Accepted Manuscript Porous microspheres of amorphous calcium phosphate: block copolymer templated microwave-assisted hydrothermal synthesis and application in drug delivery Guan-Jun Ding, Ying-Jie Zhu, Chao Qi, Bing-Qiang Lu, Jin Wu, Feng Chen PII: DOI: Reference:
S0021-9797(14)00948-5 http://dx.doi.org/10.1016/j.jcis.2014.12.004 YJCIS 20047
To appear in:
Journal of Colloid and Interface Science
Received Date: Accepted Date:
1 October 2014 1 December 2014
Please cite this article as: 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, Journal of Colloid and Interface Science (2014), doi: http://dx.doi.org/10.1016/j.jcis.2014.12.004
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Porous microspheres of amorphous calcium phosphate: block copolymer templated microwave-assisted hydrothermal synthesis and application in drug delivery
Guan-Jun Ding, Ying-Jie Zhu*, Chao Qi, Bing-Qiang Lu, Jin Wu, Feng Chen
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China
__________________________________________ * Corresponding author. Tel: 0086-21-52412616; Fax: 0086-21-52413122; E-mail address: [email protected] (Y. J. Zhu)
1
Abstract Amorphous calcium phosphate (ACP) microspheres with a porous and hollow structure have been prepared using an aqueous solution containing CaCl2 as a calcium source, adenosine triphosphate disodium salt (Na2ATP) as a phosphorus source in the presence
of
a
block
glycol)-block-poly(D,L-lactide)
copolymer (mPEG-PLA)
methoxyl by
the
poly(ethylene
microwave-assisted
hydrothermal method. The effects of microwave hydrothermal temperature and the concentrations of CaCl2 and Na2ATP on the crystal phase and morphology of the product are investigated. The as-prepared ACP porous hollow microspheres have a relatively high specific surface area of 232.9 m2 g–1 and an average pore size of 9.9 nm. A typical anticancer drug, docetaxel, is used to evaluate the drug loading ability and drug release behavior of ACP porous hollow microspheres in phosphate buffered saline (PBS) with different pH values of 4.5 and 7.4. The experiments reveal that the ACP porous hollow microspheres have a high drug loading capacity and favorable pH-responsive drug release property, and the ACP porous hollow microsphere drug delivery system shows a high ability to damage tumor cells. It is expected that the as-prepared ACP porous hollow microspheres are promising for the applications in various biomedical fields such as drug delivery.
Keyword:
microwave;
amorphous
calcium
nanostructures; drug delivery
2
phosphate;
porous
materials;
Introduction
Calcium phosphate (CP) materials, as the most important inorganic biomaterials with excellent biocompatibility and biodegradability in biological systems, have received much interest in the field of drug and gene delivery [1–5]. Among a variety of CP materials, amorphous calcium phosphate (ACP) is regarded as a metastable phase with a short range order formed from a supersaturated aqueous solution at the early stage of the reaction between calcium ions and phosphate ions. ACP nanostructured materials are promising drug carriers owing to their advantages including large specific surface area, high drug loading capacity and controlled drug release behavior [6,7]. Furthermore, ACP has a good biodegradability and an ability to promote osteoblast adhesion and osteconductivity [8,9].
Block copolymers have been demonstrated to be effective for the template-directed synthesis of CP porous materials. Various block copolymers such as poly(ethylene glycol)-block-polylactide (PEG-PLA), triblock copolymer (P123), and polymer micelles composed of pluronic P123 and Tween-60, were coated with CP nanoparticles or nanosheets for the application in drug loading and release [10–13]. However, to the best of our knowledge, there have been few studies on the synthesis and properties of ACP porous nanostructured materials using a block copolymer as the template by the microwave-assisted hydrothermal method.
3
Adenosine triphosphate (ATP), the most common energy carrier of cells in biology, is recognized as a good organic phosphate source and an effective stabilizer for ACP to inhibit the transformation from ACP to hydroxyapatite (HAP) in aqueous solution via poisoning heterogeneous nucleation sites and/or binding to embryonic HAP nucleus to prevent their growth [14–17]. Qi et al. [17] reported the microwave-assisted hydrothermal preparation of highly stable amorphous calcium phosphate porous nanospheres with a relatively uniform size using ATP as the phosphorus source and stabilizer. The as-prepared ACP porous nanospheres had a high stability in the PBS solution and were efficient for anticancer drug loading and sustained release. The ACP porous nanosphere drug-delivery system showed a high ability to damage tumor cells after loading docetaxel.
In recent years, the microwave-assisted synthesis of nanostructured materials has attracted much interest because of its advantages such as rapidness, high efficiency, low-cost and energy-saving [18]. Herein, we report a facile microwave-assisted hydrothermal synthesis of ACP porous hollow microspheres using calcium chloride as a calcium source and Na2ATP as a phosphorus source in the presence of a block copolymer methoxyl poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PLA) as a soft template. The effects of the microwave hydrothermal temperature and concentrations of CaCl2 and Na2ATP on the crystal phase and morphology of the product are investigated. The prepared ACP porous hollow microspheres have a relatively high specific surface area and are efficient for drug loading and release
4
using docetaxel as a model anticancer drug. The as-prepared ACP porous hollow microspheres loaded with docetaxel show a high ability to damage tumor cells, thus, they are promising for the application in drug delivery.
