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Research Paper

Journal of Pharmacy And Pharmacology

Realgar nanoparticle-based microcapsules: preparation and in-vitro/in-vivo characterizations Feng Shia,b, Nianping Fenga and Emmanuel Omari-Siawb a Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai, bSchool of Pharmacy, Jiangsu University, Zhenjiang, China

Keywords antitumour efficacy; bioavailability; in-vitro release; microcapsules; realgar nanoparticles Correspondence Nianping Feng, Department of Pharmaceutical Sciences, School of Pharmacy, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Zhangjiang Hi-Tech Park, Pudong New District, Shanghai 201203, China. E-mail: [email protected] Received June 15, 2014 Accepted July 27, 2014 doi: 10.1111/jphp.12314

Abstract Objectives The aim of this study was to prepare microcapsules for the oral delivery of realgar nanoparticles (RN) that are also capable of improving its stability. Methods RN and RN-based microcapsules (RNM) were prepared using ball milling and solvent evaporation techniques, respectively. Properties such as particle size, ζ-potential (ZP), morphology and X-ray diffractometer (XRD) were investigated. In addition, drug release, bioavailability and antitumour studies were also performed. Key findings The nanoparticles appeared round or elliptical in shape with a mean size of 85.4 ± 3.5 nm and a ZP of −34.3 ± 1.7 mV. The obtained RNM appeared spherical and not aggregated with a relatively narrow size distribution. XRD analysis revealed that ball milling technique did not change the crystallinity of the realgar powder. RN and RNM exhibited considerable higher release of As2S2, bioavailability and antitumour efficacies compared with crude realgar. Furthermore, RNM could protect RN directly exposed to the air and light, and therefore increased the stability of the RN. Conclusions The developed RNM demonstrated a greater potential as a delivery system for realgar.

Introduction Realgar (As2S2), also called Xiong Huang in China, has been traditionally used for many centuries. The first medicinal use of realgar was historically recorded in Shen Nong Ben Cao Jing, which was the first Chinese Materia Medica before 100 BC.[1] In ancient China, realgar was applied for the treatment of insect bites, abdominal pains, carbuncles, scalds and burns, infantile convulsions and psoriasis, among others.[2] Recently, with the confirmation of its clinical effectiveness for the treatment of some varieties of refractory or relapsed tumour/cancer, realgar has attracted increasing attention in the biomedical field.[3,4] Despite the pharmacological functions, realgar is insoluble in water and most organic solvents, resulting in poor bioavailability and limited clinical use.[5] It has been demonstrated that a particle-size reduction might significantly improve its solubility and extent of absorption.[6] To this end, realgar nanoparticles (RN) and several strategies have been proposed over the past few years. However, according to previous studies, the disintegration

of realgar particles was accompanied by an obvious increase in arsenic trioxide (As2O3),[7] which is known to be the most toxic arsenic species and can cause severe side effects when used clinically. In this regard, the amount of As2O3 in RN should be critically considered.[8] Besides the problem of high content of As2O3 in RN, the stability of the particle size also requires urgent attention. From our pre-experiments, it was found that the particle size increased constantly during storage. Therefore, a reasonable formulation is highly desired to overcome the aforementioned problems of RN. Microencapsulation has been used in the pharmaceutical industry for the conversion of liquids to solids, taste-masking of bitter drugs, acquiring prolonged or sustained payload (drug) release and stability enhancement of unstable drugs.[9] Given these features, we hypothesized that microcapsules were ideal delivery systems for RN, which might protect RN from being oxidized to As2O3 while enhancing its particle size stability.

© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 67, pp. 35–42

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Realgar nanoparticle-based microcapsules

Feng Shi et al.

Therefore, the aim of this study was to prepare microcapsules for the oral delivery of RN that are capable of improving its stability. In this study, both RN and RN-contained microcapsules were prepared, and their properties, such as particle size, ζ-potential (ZP), drug encapsulation efficiency (EE), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were evaluated. In addition, the in-vitro release, in-vivo bioavailability and antitumour efficacy were investigated.

Materials and Methods Animals Kunming strain mice (20 ± 2 g) and Wistar rats (200 ± 20 g) were purchased from the Laboratory Animal Center of the Shanghai University of Traditional Chinese Medicine. All the animals were acclimatized at a temperature of 25 ± 2°C and a relative humidity of 70 ± 5% under natural condition for 1 week with food and water provision. All experimental procedures were abided by the ethics of the Animal Ethical Committee, Shanghai University of Traditional Chinese Medicine (permit number SYXK (Hu) 2009-0069).

