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Preparation, characterization, release and antioxidant activity of curcumin-loaded amorphous calcium phosphate nanoparticles
Journal of Non-Crystalline Solids xxx (xxxx) xxx–xxx
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Preparation, characterization, release and antioxidant activity of curcuminloaded amorphous calcium phosphate nanoparticles Xia Guoa,b, Wenfeng Lia,b, Heping Wanga,b, Yan-Ying Fand, Huifang Wanga,b, Xianghua Gaoa,b, ⁎⁎ ⁎ Baolong Niua,b,d, , Xuechen Gongc, a
Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan 030024, PR China College of Materials Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China c Agriculture and Forestry Technologically College, Hebei North University, Zhangjiakou 075000, PR China d School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amorphous calcium phosphate Curcumin Drug release Antioxidant properties
In the study, amorphous calcium phosphate (ACP) nanoparticles were prepared by the coprecipitation method to optimize release profile of curcumin (Cur) and avoid burst releases, which were used to overcome the weakness of Cur, such as poor chemical stability and bioavailability. To find the best preparation condition, the influence of reaction concentration, temperature, time and pH on crystal phase of the samples was investigated by XRD and FTIR. The results showed that amorphous calcium phosphate (ACP) was obtained, when pH value and the concentration of PO43− were 8 and 0.024 mM, prepared at 30 °C for 10 min. In vitro drug release assay, ACP nanoparticles showed high loading capacity of Cur and favorable pH-responsive drug release properties. Furthermore, the Cur-load ACP nanoparticles showed an excellent ability to scavenge free radical and damage A549 cells, resulting in a high antioxidant properties and low cell viability. Therefore, the as-prepared nanoparticles have promising applications in the food and biomedical fields.
1. Introduction Although reactive oxygen species (ROS) is beneficial for human body at low/moderate concentrations, such as defence against infectious agents and contribution to cellular signalling systems, it also exerts many harmful effects to living systems [1]. Oxidative stress caused by excess ROS disturbs the equilibrium status of prooxidant/ antioxidant reactions in living organisms, which damages cellular lipids, proteins or DNA and inhibits their normal functions [2]. Therefore, scavenging ROS is one of the most effective means of decreasing the level of oxidative stress, which may be achieved using antioxidant compounds extracted from natural plants, such as catechins, isoflavones, anthocyanins, phenolic acids and vitamins. Particularly, antioxidant compounds extracted from natural plants gradually have attracted more attention [2]. Curcumin (Cur), a natural lipophilic polyphenol found in the rhizomes of turmeric [3], shows a wide variety of pharmacological properties, including antioxidant, anti-inflammatory and anticancer [4], which endows it with applications in antioxidant therapy. However, its bioavailability is significantly
restricted because of its poor chemical stability. Employing carriers is a feasible strategy to overcome the drawbacks of Cur. Therefore, various efficient carriers have been appiled, such as dendrimers, liposome, and micro/nano-particles [5]. Compared with other delivery systems, nanoparticle delivery system has been widely investigated due to their distinct advantages, such as high encapsulation efficiency, slower degradation rate, small particle size and effective penetration ability [6]. Amorphous calcium phosphate (ACP), one of the calcium phosphate materials, is regarded as a metastable phase with a short range order [7]. Compared with other calcium phosphate materials, ACP nanoparticles are promising drug carriers owing to their advantages including large specific surface area, high drug loading capacity and controlled drug release behavior [8]. Furthermore, ACP nanoparticles have good biodegradability and could promote osteoblast adhesion and osteconductivity [9]. In previous studies, ACP nanoparticles were prepared as cements, which were used in bone consolidation or reconstruction and were commercially available since the 1990s [10]. Moreover, the solubility of synthetic ACP nanoparticles increases with the decrease of pH value in aqueous solution [11]. Consequently, the
⁎
Corresponding author at: Key Laboratory of Interface Science and Engineering in Advanced Materials, College of Materials Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China. ⁎⁎ Corresponding author. E-mail address: [email protected] (B. Niu). https://doi.org/10.1016/j.jnoncrysol.2018.08.015 Received 17 May 2018; Received in revised form 17 July 2018; Accepted 12 August 2018 0022-3093/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Guo, X., Journal of Non-Crystalline Solids, https://doi.org/10.1016/j.jnoncrysol.2018.08.015
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drug in the supernatant was determined by UV–vis spectrophotometer. The drug concentration in the supernatant was quantified on the basis of a linear standard curve by ultraviolet (UV) absorption at 424 nm, a strong absorption band of Cur. The encapsulation efficiency (EE) and loading capacity (LC) were calculated according to equation [3, 16]:
ACP nanoparticles could be designed and used as pH sensitive drug nano-carriers, which give an almost entire prevention of premature drug release in physiological condition of plasma (pH 7.4). Above all, nanoparticle delivery system could lower or annihilate the plasmatic drug concentration, thus avoiding secondary effects or general toxicity [12]. In recent years, much attention has been paid to the preparation of ACP nanoparticles using different methods. Yang et al. [13] investigated the synthesis of novel gallium-doped ACP nanoparticles by sol-gel method. Zhu et al. [14] fabricated ACP porous nanospheres by using a microwave-assisted hydrothermal method with adenosine 5′triphosphate disodium salt (ATP) as the phosphorus source and stabilizer. The co-precipitation method to synthesize nanostructured materials has attracted much interest and been growing fast because of its excellent advantages such as rapidness, facile productive process and low-cost. Moreover, ACP nanoparticles have been used for the investigation of silybin loading and release [15]. However, to date, a few studies have been reported about the specific preparation by co-precipitation method as well as and release, antioxidant activity and anticancer activity of Cur-loaded ACP nanoparticles, which is referred in this paper. The present study aimed to synthesize ACP nanoparticles as novel delivery systems to encapsulate, stabilize and slowly release Cur by coprecipitation method. In addition to the encapsulation efficiency and loading efficiency of Cur and the micro-morphology of the ACP nanoparticles were evaluated. Furthermore, the ability of ACP nanoparticles to control the release of Cur was also investigated in this work, which was useful for the development of potential carriers for bioactive compounds. The effects of the temperature, time, pH and concentrations of (NH4)2HPO4 on the crystal phase of the product were researched. The prepared ACP nanoparticles have relatively high specific surface area and are efficient for drug loading and release using Cur as a model drug. The Cur-load ACP nanoparticles show an outstanding ability to damage free radical and cancer cells. Hence, they are promising for the application in drug delivery.
EE =
LE =
total wei ght of Cur − weight of unloaded Cur ∗ 100% total weight of Cur total weight of Cur − weight of unloaded weight of microspheres
Cur ∗
100%
(1)
(2)
2.3. Characterization of nanoparticles The X-ray powder diffraction (XRD) of the sample was characterized using X-ray diffractometer (Rigaku D/max 2500 V, Cu Kα radiation, k = 1.54178 Å). Fourier transform infrared (FTIR) spectra were recorded using a Fourier Transform IR Spectrometer (Model: Nicolet-710 spectrometer) by the KBr pellet at wavelengths ranging from 400 to 4000 cm−1. The UV–vis spectroscopy was carried out on a UV–vis spectrophotometer (UV1800, Shimadzu Corporation) in the wavelength range of 200-500 nm. The morphology of the samples was examined by a scanning electron microscope (SEM, FEI Magellan 400, USA) and a transmission electron microscope (TEM, JEM2100F, Japan). Energy Dispersive Spectrometer (EDS) was used to estimate the Ca/P molar ratio of the ACP nanoparticles. Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were obtained by a surface area and pore size analyzer (V-Sorb 2800P, Gold APP China). Dynamic light scattering (DLS) was used to measure diameter of sample (Malvern, UK). 2.4. In vitro drug release assay The drug release was studied using the membrane dialysis method against phosphate buffered saline (PBS, pH 7.4) and acetate buffers (pH 5.4) at 37 °C, which were used as the drug-release media to simulate normal blood/tissue and tumor environments. Before the experiment, the Cur-loaded ACP nanoparticles were re-suspended with PBS (pH 7.4, 5.4). Firstly, 5 mL of the Cur-loaded ACP nanoparticles (2 mg/ mL) was placed in dialysis bags with a molecular weight cutoff of 34 kDa. Next, the dialysis bags were immersed in 100 mL of the beakers, which was shaken (70 rpm) at 37 °C while shielded from light. The release medium was withdrawn at various intervals and replenished with an equal volume of fresh medium. The amount of released Cur was estimated by measuring the absorbance at 424 nm.
