly(melamine formaldehyde) (PMF) hybrid shells by O/W Pickering emulsion templates. First, we described the synthesis and characterization of HAp nanoparticles and their use as the particulate emulsifier to prepare O/W AAO Pickering emulsions. Then, we demonstrated the preparation of AAO-loaded microcapsules by in situ polymerization of melamine formaldehyde pre-polymer (pre-MF) on the surface of the AAO Pickering emulsion droplets. Finally, the release behavior and antimicrobial activity of the AAO-loaded microcapsules were investigated. 2. Experimental 2.1. Materials Calcium nitrate (Ca(NO3 )2 ·4H2 O) was obtained from Taishan Yueqiao Reagent and Plastic Co., Ltd., China. Diammonium hydrogen phosphate ((NH4 )2 HPO4 ) was supplied by Tianjin Fuchen Chemical Reagent Co., Ltd., China. AAO, citronella oil and patchouli oil were bought from Jiangxi Jishui Kangshen Natural Medicinal Oil Refinery, China. Radix isatis oil was extracted by ethanol extraction method using a Soxhlet apparatus at 90 ◦ C for 6 h. Melamine was obtained from Sinopharm Chemical Reagent Co. Ltd., China. Formaldehyde aqueous solution (37%) was supplied by Guangzhou Donghong Chemical Factory, China. Polyvinyl alcohol (PVA), 25% ammonia aqueous solution, triethanolamine (TEA), acetic acid and hexane were supplied by Guangzhou Chemical Reagent Factory, China. All chemicals were used as received. Water used in all experiments was deionized and filtrated using a Millipore purification apparatus (MA, USA) to a resistivity higher than 18.0 M cm. 2.2. Synthesis of HAp nanoparticles HAp nanoparticles were synthesized by wet chemical precipitation method reported by Fujii et al. [25,29] with slight modification. The detailed synthesis procedures were as follows. Ca(NO3 )2 aqueous solution (21 mM, 400 mL) and (NH4 )2 HPO4 aqueous solution (50 mM, 100 mL) were prepared respectively, and their pH values were both adjusted to 12.0 by addition of 25% ammonia solution. Then, Ca(NO3 )2 aqueous solution was poured into a 500 mL threeneck flask equipped with an inlet of N2 and a magnetic stirrer. After the temperature in the flask had been equilibrated at 25 ◦ C,

(NH4 )2 HPO4 aqueous solution was added into the flask within 20 s, and the system was stirred continuously for another 10 h at 25 ◦ C. After reaction, the resultant suspension was aged for 12 h at 25 ◦ C. The resulted HAp dispersions were obtained by centrifugation and washed with water three times. 2.3. Preparation of HAp nanoparticle-stabilized AAO Pickering emulsions First, 8 mL of HAp nanoparticle dispersion (1.0 wt%, pH = 8) was prepared by ultrasonication at room temperature. Thereafter, 2 mL of AAO as the oil phase was added to the above HAp nanoparticle dispersion and the mixture was emulsified at 10,000 rpm using IKA Ultra Turrax T25 homogenizer for 2 min at room temperature. The fabrication strategy is illustrated in Fig. 1. 2.4. Synthesis of pre-MF Pre-MF was synthesized based on the method reported by Meng et al. [30] with some modification. Melamine of 0.03 mol, formaldehyde of 0.1 mol, and water of 10 mL were mixed in a 50 mL three-neck flask equipped with a magnetic stirrer. Then, the pH value of the system was adjusted to 8.5–9.0 by adding triethanolamine (TEA), meanwhile the system was heated to 60 ◦ C. After 30 min of continuous agitation, the pre-MF solution was obtained. 2.5. Preparation of AAO-loaded microcapsules The above-mentioned pre-MF solution was added into a 100 mL three-neck flask containing 20 mL of 0.1 wt% PVA aqueous solution, in which PVA was used as a protective colloid. Then, the pH value of the solution was adjusted to 5.5–6.0 by adding acetic acid, while the solution was heated to 50 ◦ C. Afterwards, AAO Pickering emulsion was added to the above solution, and the reaction mixture was stirred continuously for 3 h. The obtained microcapsules were washed with water to remove free PMF particles suspended in water solution [8]. Finally, the resultant microcapsules were dried at room temperature for 24 h in a sealed desiccator containing phosphorus pentoxide and saturated AAO vapor. The fabrication strategy is illustrated in Fig. 1. 2.6. AAO loading capacity and encapsulation efficiency The AAO loading capacity and encapsulation efficiency of the microcapsules were determined by the extraction method as follows. First, W0 g of AAO was used to fabricate the AAO-loaded microcapsules as the above process. The obtained microcapsules were weighted (W1 g). They were ground in a mortar with a pestle at room temperature. Then, hexane was added in the ground