Drugs and chemicals Realgar was purchased from Shimeng, Hunan. As2O3 was provided by National Standard Material Center.

Assay of the arsenic trioxide content As2O3 content was assayed by the classical Silver diethyldithiocarbamate (Ag-DDC) method.[7] In brief, arsenic was reduced to arsenic trihydride by Zn in a solution of dissolved potassium iodide and stannous chloride. To remove hydrogen sulfide in the gas, the arseniuretted hydrogen was passed through the acetate solution, which was absorbed by cotton. Afterwards, the remaining pure gas was passed through the mixed solution of silver diethyldithiocarbamate and trichloromethane. Standard arsenic solution was used as the blank to assay the content of elemental As by the spectrophotometer.

Assay of the realgar content As2S2 content was indicated by the total arsenic content and was assayed by using hydride generation atomic absorption spectrometer (HGAAS).[10] An electrodeless discharge lamp for arsenic operated at 6 mA was used, and the determination wavelength was set to 193.7 nm. Typical analytical conditions were as follows: flow rate at 200 ml/min, burner height at 10 mm and burner location at 3 mm.

Preparation and characterization of realgar nanoparticles The ball milling equipment (PM 100, Retsch, Haan, Germany) was used to prepare RN. Realgar powder was 36

added to grinding vials in the equipment using saturated sodium dodecyl sulfate (SDS) aqueous solution as the milling medium. The weight rate of milling ball and realgar powder was set to 40 : 1. Rotation rate and time were 13.44 g and 30 min, respectively. The prepared RN was processed by water cleaning. Particle size distribution and ZP were determined by a Zetasizer (Mastersizer 2000; Malvern Instruments, Malvern, UK). The testing conditions were as follows: the dispersed medium was ultrapure water; RN concentration was 0.25 mg/m; the suspension was sonicated for 5 min before measuring; and the sample prepared with 30 min was analysed. A scanning electron microscope (JEM-2010 HR SEM; JOEL, Tokyo, Japan) was used to examine the morphology of the ground RN and also to physically measure the size of the ground RN.

Preparation and characterization of realgar nanoparticle-based microcapsules Realgar nanoparticle-based microcapsules (RNM) was prepared by the emulsify/solvent evaporation technique.[11] Gelatin was dissolved in 20 ml of distilled water, and RN was added at the set ratio of capsule core to material. The drug-material mixture was mixed well and then slowly emulsified in 50 ml of liquid paraffin containing 1 g of Span 60 (China National Pharmaceutical Group Corporation, Shanghai, China) as an emulsifier. The whole system was continuously stirred and then cooled promptly below 4°C. Twenty millilitres of formaldehyde was added to solidify the microcapsules, which were subsequently separated from the solution by filtration. The filtered microcapsules were washed three times with 50 ml of n-hexane to remove the residual oil. Finally, the microcapsules were collected, dried overnight at room temperature and stored in a desiccator. Formulation optimization of RNM was performed using orthogonal experiment. Based on the pre-experiments, four influential factors including the weight ratio of capsule core to material (C/M, w/w), preparation temperature, stirring speed and blank factor were optimized through L9 (34) orthogonal experiment taking the EE as the index. The four factors and their three levels are listed in Table 1a. RNM (0.1 g) was put into the mortar and then grinded with simulated intestinal fluid to degraded the structure of microcapsules. The content of As2S2 was assessed by HGAAS. The EE and drug loading (LD) was calculated according to the following equation:

EE (%) = Drug content in the microcapsules Added drug content ×100%


Feng Shi et al.

LD (%) = Drug content in the microcapsules microcapsules content ×1000% The particle size and distribution of microcapsules were evaluated using the sieve method and were unified with the standard of Chinese Pharmacopoeia (CP). The morphology of RNM was observed by light microscope. X-ray diffraction measurement (XRD, Rigaku Corporation, Tokyo, Japan) and Fourier transform infrared spectrometer (Nicolet-5700 FTIR; Nicolet, Waltham, MA, USA) were used to study the changes in phase and chemical composition of the RN and RNM. The diffraction pattern was measured at 2θ value of 2–50° at a scanning rate of 5°/min with a Cu-Kα radiation source.