2. Experiment 2.1. Materials Curcumin (99%), 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2, 2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Butyl hydroxy anisd (BHA), butylated hydroxytoluene (BHT) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Calcium nitrate (Ca(NO3)2), ammonium hydrogen phosphate ((NH4)2HPO4) and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Co. Ltd. All the other reagents obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. were analytical grade and all solutions were prepared by distilled water.
2.5. Cur protection To elucidate the effect of encapsulation on the stability of Cur against external severe processing, we compared with remnant content of free Cur with that of entrapped Cur in ACP nanoparticles after thermal treatment and ultraviolet radiation. Free and encapsulated Cur with quality of 20 mg by thermal treatment (60 °C, 30 min) and ultraviolet radiation was taken into account for the protective effect. For free Cur and Cur-loaded ACP nanoparticles, ethyl alcohol was added and stirred for 30 min [17]. The existent Cur was calculated through the absorption value at 424 nm.
2.2. Synthesis ACP nanoparticles were formed by coprecipitation method [15]. Briefly, Ca(NO3)2·4H2O was dissolved in 29 mL deionized water to form 1 mM aqueous solution. 29 mL (NH4)2HPO4 phosphate solution was added dropwise to the above solution and the aqueous solution quickly turned to white suspension. The raw materials were designed with a Ca/P molar ratio from 1.5 and the pH value was maintained within 8 by addition of 1 M NaOH solution. The resulting mixture was agitated at 30 °C for 10 min. Finally, the obtained nanoparticles were washed three times with deionized water to remove any residual ions, and the sample was collected by centrifugation and freeze-dried. Drug loading: Cur (5 mg/mL) was added to the solution of Ca (NO3)2. The mixture was stirred for 1 h, followed by slow addition of an aqueous solution of Na2HPO4 (0.024 mM). The mixture was gently stirred at 30 °C for another 15 min. After the suspension was centrifuged at high speed to collect the products, the concentration of unloaded
2.6. Determination of antioxidant activity in vitro 2.6.1. 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) radical-scavenging activity assay The antioxidant properties of Cur and Cur-load ACP nanoparticles were evaluated by determination of scavenging effect on DPPH radicals, and scavenging capacity of the samples was determined using a method reported previously [18, 19]. In this assay 1 mL samples at 2
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multiple concentrations (2, 4, 6, 8, 10 μg/mL) were mixed with 2 mL of 0.1 mM DPPH· ethanol solution. Then mixtures were stored in dark for 30 min at room temperature and the absorbance of the reaction solution was measured at 517 nm. The same reaction condition was applied for the blank and standard solution. The free radical scavenging activity was calculated using Eq. (3):
Inhibition (%) =
A0 − A1 ∗ 100% A0
3. Results and discussion 3.1. Preparation of ACP nanoparticles There is no doubt that ACP is a metastable phase of hydroxyapatite (HA), and it has been demonstrated that amorphous calcium phosphate has better biodegradability than hydroxyapatite in vivo [8]. Consequently, to explore the effect of concentration, pH, temperature and time on the formation of ACP nanoparticles, a rapid and simple method was adopted to synthesize the carrier. Fig. 2a reveals the effect of concentration on the ACP nanoparticles. According to Fig. 2a, only the sample 1 (0.024 mM) shows a broad diffraction line at 30° that is exhibited typical amorphous phase of calcium phosphate [21]. When the PO43− concentration increased, sample had crystallized diffraction peaks located at 28.33° and 31.69°, which were attributed to (102) and (211) of HA (JCPDSNo.74-0566). Effects of different pH on ACP nanoparticles are shown in Fig. 2b. Amorphous phase was achieved when pH was 8. Crystalline peaks at 30.4, 40.1, 47 and 49° ascribed to HA (JCPDSNo.74-0566). The transformation of ACP into crystalline apatite in the aqueous solution can be described as follows: when pH > 8, ACP converts into apatite directly by rearrangement of the atomics [22]. Fig. 2c shows the sample prepared at 25 °C had two XRD pattern with HA diffraction peaks. However, the peak at 30° tended to become sharp when temperature increased, only the sample at 30 °C had the perfect characteristics of amorphous XRD pattern. The sample with reaction time for 10 min had no obvious diffraction peaks, indicating that the product consisted of the ACP phase. However, when the reaction time increased to 3 h (Fig. 2d), the product contained a small amount of HA (JCPDSNo.74-0566), which is due to the partial transformation from ACP to HA [23]. The experimental results indicated that the product prepared at 30 °C for 10 min is amorphous calcium phosphate (ACP) when PO43− concentration is 0.024 mM and pH value is 8. The formation of ACP nanoparticles was further investigated using the FTIR spectroscopy. As shown in Fig. 3, the broad unresolved absorption bands located at around 572 and 1047 cm−1 were ascribed to the characteristics of PO43−. While the brands at around 3430 and 1636 cm−1 was assigned to the adsorbed water [24]. The small band at 875 cm−1 proved the presence of HPO42− in ACP nanoparticles. The amount of HPO42− and consequently the Ca/P ratio strongly depends on the solution acidity: The lower pH of the synthesis medium (in this case, DES), the greater HPO42− content, and consequently the lower Ca/P ratio. The small band at 873 cm−1 was υ3 bands of carbonate groups, due to carbonate ions located PO43− in ACP nanoparticles [25, 26]. The peak at 1412 cm−1 was attributed to the carbonate bonds, indicating that the presence of CO32– group, which may be derived from the dissolved CO2 from atmosphere [27-29]. The particle size distributions of the ACP nanoparticles was characterized by DLS, as shown in Fig. 4, from which one can see that two
(3)
where A0 is the absorbance of the control solution, and A1 is the absorbance of the samples.
2.6.2. ABTS•+ scavenging activity assay The ABTS·+ scavenging activity was estimated according to one described previously [1]. Briefly, 7 mM of ABTS and 4.9 mM of potassium persulphate (K2S2O8) at the volume ratio of 1:1 were mixed in beaker and stood in a dark at room temperature for 16 h to generate the free radical of ABTS, then 5 mL of ABTS•+ solution diluted with 250 mL absolute ethyl alcohol to give an absorbance of 0.70 ± 0.02 at 734 nm. After that, 1 mL samples with different concentrations (2, 4, 6, 8, 10, 12 μg/mL) were added to 4 mL of ABTS•+ solution and the reaction mixture was continued at room temperature for 20 min and the absorbance at 734 nm was then measured. The scavenging activity was calculated according to Eq. (3).