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microcapsules to extract AAO. After centrifugation at 8000 rpm for 5 min, the sediment was collected. This procedure was repeated three times. Finally, after vacuum-drying at room temperature for 24 h, the sediment was weighed (W2 g). All experiments were carried out in triplicate, and the mean values were calculated. The loading capacity (LC) and encapsulation efficiency (EE) were calculated according to the following equations, respectively: LC =

EE =

W − W  1 2 W1

W − W  1 2 W0

× 100%

(1)

× 100%

(2)

2.7. In vitro release In the in vitro release experiments, the required portions of AAO-loaded microcapsules, each weighing Wa g and containing Wao g AAO, were placed in an oven with a specific temperature to release the AAO. At predetermined time intervals, the samples were taken out and weighed to obtain the residual weight of the microcapsules (Wb g). All determinations were carried out in triplicate, and the mean values were calculated. The cumulative release rate (CR) was determined by the following equation: CR =

W − W  a b Wao

× 100%

(3)

2.8. Antibacterial activity of AAO-loaded microcapsules The antibacterial activity of the microcapsules was performed against both Staphylococcus aureus and Escherichia coli by the plate counting method as follows. Firstly, the inoculation bacterial suspensions containing about 106 –107 CFU/mL of the bacterial strains were obtained by suspending bacteria in sterilized broth culture. Then, 0.08 g of AAO-loaded microcapsules and 0.1 mL of the inoculation bacterial suspension were added to 10 mL of sterilized PBS solution. The mixture was incubated at 37 ◦ C for 24 h by a shaker. Afterwards, the suspension was gradient diluted with PBS solution based on the 10-fold dilution method for several times. 0.1 mL of the mentioned suspension at proper dilution degree was taken out and plated on an agar plate. The inoculated plate was cultured at 37 ◦ C for 24 h, and the number of bacteria colonies was counted. The control suspension containing bacteria and devoid of the microcapsules was used as blank control. Each experiment was carried out in triplicate, and mean values were calculated. The degree of antibacterial effect was considered as the inhibition rate of the bacteria growth by the following equation [31]: R (%) =

A − B A

× 100

(4)

where R is the percentage inhibition rate, A represents the number of bacterial colonies from the control suspension (without the microcapsules), and B is the number of bacterial colonies from the sample suspension (with the microcapsules). 2.9. Characterization The average diameter and zeta potential of HAp nanoparticles dispersed in water with different pH values were determined with a Malvern Zetasizer Nano ZS90. The X-ray diffraction (XRD) pattern of HAp nanoparticles was obtained using an X’pert PRO diffractometer (40 kV and 40 mA) equipped with a Cu K␣ radiation (wavelength 0.154 nm) at room temperature. The diffractogram was obtained in the range from 15◦ to 80◦ at scanning speed of 10◦ /min. The HAp nanoparticles were observed using a JEM-100CXII transmission

Fig. 2. (a) TEM image of HAp nanoparticles. (b) Zeta potential and mean particle size of HAp nanoparticles versus the pH value of the HAp nanoparticle aqueous dispersion. Error bars indicate SD (n = 3).