Stability The particle size stability of RN was assessed under shade environment at room temperature. The freshly made RN was stored in the conditions mentioned for 10 days. Changes of the particle size was measured as previously described. The As2O3 content stability of RN and RNM was assessed at room temperature under open condition for 90 days. Changes of the As2O3 content was measured by Ag-DDC method.

In-vitro release The in-vitro drug release of As2S2 from crude realgar (CR), RN and RNM was carried out using CP paddle apparatus at 100 rpm and 37 ± 0.5°C.[12] Artificial gastric juice was used as the dissolution medium. At 5, 10, 15, 20, 30, 40, 50, 60 and 80 min, 2 ml dispersion samples was withdrawn and replaced with the same volume of fresh medium. The amount of As2S2 was determined by the established HGAAS method.

In-vivo bioavailability Pharmacokinetic studies were performed in normal, healthy, male Wistar rats.[13] The rats were divided into three groups (n = 6), and were administered CR, RN and RNM suspensions orally at a dose of 50 mg As2S2/kg body weight using a gavage needle. After administration, about 0.5 ml blood sample was collected from the ocular vein into a haeparinized tube in accordance with a programmed schedule at 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36 and 48 h. The collected samples were dispelled using microwave and then assessed by HGAAS method. Pharmacokinetic parameters such as the Cmax, CL, and AUC were calculated using the pharmacokinetic software, 3P97 (Mathematics Pharmacological Committee of the Chinese Academy of Pharmacology, Beijing, China).


Antitumour efficacy Evaluation of antitumour efficacy in vivo was performed as previously reported.[14] Briefly, Kunming strain mice were divided into five groups (10 mice/group) and inoculated subcutaneously with H22 tumour cells into the right axilla at a concentration of 0.2 ml (2 × 106/ml). After inoculation, mice in group 1 were administered via tail veins with fluorouracil (reference drug) once every 2 days at a dose of 25 mg/kg. Mice in groups 2, 3, 4 and 5 were continuously administered intragastrically with saline (control drug), CR, RN and RNM (As2S2 equivalent dose of 75 mg/kg), respectively. Ten days later, all animals were killed by cervical dislocation, and tumours were segregated and weighed. The tumour growth inhibition rate was calculated according to the following equation:

Tumour inhibitory rate % =

C −T ×100 C

where C and T are the tumour weight average of the control group and treated groups.

Statistical analysis Quantitative data is expressed as mean ± SD and analysed by one-way anova. Multiple comparisons between the groups were performed using Student-Newman-Keuls method. Statistical significance was set at a level of P < 0.05.

Results and Discussion Characterization of realgar nanoparticles To achieve a good effect, RN was dispersed into the glycol. Dynamic Light Scattering analyses revealed that the mean diameter of the freshly prepared RN was 85.4 ± 3.5 nm with a particle diameter size range of 50–300 nm. RN exhibited a ZP of −34.3 ± 1.7 mV. The morphological structure of the RN was analysed by TEM (Figure 1). The nanoparticles appeared round or elliptical in shape with no obvious particle aggregation observed.

Formulation optimization of realgar nanoparticle-based microcapsules Currently, there are several techniques for the preparation of microcapsules namely emulsify/solvent evaporation, spray drying and the non-solvent addition process.[15–17] In this study, emulsify/solvent evaporation was used to prepare RNM. Formulation optimization of RNM was performed using the orthogonal experiment. On the basis of the preexperiments, three influential factors including the weight


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Feng Shi et al.

Table 2

EE and LD of RNM

Sample number

EE (%)

LD (%)

1 2 3 Average

65.52 64.79 68.30 66.20 ± 1.85

26.05 25.31 27.54 26.30 ± 1.14

EE, encapsulation efficiency; LD, drug loading; RNM, realgar nanoparticles-based microcapsules.