2.7. In vitro cytotoxicity tests The cytotoxic activity of ACP nanoparticles and Cur-load ACP nanoparticles was evaluated using A549 cells. Typically, cells were seeded in 96-well plate (5000 cell per well) and cultured in 5% CO2 at 37 °C for 24 h. The different serial concentrations of materials were added into the wells and co-cultured with the cells for 48 h. Subsequently, each well was treated with MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) for another 4 h. The formed formazan crystals were dissolved in DMSO and absorbance was measured at 490 nm. The data was representative as the main value of five parallel experiments. Cell images of A549 cells treated with different concentrations of materials were obtained using an optical microscope [8, 20].
2.8. Statistical analysis All data were conducted in triplicates and presented as averages and standard deviations. Statistical analysis was performed using one-way analysis of variance (ANOVA) using SPSS18.0 software. The level of significance was set at 0.05 (Fig. 1).
Fig. 1. Schematic diagrams: formation of ACP nanoparticles synthesized by coprecipitation method. 3
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Fig. 2. XRD patterns of the ACP nanoparticles: a) different PO43− concentration: sample 1(0.024 mM), sample 2(0.028 mM), sample 3(0.034 mM), sample 4(0.047 mM), sample 5(0.07 mM), sample 6(0.14 mM); b) different pH: 8,9,10,10.5; c) different temperature: 25 °C, 30 °C, 35 °C, 40 °C; d) different time: 10 min,1 h,2 h,3 h.
of the ACP nanoparticles determined by DLS was 189 nm. The difference in particle size determined by SEM, TEM and DLS might be caused by the different preparations of the samples. DLS is a method that allows the direct observation of the polymeric aggregates in aqueous solutions, while SEM measures particles in the dried state [31]. EDX (Fig. 5c) shows the presence of Ca, P, O and C elements, and elemental analysis indicated a Ca/P molar ratio of 1.38 ± 0.1 in the region of the ACP nanoparticles, which was below the stoichiometric value of 1.5 for amorphous calcium phosphate. Deviation from the stoichiometic value is known in the literatures and due to the presence of small amounts (< 15%) of HPO42− ions, surface-adsorbed soluble phases which can be washed away or cationic substitutions at the Ca2+ sites by Mg2+ or Na+ or anionic substitution at PO43− sites by CO32– or HPO42− or a combination of these substitutions [24, 32-34]. The porosity, one of the most attractive properties of ACP nanoparticles, is highly desirable for drug or protein loading. In this study, large pore sizes are also expected to exit in this ACP nanoparticles, which is appropriate for small molecules to pass through without limitation. As shown in Fig. 6, products exhibited a type IV isotherm with H1 hysteresis loop, typical of mesostructured materials with a mixture of mesopore sizes [35]. The BET specific surface area of the ACP nanoparticles was about 41.414 m2/g. Total pore volume and the average pore size of ACP nanoparticles were about 0.1656 cm3/g and 10.5 nm, respectively. A similar result was reported by Zhu [14], who found that specific surface area of the as-prepared ACP porous nanospheres was about 25 m2/g.
Fig. 3. FTIR spectra infrared spectra of ACP nanoparticles.
sets of size distributions of ACP nanoparticles are observed: one is located at approximately 189 nm and the other is around 5.9 μm. This result was caused by the aggregation of ACP nanoparticles [30]. The morphology of the ACP nanoparticles was analyzed by SEM and TEM as shown in Fig. 5a and b. The spherical and aggregated nanoparticles with smooth surface were observed from SEM and TEM. Most of the particles ranged from 80 to 120 nm. However, the mean diameter 4
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Fig. 4. Particle size distribution of ACP nanoparticles by DLS.
a rapid degradation after being exposed to alkaline solution, ACP nanoparticles could achieve to a controlled and sustained release of Cur in simulated blood environment, which avoids degradation of Cur at pH of 7.4. Therefore, ACP carrier could overcome rapid release of Cur in vivo and prolong their effect in the body.
3.2. In vitro drug release Fig. 7 displays the drug in vitro release behaviors of Cur-loaded ACP nanoparticles under different pH at 37 °C. Clearly, there was no burst release for the Cur-loaded ACP nanoparticles in PBS buffer solution (pH 7.4 and pH 5.4). At the beginning of release (approximately 5 h), there was no significant difference in the drug releases in buffer solutions with different pH. However, with the increase of time, the release amount of Cur from the ACP nanoparticles in an acidic environment was accelerated compared with that in a neutral environment, indicating the ACP nanoparticles are a promising drug delivery system that could reduce the concentration of free chemotherapeutics in nontargeted sites. At the release time of 52 h, the accumulative released percentages of Cur were approximately 85.84 and 64.6789% in pH 5.4 and pH 7.4 buffer solutions, respectively. The drug release behavior of Cur-loaded ACP nanoparticles was very similar to the result of calcium phosphate prepared in the absence of the block copolymer in a previous study [15]. According to the result above, compared with pure Cur with
3.3. Cur protection As we know, a variety of factors, including pH, temperature and ultraviolet radiation, have been reported to affect chemical stability of Cur [18]. Fig.8 shows that ACP-loaded strategy was a feasible and effective method to protect Cur against thermal and ultraviolet treatment. The amount of free Cur was reduced from 20 to 5 mg and 6 mg when the solution suffered from 60 °C treatment for 30 min and ultraviolet radiation for 1 h, respectively. The preserved Cur could reach 9 mg when subjected treatment at 60 °C for 1 min, while achieved 11 mg for 1 h ultraviolet radiation, respectively. The drastically reduced phenomenon was consistent with previous study [36]. Compared with free
Fig. 5. SEM (a), TEM (b) and EDS (c) micrographs of ACP nanoparticles synthesized by using Ca(NO3)2 as the calcium source and (NH4)2HPO4 as the phosphorus source by the coprecipitation method. 5
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Fig. 9. DPPH radical scavenging activity of Cur, BHA, BHT and Cur-loaded ACP nanoparticles.
Fig. 6. Characterization of ACP nanoparticles by N2 adsorption–desorption isotherms and BJH pore size distribution (inset).
Fig. 10. ABTS·+ radical scavenging activity of Cur, BHA, BHT and Cur-loaded ACP nanoparticles.
Fig. 7. Drug release profiles of Cur-loaded ACP nanoparticles under different pH environment.
Fig. 11. Cytotoxicity tests of the as-prepared ACP nanoparticles prepared with and without anticancer drug Cur loading using A549 cells.
Fig. 8. Cur protection by ACP nanoparticles in the heat and ultraviolet treatment.
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Fig. 12. The optical images of A549 cells treated with different concentrations of ACP nanoparticles without Cur loading (the first row images) and with Cur loading (the second row images).
all samples showed dose-dependent ABTS·+ scavenging capacity in the concentration range of 0–12 μg/mL. At 12 μg/mL, the scavenging activities of Cur-loaded ACP nanoparticles, Cur, BHA and BHT on ABTS·+ were 85.12, 76.85, 64.56, and 62.71%, respectively, which suggested that the ABTS·+ scavenging capacity of encapsulated Cur was maximum. The results were consistent with the DPPH radical scavenging ability. Similar results have also been reported by other authors who used different formulation for Cur encapsulation to enhance its antioxidant activity. [39, 40]. It was seen that the antioxidant activity of encapsulated Cur was superior for the free Cur. Yoncheva et al. [41] recently prepared Cur encapsulated cationic triblock copolymer micelles and determined its antioxidant activity. Their results showed that the ability of the micellar Cur to scavenge the free radical was higher than that of the free Cur.