electron microscopy (TEM) at an accelerating voltage of 200 kV. The Pickering emulsions and AAO-loaded microcapsules were observed with an optical microscope (Carl Zeiss, German). The average diameter of the Pickering emulsion was determined with a Malvern Mastersizer 2000. The morphology images of HAp nanoparticles and the microcapsules were obtained using a Zeiss EVO 18 scanning electron microscope (SEM) operating at 10 kV. Fourier transform infrared (FTIR) spectra of the samples were recorded using a German Vector-33 IR instrument. The thermal gravimetric analysis (TGA) of the samples were performed using a thermo-analyzer (TG 209, NETZCH Co.) from 40 to 600 ◦ C at a heating rate of 10 ◦ C/min under nitrogen atmosphere. Photographs were captured by a digital camera (IXUS 220HS, Canon). 3. Results and discussion 3.1. Preparation of HAp nanoparticles HAp nanoparticles were used as the particulate stabilizer to form the O/W Pickering emulsions. The XRD pattern indicated that HAp nanoparticles prepared at 25 ◦ C had low crystallinity (see Fig. S1a), which was in agreement with the results reported in the literature [25,32]. TEM and SEM observations of the dried HAp nanoparticles showed that HAp nanoparticles had an approximately spherical shape, and the particle diameters were mostly in the range between 30 and 70 nm (Figs. 2a and S1b). Zeta potential and dynamic light scattering (DLS) studies of highly dilute aqueous dispersions (0.01 wt%) of HAp nanoparticles were carried out in a wide range of pH between 4.0 and 11.0 using a Malvern Zetasizer. The

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zeta potential curve of HAp nanoparticles had a classic “L” shape, and the isoelectric point was at pH approximately 5.8 (see Fig. 2b). The value of the zeta potential reflected the surface charge of HAp nanoparticles which could affect the aggregation behavior of HAp nanoparticles in the aqueous dispersions. The low zeta potential can lead to an increase in the effective HAp particle size by flocculation. DLS studies on the HAp nanoparticles indicated the intensityaverage diameters were over 190 nm (see Fig. 2b) at pH from 4.0 to 11.0 even after intensive ultrasonication for 30 min. From Fig. 2b, it was also observed that the nearer to pH 5.8, the bigger intensityaverage diameter of HAp nanoparticles. The local maximum of particle size coincided with isoelectric point, which indicated that the electrostatic interaction between the HAp nanoparticles played a critical role in the stability of the suspension. Therefore, this colloidal instability should be mainly attributed to the low zeta potential, which can easily lead to flocculation. It has been reported that the particles in their weakly flocculated states have good emulsion stability [33,34]. Therefore, the weakly flocculated HAp nanoparticles would work as the particulate emulsifier and were expected to play a significant role in the preparation of stable emulsions. 3.2. Formation of AAO Pickering emulsions Colloidal particle interfacial self-assembly at oil–water interface to form Pickering emulsions has been well investigated in the literature [35–39]. Herein, HAp nanoparticle-stabilized AAO Pickering emulsions were prepared in the aqueous media without any traditional molecular surfactants. The HAp nanoparticles self-assembling at AAO–water interface could strongly reduce the system interfacial free energy and form a rigid particle interfacial layer [40], which could effectively stabilize AAO Pickering emulsions. A typical optical micrograph of the emulsion is shown in Fig. 3a. The type of the prepared emulsion was measured by ‘drop test’. Four drops of the emulsion were added to water or AAO. The emulsion drops dispersed rapidly in water, while the emulsion drops aggregated in AAO and sedimented at the bottom of the vessel (see Fig. S2). As we know, the diluent in which the emulsion drops dispersed quickly is the continuous phase of the emulsion. On the basis of what we just mentioned, the type of the prepared Pickering emulsion was an O/W emulsion. The stable emulsion can be stored over 5 months (see Fig. S3). It is well known that the formation of the Pickering emulsion is significantly influenced by many factors, such as the pH value, the volume ratio of oil to water, the kind of oil, and the particulate stabilizer concentration. Herein, the effect of initial pH of the HAp nanoparticle dispersion on the emulsion stability was investigated (see Fig. S4). The emulsions were prepared above pH 4.9, while no stable emulsion was obtained below pH 4.0. According to the literature [25], Ca2+ and PO4 3− ions derived by acid-dissolution of HAp (Ca10 (PO4 )6 (OH)2 ), and/or their small clusters such as Ca9 (PO4 )6 cannot act as efficient emulsifiers. Therefore, no stable emulsion obtained below pH 4.0 should be ascribed to the increasing solubility of HAp. This indicated that HAp nanoparticles worked as effective Pickering emulsifiers should be in their non-dissolved states. The optical micrographs of the corresponding emulsions are shown in Fig. S5. The effect of the volume ratio of oil to water on the formation of AAO Pickering emulsions was also studied, and the optical micrographs of the corresponding emulsions are illustrated in Fig. S6. At all volume ratios of oil to water (1:1, 1:2, 1:4, and 1:8), the stable emulsions could be obtained. In addition, four oils extracted from Chinese medicinal herbs (AAO, citronella oil, patchouli oil, radix isatis oil) were selected for preparing Pickering emulsions. The above-mentioned four oils all exhibit some excellent properties, such as antibacterial, anti-inflammatory and antifungal effect. However, they are unstable in the presence of air, light and heat. As shown in Fig. S7, in all four cases, stable