100 nm

Figure 1 Transmission nanoparticles (RN).

scanning

microscope

image

of

realgar

Table 1a Four factors and their three levels set for orthogonal experiment Factors/levels

C/M (w/w)

PT (°C)

SS (g)

BF

1 2 3

1:4 2:4 3:4

40 50 60

1.4 2.0 2.7

1 2 3

C/M refers to the weight ratio of capsule core to material, PT refers the preparation temperature, SS refers to the stirring speed and BF refers to the blank factor. Table 1b

Results of L9 (34) orthogonal experiment EE

Factors

C/M (w/w)

PT (°C)

SS (g)

BF

(%)

1 2 3 4 5 6 7 8 9 K1 K2 K3 Rj

1:4 1:4 1:4 2:4 2:4 2:4 3:4 3:4 3:4 69.17 67.65 47.73 21.44

40 50 60 40 50 60 40 50 60 60.60 64.08 59.86 4.22

1.4 2.0 2.7 2.0 2.7 1.4 2.7 1.4 2.0 60.86 60.26 63.43 3.17

1 2 3 3 1 2 2 3 1 61.09 61.46 61.99 0.91

67.17 70.43 69.91 65.96 71.70 65.24 48.67 50.12 44.39

ratio of capsule core to material (C/M, w/w), preparation temperature and stirring speed were optimized using the L9 (34) orthogonal experiment taking the EE as the index. Four factors and their three levels are listed in Table 1a, and the results are shown in Table 1b. The Rj value of C/M 38

was the highest among the four factors, indicating its effect on EE. EE was enhanced with a decrease in C/M. The optimal formulation (which was dependent on Rj and K) was as follows: the C/M, preparation temperature and the stirring speed was 1 : 4, 50°C and 2.7 g, respectively. Because there was no obvious difference of EE when C/M was set to 2 : 4 or 1 : 4, C/M was established at 2 : 4 to increase the DL and reduce the administration dosage. In addition, because the preparation temperature did not largely affect the EE, the temperature was set at 40°C to increase the yield of microcapsules. The optimized formulation was performed in triplicates. EE and LD results are shown in Table 2.

Characterization of realgar nanoparticle-based microcapsules The obtained microcapsules were visually observed as light yellow powder. Light microscope photographs of RNM revealed that the surface structure of RNM appeared spherical and not aggregated (Figure 2). Smaller particles were observed in the internal core after amplifying a single microcapsule. Because RN was not dissolved in the gelatin, the smaller particles were deduced to be RN. More than 70% of RNM were distributed between 90 and 125 μm, indicating a relatively narrow size distribution (Figure 3). To study the crystallinity change of the realgar powder after ball milling, the XRDs of the respective realgar powder were scanned. The X-ray patterns of the CR, RN and RNM are displayed in Figure 4. The XRD patterns of CR and RN showed a lot of overlapping peaks, and the sharp diffraction peaks indicated good crystallization of the as-prepared As2S2 nanocrystals. The characterized diffraction angles observed in CR were in agreement with that of the RN powder. The results confirmed that the selected ball milling technique did not change the crystallinity of the realgar powder.[18] However, the crystallinity of RNM was different from CR or RN, and was in a less ordered structure, which indicated that RN was successfully encapsulated by microcapsules.

Stability Recently, RN has received substantial attention since their successful clinical application for the treatment of


Feng Shi et al.

Figure 2


Morphology of realgar nanoparticles-based microcapsules (RNM).

45

Percentage (%)

40 35 30 25 20 15 10 5 0 < 75 um

90–109 um 109–120 um 120–150 um 150–180 um

> 180 um

Particle distribution of realgar nanoparticles-based microcapsules (RNM).

CR RNM RN

250

Particle size (nm)

Figure 3

75–90 um

200 150 100 50 0

1

2

3

10

Time (days) Figure 5

0

5

10

15

20

25 30 2θ (deg.)

35

40

45

50

Figure 4 X-ray patterns of crude realgar (CR), realgar nanoparticles (RN) and realgar nanoparticles-based microcapsules (RNM).

hematopoietic malignancies. At present, many experts are still carrying out studies on RN. RN could improve the bioavailability, thereby increasing its clinical curative effect of realgar.[19–21] But the study on the preparation of RN

The particle size stability of realgar nanoparticles (RN).

ignores two important aspects. Firstly, in the milling process, a large amount of mechanical energy was transmissed to the nanoparticles, which leads a possible reunion. Secondly, the particle size will decrease after milling, and the surface exposure to air or light will increase sharply. This can easily facilitate the oxidation of As2S2 to As2O3. Therefore, based on the consideration of the aforementioned points, this paper further studies RNM. As shown in Figure 5, particle size of RN had a gradually increasing trend with the passage of time. Particle size


39

25

CR

RN

Feng Shi et al.