Cur at the same treatment, the amount of ACP nanoparticles protected Cur was enhances. The results demonstrated Cur could be protected and improve its stability during food processing, which is helpful to broaden Cur potential application in food field. 3.4. Antioxidant analysis 3.4.1. DPPH· scavenging capacity The absorbance of DPPH can be reduced by hydrogen atom transferred from H-donors (antioxidants) which led to the disappearance of the visible band in DPPH [37]. The DPPH radical scavenging ability of Cur-loaded ACP nanoparticles, Cur, BHA and BHT were compared in Fig. 9. DPPH radical scavenging ability was enhanced with the increase of concentrations for all samples. At the concentration of 10 μg/mL, the DPPH radical scavenging capacity of free Cur was 46.5%. However, the DPPH scavenging ability of Cur was raised to 49.5% when encapsulated with ACP nanoparticles. These results indicated that encapsulated Cur is more efficient at scavenging free radicals than free Cur. The reason above could be attributed to the low solubility of Cur, which could lower the amount of reactant molecules. However, the increase of water dispesity and larger surface area after encapsulated Cur due to the smaller mean particle diameter facilitated the interaction between Cur and radicals [16, 19]. Previous studies have reported by Huang et al. [1] that Cur encapsulated within these core–shell protein–polysaccharide nanoparticles exhibited higher antioxidant and radical scavenging activities than Cur. For BHA and BHT, the DPPH radical scavenging activity was poorer than Cur, which is in good agreement with the previous reports that scavenging activity of Cur was stronger [38]. Anyhow, the antioxidant activity enhancement was beneficial to improving the economic and health value of the active product in functional foods.
3.5. Cytotoxicity tests To evaluate the biocompatibility of the ACP nanoparticles, the in vitro cytotoxicity was assessed on by standard MTT assay on A549 cells. As shown in Fig. 11, the cell viability was still higher than 90% when the cells were co-cultured with ACP nanoparticles at high concentration (32 μg/mL), indicating good biocompatibility of ACP nanoparticles. On the other hand, the cell viability gradually decreases with increasing concentration of Cur-loaded ACP nanoparticles. The cell viability was only 39% when the concentration of Cur-loaded ACP nanoparticles was 32 μg/mL. This implied that the cell viability was significantly inhibited by the Cur released from ACP nanoparticles. The as-prepared ACP nanoparticles are predicted to be used as drug carriers for cancer. Fig. 12 shows the optical images of A549 cells co-cultured with different concentrations of ACP nanoparticles with and without Cur loading. The experiments showed that the cells could maintain spindle morphology, implying that the cells still have a good physiological state after being treated with ACP nanoparticles at the concentrations in the range of 1–16 μg/mL. However, with the increase of Cur-loaded ACP nanoparticles concentration, the cells obviously changed to be spherical,
3.4.2. ABTS·+ scavenging capacity The ABTS·+ scavenging capacity of Cur encapsulated in ACP nanoparticles, BHA and BHT were also investigated (Fig. 10). Evidently, 7
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which is significantly different from the ACP nanoparticles without Cur loading. The spherical morphology of the cells was caused by the damage or killing of the cells, which was attributed to release of Cur from ACP nanoparticles. These results suggested that the as-prepared Curloaded ACP nanoparticles have a high cytotoxicity to cancer cells, which is consistent with the MTT assay results [8, 23].
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Mendoza, Characterization, release and antioxidant activity of curcumin-loaded
4. Conclusion A facile, rapid, surfactant-free, and environmentally friendly strategy was developed for the synthesis of amorphous calcium phosphate (ACP) nanoparticles, where an aqueous solution containing Ca (NO3)2 was used as a calcium source, and (NH4)2HPO4 as a phosphorus source. The reaction temperature, time, pH and the concentrations of (NH4)2HPO4 have significant effects on the crystal phase of the product. When reaction temperature, time, pH value and concentration was 30 °C, 10 min, 8 and 0.024 mM, respectively, the product was composed of amorphous calcium phosphate. The as-prepared ACP nanoparticles have a high specific surface area. The experiments showed that the ACP nanoparticles have a high Cur drug loading capacity and favorable pHresponsive drug release property. The Cur-loaded ACP nanoparticles have superior radical scavenging activities and show a high ability to damage tumor cells. Thus, the as-prepared ACP nanoparticles are promising for the applications in various biomedical and pabular fields such as drug delivery, functional foods and beverages. Acknowledgments The work was financially supported by Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (172040098-S), National Natural Science Foundation of China (31700689), Shanxi Scholarship Council of China (2015-033), and the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province. References [1] X. Huang, X. Huang, Y. Gong, H. Xiao, D.J. McClements, K. Hu, Enhancement of curcumin water dispersibility and antioxidant activity using core–shell protein–polysaccharide nanoparticles, Food Res. Int. 87 (2016) 1–9. [2] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int. J. Biochem. Cell Biol. 39 (2007) 44–84. [3] C. Sun, C. Xu, L. Mao, D. Wang, J. Yang, Y. Gao, Preparation, characterization and stability of curcumin-loaded zein-shellac composite colloidal particles, Food Chem. 228 (2017) 656–667. [4] Z. Li, Y. Wang, Y. Pei, W. Xiong, C. Zhang, W. Xu, S. Liu, B. Li, Curcumin encapsulated in the complex of lysozyme/carboxymethylcellulose and implications for the antioxidant activity of curcumin, Food Res. Int. 75 (2015) 98–105. [5] B.R. Shah, C. Zhang, L. Yan, B. Li, Bioaccessibility and antioxidant activity of curcumin after encapsulated by nano and pickering emulsion based on chitosan-tripolyphosphate nanoparticles, Food Res. Int. 89 (2016) 399–407. [6] B. Abdous, S.M. Sajjadi, L. Ma'mani, β-Cyclodextrin modified mesoporous silica nanoparticles as a nano-carrier: response surface methodology to investigate and optimize loading and release processes for curcumin delivery, J. Appl. Biomed. 15 (2017) 210–218. [7] G.J. Ding, Y.J. Zhu, C. Qi, B.Q. Lu, F. Chen, J. Wu, Porous hollow microspheres of amorphous calcium phosphate: soybean lecithin templated microwave-assisted hydrothermal synthesis and application in drug delivery, J. Mater. Chem. B 3 (2015) 1823–1830. [8] G.J. Ding, Y.J. Zhu, C. Qi, B.Q. Lu, J. Wu, F. Chen, Porous microspheres of amorphous calcium phosphate: block copolymer templated microwave-assisted hydrothermal synthesis and application in drug delivery, J. Colloid Interface Sci. 443 (2015) 72–79. [9] Z.F. Zhou, T.W. Sun, F. Chen, D.Q. Zuo, H.S. Wang, Y.Q. Hua, Z.D. Cai, J. Tan, Calcium phosphate-phosphorylated adenosine hybrid microspheres for anti-osteosarcoma drug delivery and osteogenic differentiation, Biomaterials 121 (2017) 1–14. [10] M. Parent, H. Baradari, E. Champion, C. Damia, M. Viana-Trecant, Design of calcium phosphate ceramics for drug delivery applications in bone diseases: a review of the parameters affecting the loading and release of the therapeutic substance, J. Control. Release 252 (2017) 1. [11] Y. Lv, H. Huang, B. Yang, H. Liu, Y. Li, J. Wang, A robust pH-sensitive drug carrier: aqueous micelles mineralized by calcium phosphate based on chitosan, Carbohydr.