Fig. 3. (a) Optical micrograph of AAO Pickering emulsion prepared by the HAp nanoparticle concentrations of 1.0 wt%. (b) The size distribution graphs of the AAO Pickering emulsions prepared by the HAp nanoparticle concentrations of 0.1, 0.5, 1.0 and 2.0 wt%. The AAO to water ratio is 1:4, pH of the HAp nanoparticle dispersion is 8.

Pickering emulsions could be easily obtained regardless of different properties of these oils (e.g. viscosity). We also investigated the effect of HAp nanoparticle concentrations (0.1, 0.5, 1.0, and 2.0 wt%) on the formation of the AAO Pickering emulsions. The optical micrographs of the corresponding emulsions showed that polydisperse spherical emulsion droplets were prepared in each case (Fig. S8a1 –d1 ). And the number-average diameter of the emulsion droplets ranged from 23.9 ␮m to 46.6 ␮m (see Fig. 3b). It was observed that increasing the HAp nanoparticle concentration in the aqueous phase lead to the decrease in the mean droplet diameter. This result is consistent with the previous report [41]. Therefore, the AAO Pickering emulsions with different sizes can be obtained easily by tuning the HAp nanoparticle concentrations. 3.3. Fabrication of microcapsules The AAO-loaded microcapsules were facilely and high effectively fabricated by in situ polymerization of pre-MF at the interface of the HAp nanoparticle-stabilized AAO Pickering emulsions. After adding AAO Pickering emulsions into pre-MF solution, the HAp particle interfacial layer became thicker as a result of the aggregation of pre-MF onto the HAp nanoparticle surfaces [42], which was caused by the electrostatic attraction [43] between the negative charges on the HAp nanoparticle surfaces and the positive charges of pre-MF.

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Fig. 4. SEM images of AAO-loaded microcapsules (a) and a ruptured microcapsule (b). The insets in micrographs (b) show magnified image. The HAp nanoparticle concentration is 1.0 wt%, the AAO to water ratio is 1:4, and pH of the HAp nanoparticle dispersion is 8.

With the above rigid interfacial layer, these AAO Pickering emulsions were extremely stable in the procedure of shell-forming to protect the core materials. If the resultant microcapsule suspension was set for 1 h, the microcapsules would sediment in the aqueous medium due to gravity. However, the microcapsules could easily redisperse in the aqueous medium by shaking. The morphology images of the microcapsules were observed using optical microscopy (see Fig. S8a2 –d2 ) and SEM images (see Fig. 4a). The images showed that the microcapsules maintained a spherical shape and possessed a rough surface. The number-average diameter of the microcapsules was a little larger than the diameter of the corresponding emulsion droplets. This difference should be attributed to the thickness of PMF shells formed by the self-crosslinking of pre-MF on the surface of the emulsion drops. Moreover, some of the ruptured microcapsules were observed by SEM as shown in Fig. 4b. The result directly conformed that the microcapsules had a core-shell structure. Loading capacity and encapsulation efficiency of the drug are important indices for the preparation of drug-loaded microcapsules. The effect of different HAp nanoparticle concentrations on the loading capacity and encapsulation efficiency of the AAOloaded microcapsules were investigated. The loading capacities of the microcapsules prepared at 0.1 wt%, 0.5 wt%, 1.0 wt% and 2.0 wt% of HAp nanoparticle concentrations were 16.7 ± 0.9%, 22.5 ± 1.1%, 26.6 ± 1.5%, 28.4 ± 0.7%, respectively, and the corresponding