RNM

20 15 10 5 0

0

20

40 60 Time (days)

80

100

Release content (mg/g)

Figure 6 The arsenic trioxide (As2O3) content stability of realgar nanoparticles (RN) and realgar nanoparticles-based microcapsules (RNM).

10 9 8 7 6 5 4 3 2 1 0

CRr

0

20

NR

Plasma concentration (ng/ml)

Content of AS2O3 (mg/g)


1600 1400 1200 1000 800 600 400 200 0

CR

0

12

24 36 Time (h)

RN

RNM

48

60

Figure 8 Mean plasma concentration of realgar (As2S2) vs time curves after an administration of crude realgar (CR), realgar nanoparticles (RN) and realgar nanoparticles-based microcapsules (RNM) suspension. Each value represents the mean and standard deviation (SD) (n = 6).

RNM

40 60 Time (min)

80

100

Figure 7 In vitro drug release of realgar (As2S2) from crude realgar (CR), realgar nanoparticles (RN) and realgar nanoparticles-based microcapsules (RNM) (n = 3).

As2S2 is one of mineral drugs with poor solubility in both aqueous and organic phases because of its unique properties. In our pre-experiment, we investigated the dissolution conditions in various media, namely simulated gastric fluid, simulated intestinal fluid and 0.5% SDS solution. It was found that none of these could dissolve As2S2 as an ideal solvent. This study selected simulated gastric fluid as a dissolution medium to simulate the dissolution process in-vitro release better.

In-vivo bioavailability increased more than twofold after 10 days, indicating the agglomeration during storage. Figure 6 revealed that the content of As2O3 in RN increased rapidly in 90 days, which was not observed in RNM. This suggests that microencapsulation could protect RN against direct exposure to air and light, and therefore increase the stability of the RN.

In-vitro release The dissolution curves of CR, RN and RNM are shown in Figure 7. As indicated, As2S2 released very slowly from CR, and almost no dissolution was observed before 20 min with the release content barely reaching 1 mg/g after 80 min. However, As2S2 released rapidly from RN, the release content reached 2 mg/g after 10 min and was 7.3 times higher than that of CR after 80 min. The release of As2S2 compared with RN, reflects a slow-release effect, especially within 5–20 min and 30–80 min, suggesting that RN took some time to be released from the microcapsules to the dissolution medium. The quick release observed from 20 to 30 min in RNM dissolution profile might be ascribed to the complete disruption of microcapsules. 40

Numerous studies have shown that particle size is crucial for uptake and transport of drugs molecules across the gastrointestinal tract mucosal barrier. It is known from microparticles size-dependent exclusion phenomena in the gastrointestinal mucosal tissue that particles less than 100 nm size show significant tissue uptake than the larger particles. Colloidal carriers such as nanoparticles can reduce adverse effects of a drug associated with its use under conventional pharmaceutical dosage forms and improve its bioavailability.[6] Pharmacokinetic studies were performed by determining the drug concentration in rat plasma up to 48 h after administration. The plasma concentration-versus-time curves shown in Figure 8 displayed a one-compartment model with a weight factor of 1/cm3. The main pharmacokinetic parameters examined in this study are summarized in Table 3. Two parameters, including Cmax and AUC, showed significant differences between the CR and RN or RNM (P < 0.05), indicating the enhancement of bioavailability after nanocrystallization. It could be deducted that increase in the solubility of As2S2 enhances the absorption of As2S2. No significant difference of t1/2 was observed between CR and RN (P > 0.05). However, the RN group demonstrated a 4.66-fold, 1.82-fold, 0.84-fold and


Feng Shi et al.

Table 3


Pharmacokinetic parameters in rats after administration of CR, RN and RNM suspension

Parameters

Unit

CR

RN

RNM

A Ka Ke T1/2 CL/F Tmax Cmax AUC(0 – t)

ug/l 1/h 1/h h l/h h ug/l μg h/l

308.57 ± 57.86 1.93 ± 0.24 0.091 ± 0.023 8.09 ± 2.27 11.26 ± 2.27 2.17 ± 0.41 287.80 ± 58.84 4018.72 ± 512.19

1439.78 ± 202.70* 3.51 ± 1.17* 0.076 ± 0.029 10.13 ± 3.19 2.15 ± 0.63* 2.13 ± 0.56 1290.14 ± 128.52* 20213.62 ± 467.33*

1923.31 ± 683.30* 0.40 ± 0.26*,** 0.048 ± 0.012* 15.20 ± 3.95*,** 1.26 ± 0.13*,** 6.00 ± 1.189*,** 1151.17 ± 146.86* 27351.88 ± 4967.01*,**

CR, crude realgar; RN, realgar nanoparticles; RNM, realgar nanoparticles-based microcapsules. The data are mean ± standard deviation (n = 6). *P < 0.05 vs CR group; **P < 0.05 vs RN group.