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Contents lists available at ScienceDirect
Journal of Non-Crystalline Solids journal homepage: www.elsevier.com/locate/jnoncrysol
Preparation, characterization, release and antioxidant activity of curcuminloaded amorphous calcium phosphate nanoparticles Xia Guoa,b, Wenfeng Lia,b, Heping Wanga,b, Yan-Ying Fand, Huifang Wanga,b, Xianghua Gaoa,b, ⁎⁎ ⁎ Baolong Niua,b,d, , Xuechen Gongc, a
Key Laboratory of Interface Science and Engineering in Advanced Materials, Taiyuan University of Technology, Ministry of Education, Taiyuan 030024, PR China College of Materials Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China c Agriculture and Forestry Technologically College, Hebei North University, Zhangjiakou 075000, PR China d School of Basic Medical Sciences, Shanxi Medical University, Taiyuan 030001, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amorphous calcium phosphate Curcumin Drug release Antioxidant properties
In the study, amorphous calcium phosphate (ACP) nanoparticles were prepared by the coprecipitation method to optimize release profile of curcumin (Cur) and avoid burst releases, which were used to overcome the weakness of Cur, such as poor chemical stability and bioavailability. To find the best preparation condition, the influence of reaction concentration, temperature, time and pH on crystal phase of the samples was investigated by XRD and FTIR. The results showed that amorphous calcium phosphate (ACP) was obtained, when pH value and the concentration of PO43− were 8 and 0.024 mM, prepared at 30 °C for 10 min. In vitro drug release assay, ACP nanoparticles showed high loading capacity of Cur and favorable pH-responsive drug release properties. Furthermore, the Cur-load ACP nanoparticles showed an excellent ability to scavenge free radical and damage A549 cells, resulting in a high antioxidant properties and low cell viability. Therefore, the as-prepared nanoparticles have promising applications in the food and biomedical fields.
1. Introduction Although reactive oxygen species (ROS) is beneficial for human body at low/moderate concentrations, such as defence against infectious agents and contribution to cellular signalling systems, it also exerts many harmful effects to living systems [1]. Oxidative stress caused by excess ROS disturbs the equilibrium status of prooxidant/ antioxidant reactions in living organisms, which damages cellular lipids, proteins or DNA and inhibits their normal functions [2]. Therefore, scavenging ROS is one of the most effective means of decreasing the level of oxidative stress, which may be achieved using antioxidant compounds extracted from natural plants, such as catechins, isoflavones, anthocyanins, phenolic acids and vitamins. Particularly, antioxidant compounds extracted from natural plants gradually have attracted more attention [2]. Curcumin (Cur), a natural lipophilic polyphenol found in the rhizomes of turmeric [3], shows a wide variety of pharmacological properties, including antioxidant, anti-inflammatory and anticancer [4], which endows it with applications in antioxidant therapy. However, its bioavailability is significantly
restricted because of its poor chemical stability. Employing carriers is a feasible strategy to overcome the drawbacks of Cur. Therefore, various efficient carriers have been appiled, such as dendrimers, liposome, and micro/nano-particles [5]. Compared with other delivery systems, nanoparticle delivery system has been widely investigated due to their distinct advantages, such as high encapsulation efficiency, slower degradation rate, small particle size and effective penetration ability [6]. Amorphous calcium phosphate (ACP), one of the calcium phosphate materials, is regarded as a metastable phase with a short range order [7]. Compared with other calcium phosphate materials, ACP nanoparticles are promising drug carriers owing to their advantages including large specific surface area, high drug loading capacity and controlled drug release behavior [8]. Furthermore, ACP nanoparticles have good biodegradability and could promote osteoblast adhesion and osteconductivity [9]. In previous studies, ACP nanoparticles were prepared as cements, which were used in bone consolidation or reconstruction and were commercially available since the 1990s [10]. Moreover, the solubility of synthetic ACP nanoparticles increases with the decrease of pH value in aqueous solution [11]. Consequently, the
⁎
Corresponding author at: Key Laboratory of Interface Science and Engineering in Advanced Materials, College of Materials Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, PR China. ⁎⁎ Corresponding author. E-mail address: [email protected] (B. Niu). https://doi.org/10.1016/j.jnoncrysol.2018.08.015 Received 17 May 2018; Received in revised form 17 July 2018; Accepted 12 August 2018 0022-3093/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Guo, X., Journal of Non-Crystalline Solids, https://doi.org/10.1016/j.jnoncrysol.2018.08.015
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drug in the supernatant was determined by UV–vis spectrophotometer. The drug concentration in the supernatant was quantified on the basis of a linear standard curve by ultraviolet (UV) absorption at 424 nm, a strong absorption band of Cur. The encapsulation efficiency (EE) and loading capacity (LC) were calculated according to equation [3, 16]:
ACP nanoparticles could be designed and used as pH sensitive drug nano-carriers, which give an almost entire prevention of premature drug release in physiological condition of plasma (pH 7.4). Above all, nanoparticle delivery system could lower or annihilate the plasmatic drug concentration, thus avoiding secondary effects or general toxicity [12]. In recent years, much attention has been paid to the preparation of ACP nanoparticles using different methods. Yang et al. [13] investigated the synthesis of novel gallium-doped ACP nanoparticles by sol-gel method. Zhu et al. [14] fabricated ACP porous nanospheres by using a microwave-assisted hydrothermal method with adenosine 5′triphosphate disodium salt (ATP) as the phosphorus source and stabilizer. The co-precipitation method to synthesize nanostructured materials has attracted much interest and been growing fast because of its excellent advantages such as rapidness, facile productive process and low-cost. Moreover, ACP nanoparticles have been used for the investigation of silybin loading and release [15]. However, to date, a few studies have been reported about the specific preparation by co-precipitation method as well as and release, antioxidant activity and anticancer activity of Cur-loaded ACP nanoparticles, which is referred in this paper. The present study aimed to synthesize ACP nanoparticles as novel delivery systems to encapsulate, stabilize and slowly release Cur by coprecipitation method. In addition to the encapsulation efficiency and loading efficiency of Cur and the micro-morphology of the ACP nanoparticles were evaluated. Furthermore, the ability of ACP nanoparticles to control the release of Cur was also investigated in this work, which was useful for the development of potential carriers for bioactive compounds. The effects of the temperature, time, pH and concentrations of (NH4)2HPO4 on the crystal phase of the product were researched. The prepared ACP nanoparticles have relatively high specific surface area and are efficient for drug loading and release using Cur as a model drug. The Cur-load ACP nanoparticles show an outstanding ability to damage free radical and cancer cells. Hence, they are promising for the application in drug delivery.