Fig. 5. (A) FTIR spectra of (a) HAp nanoparticles, (b) pure PMF, (c) microcapsule shells, (d) AAO-loaded microcapsules, and (e) AAO. (B) TGA curves of (f) microcapsule shells, (g) AAO-loaded microcapsules, and (h) AAO.

encapsulation efficiencies were 52.7 ± 1.4%, 70.2 ± 2.5%, 83.7 ± 3.8%, 87.1 ± 1.2%, respectively. It was shown that increasing the HAp nanoparticle concentration in the aqueous phase observably increased the loading capacity and encapsulation efficiency. The reason should be ascribed to the electrostatic attraction between the negative charges on the HAp nanoparticle surfaces and the positive charges of pre-MF, which availed for the formation of PMF shells on the surface of the emulsion drops by self-crosslinking of pre-MF. FTIR studies were conducted to confirm whether the above AAO-loaded microcapsules were prepared successfully or not (see Fig. 5A). In the FTIR spectrum of the pure HAp (Fig. 5A-a), the peaks at 566 and 604 cm−1 are attributed to the bending vibration of PO4 3− . For the pure PMF (Fig. 5A-b), the peaks at 3350, 1348–1592, and 813 cm−1 are ascribed to the stretching vibration of N H and O H, the stretching vibration of C N and C N, and the stretching vibration of triazine ring, respectively. Compared with the FTIR spectrum of the pure PMF, the FTIR spectrum of the microcapsule shells has two weak peaks at 566 and 604 cm−1 (Fig. 5A-c and Fig. S9), which derive from the bending vibration of PO4 3− in HAp. This result demonstrates that the microcapsule shells are composed of PMF and HAp. Compared with the FTIR spectrum of the microcapsule shells, in the FTIR spectrum of the microcapsules (Fig. 5A-d), the new sharp peak at 1744 cm−1 appears and the peak at 2927 cm−1 becomes broader and stronger, which indicates that AAO exists in the microcapsules, because the peaks at 1744 and 2927 cm−1 are the characteristic peaks of AAO (Fig. 5A-e).

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Table 1 The results of fitting release profiles by different kinetic models. Temperature (◦ C)

Kinetic models Zero-order Qt = K0 t + C0

25 37 50

Higuchi Qt = KH t1/2 + CH

First-order ln(100-Qt ) = K1 t + C1

K0

C0

R0

K1

C1

R1

KH

CH

RH

0.0429 0.0132 0.1119

3.6541 6.6609 9.1584

0.7611 0.6810 0.6983

−0.0004 −0.0008 −0.0014

4.5677 4.5348 4.5061

0.7731 0.6993 0.7838

0.7822 1.3667 2.0708

1.1146 2.0817 2.2802

0.9328 0.8752 0.8855

Qt , the % cumulative release rate of AAO at time t. K0 , C0 , K1 , C1 , KH , CH : Constants of the corresponding kinetic models. R0 , R1 , RH : Correlation coefficients of the corresponding kinetic models.

According to the results of the FTIR spectra analysis, the AAO-loaded microcapsules with HAp/PMF shells were prepared successfully. TGA was also conducted to evaluate the thermal stability of the microcapsule shells, AAO-loaded microcapsules and AAO as shown in Fig. 5B. From the TGA curves, the weight of the microcapsule shells, the microcapsules and AAO decreased quickly from 290 to 450 ◦ C, 245 to 450 ◦ C, and 80 to 180 ◦ C, respectively. And the final residual weight of the microcapsule shells, the microcapsules, and AAO was 28.2, 16.1, and 4.1 wt%, respectively. The TGA analysis suggested that the thermal stability of the microcapsules was between the microcapsule shells and AAO. Then, it suggested that the thermal stability of AAO in the microcapsules was improved. Moreover, the loading capacity of the microcapsules was also measured using the mass data from the TGA curves according to the following equation: WMC = (1 − C)WML + CWC