Table 4

In-vivo antitumour effects in H22-bearing mice Mice body weight (g)

Drug groups

Before study

After study

Tumour weight (g)

Inhibition rate (%)

Saline 5-FU (25 mg/kg) CR RN RNM

19.70 ± 1.16 20.10 ± 1.52 19.40 ± 1.17 19.50 ± 1.27 19.00 ± 1.05

28.80 ± 2.10 22.90 ± 1.73 24.25 ± 2.31 24.17 ± 0.98 24.60 ± 2.41

1.08 ± 0.43 0.31 ± 0.12* 0.67 ± 0.15 0.51 ± 0.13*,** 0.56 ± 0.16*,**

71.33 39.50 53.14 48.55

5-FU, fluorouracil; CR, crude realgar; RN, realgar nanoparticles; RNM, realgar nanoparticles-based microcapsules. The data are mean ± standard deviation (n = 10). *P < 0.05 vs CR group; **P < 0.05 vs RN group.

0.19-fold in the A, Ka, Ke and CL respectively compared with the CR group, indicating an increase in the absorption phase and a decrease in the elimination phase. There is no significant difference of Cmax between RN group and RNM group. However, the ratio of Ka and Ke in RNM group was 0.11 : 1 and 0.63 : 1, respectively to the RN group. In addition, t1/2 and tmax of RNM group was evidently prolonged compared with RN group. Our results indicated that the release of As2S2 from RNM was in a sustainedrelease manner. It was observed during the pharmacokinetics study that some animals in RN group died before the end of the experiments. This could be attributed to the high level of As2O3 in RN group, suggesting a more toxic effect of RN.

Antitumour efficacy The pharmacokinetic investigation revealed an increase in the availability of the RN and RNM, which may offer an improved therapeutic index compared with the CR. To confirm this hypothesis, H22-tumour-bearing mice were used to assess the antitumour efficacy of RN and RNM. The tumour weight and inhibition of H22 in mice are shown in Table 4. Compared with the saline group, the other four groups significantly inhibited H22-solid tumours, and the tumours were considerably lighter (P < 0.05). In addition, the inhibition rates of CR, RN and RNM were 39.50%, 53.14% and

48.55%, respectively. Significant antitumour effects (P < 0.05) were also observed in the RN and RNM-treated groups compared with the CR group. These results suggested that the antitumour efficacy of the RN and RNM was increased, which may be attributed to the enhanced bioavailability. In addition, the improvement of efficacy correlated with smaller particle size of RN plays an important role during the cellular uptake process. It has been pointed out that drug is absorbed more easily when the particle size is less than 100 nm.[7] Therefore, the less the particle size, the more the efficacy is improved. Antitumour efficacy of RN was higher than that of RNM, which might have resulted from the higher content of As2O3. A similar problem of mice death also occurred in this study as in pharmacokinetic study. By the end of the experiment, a total of five mice had died. The death of the mice might be attributable to the toxicity of RN induced by high concentration of As2O3. In furtherance to this study, future studies will focus on the relationship between the content of As2O3 and antitumour efficacy or toxicity.

Conclusions In this study, RN and RNM were prepared using ball milling and solvent evaporation technique, respectively. The obtained RNM appeared spherical and not aggregated with a relatively narrow size distribution. RN and RNM


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Feng Shi et al.

exhibited considerable higher release of As2S2, bioavailability and antitumour efficacies compared with CR. In addition, RNM could protect RN directly exposed to the air and light and therefore increased the stability of the RN. Therefore, the developed RNM has a great potential as a delivery system for realgar.

Funding This work was supported by Subject Chief Scientist Program (10XD14303900) from the Science and Technology Commission of Shanghai Municipality and Doctoral Fund of Ministry of Education of China (No. 20123107110005).

Declarations Conflict of interest The Author(s) declare(s) that they have no conflicts of interest to disclose.

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