EE =
LE =
total wei ght of Cur − weight of unloaded Cur ∗ 100% total weight of Cur total weight of Cur − weight of unloaded weight of microspheres
Cur ∗
100%
(1)
(2)
2.3. Characterization of nanoparticles The X-ray powder diffraction (XRD) of the sample was characterized using X-ray diffractometer (Rigaku D/max 2500 V, Cu Kα radiation, k = 1.54178 Å). Fourier transform infrared (FTIR) spectra were recorded using a Fourier Transform IR Spectrometer (Model: Nicolet-710 spectrometer) by the KBr pellet at wavelengths ranging from 400 to 4000 cm−1. The UV–vis spectroscopy was carried out on a UV–vis spectrophotometer (UV1800, Shimadzu Corporation) in the wavelength range of 200-500 nm. The morphology of the samples was examined by a scanning electron microscope (SEM, FEI Magellan 400, USA) and a transmission electron microscope (TEM, JEM2100F, Japan). Energy Dispersive Spectrometer (EDS) was used to estimate the Ca/P molar ratio of the ACP nanoparticles. Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were obtained by a surface area and pore size analyzer (V-Sorb 2800P, Gold APP China). Dynamic light scattering (DLS) was used to measure diameter of sample (Malvern, UK). 2.4. In vitro drug release assay The drug release was studied using the membrane dialysis method against phosphate buffered saline (PBS, pH 7.4) and acetate buffers (pH 5.4) at 37 °C, which were used as the drug-release media to simulate normal blood/tissue and tumor environments. Before the experiment, the Cur-loaded ACP nanoparticles were re-suspended with PBS (pH 7.4, 5.4). Firstly, 5 mL of the Cur-loaded ACP nanoparticles (2 mg/ mL) was placed in dialysis bags with a molecular weight cutoff of 34 kDa. Next, the dialysis bags were immersed in 100 mL of the beakers, which was shaken (70 rpm) at 37 °C while shielded from light. The release medium was withdrawn at various intervals and replenished with an equal volume of fresh medium. The amount of released Cur was estimated by measuring the absorbance at 424 nm.
2. Experiment 2.1. Materials Curcumin (99%), 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH), 2, 2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Butyl hydroxy anisd (BHA), butylated hydroxytoluene (BHT) were obtained from Shanghai Aladdin Biochemical Technology Co., Ltd. Calcium nitrate (Ca(NO3)2), ammonium hydrogen phosphate ((NH4)2HPO4) and sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Co. Ltd. All the other reagents obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. were analytical grade and all solutions were prepared by distilled water.
2.5. Cur protection To elucidate the effect of encapsulation on the stability of Cur against external severe processing, we compared with remnant content of free Cur with that of entrapped Cur in ACP nanoparticles after thermal treatment and ultraviolet radiation. Free and encapsulated Cur with quality of 20 mg by thermal treatment (60 °C, 30 min) and ultraviolet radiation was taken into account for the protective effect. For free Cur and Cur-loaded ACP nanoparticles, ethyl alcohol was added and stirred for 30 min [17]. The existent Cur was calculated through the absorption value at 424 nm.
2.2. Synthesis ACP nanoparticles were formed by coprecipitation method [15]. Briefly, Ca(NO3)2·4H2O was dissolved in 29 mL deionized water to form 1 mM aqueous solution. 29 mL (NH4)2HPO4 phosphate solution was added dropwise to the above solution and the aqueous solution quickly turned to white suspension. The raw materials were designed with a Ca/P molar ratio from 1.5 and the pH value was maintained within 8 by addition of 1 M NaOH solution. The resulting mixture was agitated at 30 °C for 10 min. Finally, the obtained nanoparticles were washed three times with deionized water to remove any residual ions, and the sample was collected by centrifugation and freeze-dried. Drug loading: Cur (5 mg/mL) was added to the solution of Ca (NO3)2. The mixture was stirred for 1 h, followed by slow addition of an aqueous solution of Na2HPO4 (0.024 mM). The mixture was gently stirred at 30 °C for another 15 min. After the suspension was centrifuged at high speed to collect the products, the concentration of unloaded
2.6. Determination of antioxidant activity in vitro 2.6.1. 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) radical-scavenging activity assay The antioxidant properties of Cur and Cur-load ACP nanoparticles were evaluated by determination of scavenging effect on DPPH radicals, and scavenging capacity of the samples was determined using a method reported previously [18, 19]. In this assay 1 mL samples at 2
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multiple concentrations (2, 4, 6, 8, 10 μg/mL) were mixed with 2 mL of 0.1 mM DPPH· ethanol solution. Then mixtures were stored in dark for 30 min at room temperature and the absorbance of the reaction solution was measured at 517 nm. The same reaction condition was applied for the blank and standard solution. The free radical scavenging activity was calculated using Eq. (3):
Inhibition (%) =
A0 − A1 ∗ 100% A0
3. Results and discussion 3.1. Preparation of ACP nanoparticles There is no doubt that ACP is a metastable phase of hydroxyapatite (HA), and it has been demonstrated that amorphous calcium phosphate has better biodegradability than hydroxyapatite in vivo [8]. Consequently, to explore the effect of concentration, pH, temperature and time on the formation of ACP nanoparticles, a rapid and simple method was adopted to synthesize the carrier. Fig. 2a reveals the effect of concentration on the ACP nanoparticles. According to Fig. 2a, only the sample 1 (0.024 mM) shows a broad diffraction line at 30° that is exhibited typical amorphous phase of calcium phosphate [21]. When the PO43− concentration increased, sample had crystallized diffraction peaks located at 28.33° and 31.69°, which were attributed to (102) and (211) of HA (JCPDSNo.74-0566). Effects of different pH on ACP nanoparticles are shown in Fig. 2b. Amorphous phase was achieved when pH was 8. Crystalline peaks at 30.4, 40.1, 47 and 49° ascribed to HA (JCPDSNo.74-0566). The transformation of ACP into crystalline apatite in the aqueous solution can be described as follows: when pH > 8, ACP converts into apatite directly by rearrangement of the atomics [22]. Fig. 2c shows the sample prepared at 25 °C had two XRD pattern with HA diffraction peaks. However, the peak at 30° tended to become sharp when temperature increased, only the sample at 30 °C had the perfect characteristics of amorphous XRD pattern. The sample with reaction time for 10 min had no obvious diffraction peaks, indicating that the product consisted of the ACP phase. However, when the reaction time increased to 3 h (Fig. 2d), the product contained a small amount of HA (JCPDSNo.74-0566), which is due to the partial transformation from ACP to HA [23]. The experimental results indicated that the product prepared at 30 °C for 10 min is amorphous calcium phosphate (ACP) when PO43− concentration is 0.024 mM and pH value is 8. The formation of ACP nanoparticles was further investigated using the FTIR spectroscopy. As shown in Fig. 3, the broad unresolved absorption bands located at around 572 and 1047 cm−1 were ascribed to the characteristics of PO43−. While the brands at around 3430 and 1636 cm−1 was assigned to the adsorbed water [24]. The small band at 875 cm−1 proved the presence of HPO42− in ACP nanoparticles. The amount of HPO42− and consequently the Ca/P ratio strongly depends on the solution acidity: The lower pH of the synthesis medium (in this case, DES), the greater HPO42− content, and consequently the lower Ca/P ratio. The small band at 873 cm−1 was υ3 bands of carbonate groups, due to carbonate ions located PO43− in ACP nanoparticles [25, 26]. The peak at 1412 cm−1 was attributed to the carbonate bonds, indicating that the presence of CO32– group, which may be derived from the dissolved CO2 from atmosphere [27-29]. The particle size distributions of the ACP nanoparticles was characterized by DLS, as shown in Fig. 4, from which one can see that two
(3)
where A0 is the absorbance of the control solution, and A1 is the absorbance of the samples.
2.6.2. ABTS•+ scavenging activity assay The ABTS·+ scavenging activity was estimated according to one described previously [1]. Briefly, 7 mM of ABTS and 4.9 mM of potassium persulphate (K2S2O8) at the volume ratio of 1:1 were mixed in beaker and stood in a dark at room temperature for 16 h to generate the free radical of ABTS, then 5 mL of ABTS•+ solution diluted with 250 mL absolute ethyl alcohol to give an absorbance of 0.70 ± 0.02 at 734 nm. After that, 1 mL samples with different concentrations (2, 4, 6, 8, 10, 12 μg/mL) were added to 4 mL of ABTS•+ solution and the reaction mixture was continued at room temperature for 20 min and the absorbance at 734 nm was then measured. The scavenging activity was calculated according to Eq. (3).