(5)

where WMC , WML and WC are the final residual weight of the microcapsule shells, the microcapsules, and AAO, respectively. C represents the loading capacity of the microcapsules. By Eq. (5), it can be calculated that the loading capacity of the microcapsules (C) was 50%. The loading capacity of the microcapsules obtained by TGA far exceeded that measured by the extraction method (about 26.6%). It was noted that more free PMF particles had been removed from the microcapsules used for TGA method. 3.4. In vitro release study The release behavior of AAO from the microcapsules was investigated at 25, 37, and 50 ◦ C in an oven. The release profiles are shown in Fig. 6. It was seen that the release of AAO from the microcapsules was relatively fast in the first stage (within the first 72 h) followed

by an appreciably slow release rate over a study period. The initial weak “burst effect” was primarily attributed to a fast release of AAO on the surface and near the interior surface [44]. In the later phase, the slow release was ascribed to the decrease in the concentration difference of AAO between the inside and outside of the microcapsules. Furthermore, the release rate of AAO increased substantially with rising the temperature. This was ascribed to the fact that the volatility of AAO increased with increasing the temperature. In order to discern the release mechanisms, three different kinetic models (zero-order kinetic model, first-order kinetic model, and Higuchi kinetic model) were used to fit the release profiles. The results are listed in Table 1. The model with higher correlation coefficients was considered as the appropriate model for the release profiles [45]. From Table 1, it was seen that the values of correlation coefficient deriving from the linear regressions of Higuchi kinetic model were larger than that of other models. Thus, Higuchi kinetic model can well fit the release curves of AAO from the microcapsules. Higuchi kinetic model describes the release of drugs from insoluble matrix as a square root of time dependent process based on Fickian diffusion [46]. Therefore, the release of AAO from the microcapsules followed Fickian diffusion. 3.5. Antibacterial activity study The AAO-loaded microcapsules were stored in an unsealed beaker at room temperature, and their antimicrobial activities against S. aureus and E. coli at different storage times were investigated (see Table 2). The results showed that the microcapsules had strong antibacterial effect against S. aureus and E. coli, but the antimicrobial effect decreased with increasing the stored time. This was ascribed to the release of AAO from the microcapsules, which reduced the amount of antimicrobial compounds of the microcapsules. After storage for 60 days, the bacterial inhibition rate of the microcapsules against S. aureus and E. coli still kept over 86% and 83%, respectively. It demonstrated that the microcapsules had a long-term antimicrobial effect. In addition, it was observed that the antibacterial effect against E. coli was slightly lower than that against S. aureus. The reason was that compared with S. aureus, E. coli had an outer membrane outside the peptidoglycan layer, which could protect the bacteria cell from attacking by extraneous compounds in certain degree [47,48].

Table 2 Antibacterial test results of AAO-loaded microcapsules against S. aureus and E. coli.

Fig. 6. Release profiles of AAO from microcapsules. Error bars indicate SD (n = 3).

Storage time (d)

Bacterial inhibition rate (%) S. aureus

0 5 10 60

99.6 94.2 91.7 86.5

± ± ± ±

0.2 1.2 1.0 1.1

All values indicate mean ± S.D. for n = 3 independent observations.

E. coli 98.4 92.8 89.5 83.3

± ± ± ±

0.6 0.9 1.2 1.1

102

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4. Conclusions The AAO-loaded microcapsules with HAp/PMF shells were prepared by self-assembly of HAp nanoparticles at the interface of O/W emulsions and subsequent in situ polymerization of pre-MF. The prepared microcapsules had a spherical shape and rough surface. And the thermal stability of AAO in the microcapsules was higher than that of unpacked AAO. The release profiles of AAO from the microcapsules followed Higuchi kinetic model and release rate increased as the temperature rising. Moreover, the microcapsules had a long-term antimicrobial effect with bacterial inhibition rate against S. aureus and E. coli maintained as high as 83% even after storage for 60 days. Owning to the advantages of the microcapsules such as the reasonable thermal stability, the controlled release activity and the long-term antimicrobial activity of the microcapsules, we expect that the AAO-loaded microcapsules can bring up promising applications as antibacterial agents in the areas of textiles, leather, rubber and coatings.

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Acknowledgements This work was financially supported by the National Natural Basic Research Program of China (973 Program, 2012CB821500), the National Natural Science Foundation of China (21274046) and the Natural Science Foundation of Guangdong Province (S2012020011057). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.08.002.

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