2.7. In vitro cytotoxicity tests The cytotoxic activity of ACP nanoparticles and Cur-load ACP nanoparticles was evaluated using A549 cells. Typically, cells were seeded in 96-well plate (5000 cell per well) and cultured in 5% CO2 at 37 °C for 24 h. The different serial concentrations of materials were added into the wells and co-cultured with the cells for 48 h. Subsequently, each well was treated with MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) for another 4 h. The formed formazan crystals were dissolved in DMSO and absorbance was measured at 490 nm. The data was representative as the main value of five parallel experiments. Cell images of A549 cells treated with different concentrations of materials were obtained using an optical microscope [8, 20].
2.8. Statistical analysis All data were conducted in triplicates and presented as averages and standard deviations. Statistical analysis was performed using one-way analysis of variance (ANOVA) using SPSS18.0 software. The level of significance was set at 0.05 (Fig. 1).
Fig. 1. Schematic diagrams: formation of ACP nanoparticles synthesized by coprecipitation method. 3
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Fig. 2. XRD patterns of the ACP nanoparticles: a) different PO43− concentration: sample 1(0.024 mM), sample 2(0.028 mM), sample 3(0.034 mM), sample 4(0.047 mM), sample 5(0.07 mM), sample 6(0.14 mM); b) different pH: 8,9,10,10.5; c) different temperature: 25 °C, 30 °C, 35 °C, 40 °C; d) different time: 10 min,1 h,2 h,3 h.
of the ACP nanoparticles determined by DLS was 189 nm. The difference in particle size determined by SEM, TEM and DLS might be caused by the different preparations of the samples. DLS is a method that allows the direct observation of the polymeric aggregates in aqueous solutions, while SEM measures particles in the dried state [31]. EDX (Fig. 5c) shows the presence of Ca, P, O and C elements, and elemental analysis indicated a Ca/P molar ratio of 1.38 ± 0.1 in the region of the ACP nanoparticles, which was below the stoichiometric value of 1.5 for amorphous calcium phosphate. Deviation from the stoichiometic value is known in the literatures and due to the presence of small amounts (< 15%) of HPO42− ions, surface-adsorbed soluble phases which can be washed away or cationic substitutions at the Ca2+ sites by Mg2+ or Na+ or anionic substitution at PO43− sites by CO32– or HPO42− or a combination of these substitutions [24, 32-34]. The porosity, one of the most attractive properties of ACP nanoparticles, is highly desirable for drug or protein loading. In this study, large pore sizes are also expected to exit in this ACP nanoparticles, which is appropriate for small molecules to pass through without limitation. As shown in Fig. 6, products exhibited a type IV isotherm with H1 hysteresis loop, typical of mesostructured materials with a mixture of mesopore sizes [35]. The BET specific surface area of the ACP nanoparticles was about 41.414 m2/g. Total pore volume and the average pore size of ACP nanoparticles were about 0.1656 cm3/g and 10.5 nm, respectively. A similar result was reported by Zhu [14], who found that specific surface area of the as-prepared ACP porous nanospheres was about 25 m2/g.
Fig. 3. FTIR spectra infrared spectra of ACP nanoparticles.
sets of size distributions of ACP nanoparticles are observed: one is located at approximately 189 nm and the other is around 5.9 μm. This result was caused by the aggregation of ACP nanoparticles [30]. The morphology of the ACP nanoparticles was analyzed by SEM and TEM as shown in Fig. 5a and b. The spherical and aggregated nanoparticles with smooth surface were observed from SEM and TEM. Most of the particles ranged from 80 to 120 nm. However, the mean diameter 4
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Fig. 4. Particle size distribution of ACP nanoparticles by DLS.
a rapid degradation after being exposed to alkaline solution, ACP nanoparticles could achieve to a controlled and sustained release of Cur in simulated blood environment, which avoids degradation of Cur at pH of 7.4. Therefore, ACP carrier could overcome rapid release of Cur in vivo and prolong their effect in the body.
3.2. In vitro drug release Fig. 7 displays the drug in vitro release behaviors of Cur-loaded ACP nanoparticles under different pH at 37 °C. Clearly, there was no burst release for the Cur-loaded ACP nanoparticles in PBS buffer solution (pH 7.4 and pH 5.4). At the beginning of release (approximately 5 h), there was no significant difference in the drug releases in buffer solutions with different pH. However, with the increase of time, the release amount of Cur from the ACP nanoparticles in an acidic environment was accelerated compared with that in a neutral environment, indicating the ACP nanoparticles are a promising drug delivery system that could reduce the concentration of free chemotherapeutics in nontargeted sites. At the release time of 52 h, the accumulative released percentages of Cur were approximately 85.84 and 64.6789% in pH 5.4 and pH 7.4 buffer solutions, respectively. The drug release behavior of Cur-loaded ACP nanoparticles was very similar to the result of calcium phosphate prepared in the absence of the block copolymer in a previous study [15]. According to the result above, compared with pure Cur with
3.3. Cur protection As we know, a variety of factors, including pH, temperature and ultraviolet radiation, have been reported to affect chemical stability of Cur [18]. Fig.8 shows that ACP-loaded strategy was a feasible and effective method to protect Cur against thermal and ultraviolet treatment. The amount of free Cur was reduced from 20 to 5 mg and 6 mg when the solution suffered from 60 °C treatment for 30 min and ultraviolet radiation for 1 h, respectively. The preserved Cur could reach 9 mg when subjected treatment at 60 °C for 1 min, while achieved 11 mg for 1 h ultraviolet radiation, respectively. The drastically reduced phenomenon was consistent with previous study [36]. Compared with free
Fig. 5. SEM (a), TEM (b) and EDS (c) micrographs of ACP nanoparticles synthesized by using Ca(NO3)2 as the calcium source and (NH4)2HPO4 as the phosphorus source by the coprecipitation method. 5
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Fig. 9. DPPH radical scavenging activity of Cur, BHA, BHT and Cur-loaded ACP nanoparticles.
Fig. 6. Characterization of ACP nanoparticles by N2 adsorption–desorption isotherms and BJH pore size distribution (inset).
Fig. 10. ABTS·+ radical scavenging activity of Cur, BHA, BHT and Cur-loaded ACP nanoparticles.
Fig. 7. Drug release profiles of Cur-loaded ACP nanoparticles under different pH environment.
Fig. 11. Cytotoxicity tests of the as-prepared ACP nanoparticles prepared with and without anticancer drug Cur loading using A549 cells.
Fig. 8. Cur protection by ACP nanoparticles in the heat and ultraviolet treatment.
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Fig. 12. The optical images of A549 cells treated with different concentrations of ACP nanoparticles without Cur loading (the first row images) and with Cur loading (the second row images).
all samples showed dose-dependent ABTS·+ scavenging capacity in the concentration range of 0–12 μg/mL. At 12 μg/mL, the scavenging activities of Cur-loaded ACP nanoparticles, Cur, BHA and BHT on ABTS·+ were 85.12, 76.85, 64.56, and 62.71%, respectively, which suggested that the ABTS·+ scavenging capacity of encapsulated Cur was maximum. The results were consistent with the DPPH radical scavenging ability. Similar results have also been reported by other authors who used different formulation for Cur encapsulation to enhance its antioxidant activity. [39, 40]. It was seen that the antioxidant activity of encapsulated Cur was superior for the free Cur. Yoncheva et al. [41] recently prepared Cur encapsulated cationic triblock copolymer micelles and determined its antioxidant activity. Their results showed that the ability of the micellar Cur to scavenge the free radical was higher than that of the free Cur.
Cur at the same treatment, the amount of ACP nanoparticles protected Cur was enhances. The results demonstrated Cur could be protected and improve its stability during food processing, which is helpful to broaden Cur potential application in food field. 3.4. Antioxidant analysis 3.4.1. DPPH· scavenging capacity The absorbance of DPPH can be reduced by hydrogen atom transferred from H-donors (antioxidants) which led to the disappearance of the visible band in DPPH [37]. The DPPH radical scavenging ability of Cur-loaded ACP nanoparticles, Cur, BHA and BHT were compared in Fig. 9. DPPH radical scavenging ability was enhanced with the increase of concentrations for all samples. At the concentration of 10 μg/mL, the DPPH radical scavenging capacity of free Cur was 46.5%. However, the DPPH scavenging ability of Cur was raised to 49.5% when encapsulated with ACP nanoparticles. These results indicated that encapsulated Cur is more efficient at scavenging free radicals than free Cur. The reason above could be attributed to the low solubility of Cur, which could lower the amount of reactant molecules. However, the increase of water dispesity and larger surface area after encapsulated Cur due to the smaller mean particle diameter facilitated the interaction between Cur and radicals [16, 19]. Previous studies have reported by Huang et al. [1] that Cur encapsulated within these core–shell protein–polysaccharide nanoparticles exhibited higher antioxidant and radical scavenging activities than Cur. For BHA and BHT, the DPPH radical scavenging activity was poorer than Cur, which is in good agreement with the previous reports that scavenging activity of Cur was stronger [38]. Anyhow, the antioxidant activity enhancement was beneficial to improving the economic and health value of the active product in functional foods.
3.5. Cytotoxicity tests To evaluate the biocompatibility of the ACP nanoparticles, the in vitro cytotoxicity was assessed on by standard MTT assay on A549 cells. As shown in Fig. 11, the cell viability was still higher than 90% when the cells were co-cultured with ACP nanoparticles at high concentration (32 μg/mL), indicating good biocompatibility of ACP nanoparticles. On the other hand, the cell viability gradually decreases with increasing concentration of Cur-loaded ACP nanoparticles. The cell viability was only 39% when the concentration of Cur-loaded ACP nanoparticles was 32 μg/mL. This implied that the cell viability was significantly inhibited by the Cur released from ACP nanoparticles. The as-prepared ACP nanoparticles are predicted to be used as drug carriers for cancer. Fig. 12 shows the optical images of A549 cells co-cultured with different concentrations of ACP nanoparticles with and without Cur loading. The experiments showed that the cells could maintain spindle morphology, implying that the cells still have a good physiological state after being treated with ACP nanoparticles at the concentrations in the range of 1–16 μg/mL. However, with the increase of Cur-loaded ACP nanoparticles concentration, the cells obviously changed to be spherical,
3.4.2. ABTS·+ scavenging capacity The ABTS·+ scavenging capacity of Cur encapsulated in ACP nanoparticles, BHA and BHT were also investigated (Fig. 10). Evidently, 7
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which is significantly different from the ACP nanoparticles without Cur loading. The spherical morphology of the cells was caused by the damage or killing of the cells, which was attributed to release of Cur from ACP nanoparticles. These results suggested that the as-prepared Curloaded ACP nanoparticles have a high cytotoxicity to cancer cells, which is consistent with the MTT assay results [8, 23].
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4. Conclusion A facile, rapid, surfactant-free, and environmentally friendly strategy was developed for the synthesis of amorphous calcium phosphate (ACP) nanoparticles, where an aqueous solution containing Ca (NO3)2 was used as a calcium source, and (NH4)2HPO4 as a phosphorus source. The reaction temperature, time, pH and the concentrations of (NH4)2HPO4 have significant effects on the crystal phase of the product. When reaction temperature, time, pH value and concentration was 30 °C, 10 min, 8 and 0.024 mM, respectively, the product was composed of amorphous calcium phosphate. The as-prepared ACP nanoparticles have a high specific surface area. The experiments showed that the ACP nanoparticles have a high Cur drug loading capacity and favorable pHresponsive drug release property. The Cur-loaded ACP nanoparticles have superior radical scavenging activities and show a high ability to damage tumor cells. Thus, the as-prepared ACP nanoparticles are promising for the applications in various biomedical and pabular fields such as drug delivery, functional foods and beverages. Acknowledgments The work was financially supported by Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (172040098-S), National Natural Science Foundation of China (31700689), Shanxi Scholarship Council of China (2015-033), and the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province. References [1] X. Huang, X. Huang, Y. Gong, H. Xiao, D.J. McClements, K. Hu, Enhancement of curcumin water dispersibility and antioxidant activity using core–shell protein–polysaccharide nanoparticles, Food Res. Int. 87 (2016) 1–9. [2] M. Valko, D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int. J. Biochem. Cell Biol. 39 (2007) 44–84. [3] C. Sun, C. Xu, L. Mao, D. Wang, J. Yang, Y. Gao, Preparation, characterization and stability of curcumin-loaded zein-shellac composite colloidal particles, Food Chem. 228 (2017) 656–667. [4] Z. Li, Y. Wang, Y. Pei, W. Xiong, C. Zhang, W. Xu, S. Liu, B. Li, Curcumin encapsulated in the complex of lysozyme/carboxymethylcellulose and implications for the antioxidant activity of curcumin, Food Res. Int. 75 (2015) 98–105. [5] B.R. Shah, C. Zhang, L. Yan, B. Li, Bioaccessibility and antioxidant activity of curcumin after encapsulated by nano and pickering emulsion based on chitosan-tripolyphosphate nanoparticles, Food Res. Int. 89 (2016) 399–407. [6] B. Abdous, S.M. Sajjadi, L. Ma'mani, β-Cyclodextrin modified mesoporous silica nanoparticles as a nano-carrier: response surface methodology to investigate and optimize loading and release processes for curcumin delivery, J. Appl. Biomed. 15 (2017) 210–218. [7] G.J. Ding, Y.J. Zhu, C. Qi, B.Q. Lu, F. Chen, J. Wu, Porous hollow microspheres of amorphous calcium phosphate: soybean lecithin templated microwave-assisted hydrothermal synthesis and application in drug delivery, J. Mater. Chem. B 3 (2015) 1823–1830. [8] G.J. Ding, Y.J. Zhu, C. Qi, B.Q. Lu, J. Wu, F. Chen, Porous microspheres of amorphous calcium phosphate: block copolymer templated microwave-assisted hydrothermal synthesis and application in drug delivery, J. Colloid Interface Sci. 443 (2015) 72–79. [9] Z.F. Zhou, T.W. Sun, F. Chen, D.Q. Zuo, H.S. Wang, Y.Q. Hua, Z.D. Cai, J. Tan, Calcium phosphate-phosphorylated adenosine hybrid microspheres for anti-osteosarcoma drug delivery and osteogenic differentiation, Biomaterials 121 (2017) 1–14. [10] M. Parent, H. Baradari, E. Champion, C. Damia, M. Viana-Trecant, Design of calcium phosphate ceramics for drug delivery applications in bone diseases: a review of the parameters affecting the loading and release of the therapeutic substance, J. Control. Release 252 (2017) 1. [11] Y. Lv, H. Huang, B. Yang, H. Liu, Y. Li, J. Wang, A robust pH-sensitive drug carrier: aqueous micelles mineralized by calcium phosphate based on chitosan, Carbohydr.
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