Experimental Section Materials Methoxyl poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PLA, the molecular weight of PEG and PLA was 5000 and 3000, respectively) was obtained from Jinan Daigang Biomaterials Co. Adenosine triphosphate disodium salt (Na2ATP) was purchased from Sigma-Aldrich. Hydrochloric acid (analytical grade, HCl, 36.0%~38.0%), anhydrous CaCl2 (analytical grade), NaOH (analytical grade) were obtained from Sinopharm Chemical Reagent Co. Ltd. Docetaxel (98%) and powdered phosphate buffer were obtained from Sangon Biotech (Shanghai), Co. Ltd. Microwave-assisted hydrothermal synthesis of samples In a typical experiment, 0.0500 g of mPEG-PLA and 0.0550, 0.1100, 0.2200 or 0.3300 g of Na2ATP was dissolved in 30 mL of deionized water to form a clear solution, which was mixed with 5 mL aqueous solution containing 0.0555, 0.1110, 0.2220 or 0.3330 g of anhydrous CaCl2. Then, the pH value of the resulting solution was adjusted to 5.0 by addition of 2 M NaOH aqueous solution. The final volume of the solution was 40 mL with the extra addition of deionized water. The resulting solution was loaded into a 60 mL autoclave, sealed and heated in a microwave oven (MDS-6, Sineo, China) to a temperature of 120, 140, 160 or 180 oC and maintained at
5
this temperature for 30 min, and then cooled down naturally to room temperature. The product was collected by centrifugation and washed with deionized water three times, and dried by freeze drying. In vitro drug loading and release experiments The typical in vitro drug loading and release experiments were performed as follows: 0.100 g of ACP porous hollow microspheres, prepared using an aqueous solution containing 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of anhydrous CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min, was added into 10 mL of ethanol solution with a docetaxel concentration of 40 mg mL-1. The dispersion was then shaken at a constant rate of 150 rpm for 24 h in a sealed vessel at 37 oC. The drug-loaded ACP porous hollow microspheres were collected by centrifugation and dried in air at 60 oC. Then, 3.5 mg docetaxel-loaded ACP porous microspheres were dispersed in 20 mL phosphate buffered saline (PBS) with a pH value of 4.5 or 7.4 at 37 oC under shaking at a constant rate of 150 rpm. The shaking device was a desk-type constant-temperature oscillator (THI-92A, China). The docetaxel release medium (1.0 mL) was withdrawn for UV-vis absorption analysis at 230 nm at given time intervals and replaced with the same volume of fresh PBS. In vitro cytotoxicity tests The human gastric carcinoma (MGC-803) cells were cultured in a RPMI-1640 medium
supplemented
with
10%
fetal
bovine
serum
(FBS)
and
1
%
penicillin-streptomycin at 37oC for 48 h. Then the cells were seeded in 96-well flat-bottom microassay plates at a concentration of 1×104 cells per milliliter and
6
cultured for 24 h. The sterilized docetaxel-loaded ACP porous hollow microspheres or ACP porous hollow microspheres without drug loading were added into the wells at concentrations ranging from 0.1-100 μg mL-1 and were co-cultured with the cells for 48
h.
The
cell
viability
was
quantified
by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data was representative as the main value of three parallel experiments. All reagents used in cell viability experiments were purchased from Sigma-Aldrich. Cell images of MGC-803 cells treated with different concentrations of ACP porous hollow microspheres or docetaxel-loaded ACP porous hollow microspheres were obtained using an Olympus IX71 optical microscope. Characterization Transmission electron microscopy (TEM) images were obtained with a transmission electron microscope (TEM, HITACHI H-800, Japan). Scanning electron microscopy (SEM) micrographs were obtained with a field-emission scanning electron microscope (SEM, Hitachi S-4800, Japan). The thermogravimetric (TG) analysis was performed with a STA 409/PC simultaneous thermal analyzer (Netzsch, Germany) at a heating rate of 10 oC min-1 in air. UV-vis absorption spectra were taken on a UV 2300 spectrophotometer (Techcomp). Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were measured with a surface area and pore size analyzer (V-Sorb 2800P, Gold APP Instruments); Fourier transform infrared (FTIR) spectra were collected on a FTIR spectrometer (FTIR-7600, Lamdba Scientific, Australia). X-ray powder diffraction (XRD) patterns were recorded using an X-ray diffractometer
7
(Rigaku D/max 2500 V, Cu Kα radiation, λ = 1.54178 Å). Hydrodynamic size distribution and surface zeta potential of the ACP porous hollow microspheres were characterized by dynamic lighting scattering (DLS) and zeta potential analyzer (Brookhaven, Zetaplus).
Results and Discussion
Figure 1a-d shows the XRD patterns of the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min. The product prepared at 120 o
C for 30 min is amorphous calcium phosphate (ACP). The products obtained at 140,
160 and 180 oC for 30 min consist of hydroxyapatite (HAP).
We investigated the effect of microwave hydrothermal temperature on the morphology of the product. The morphologies of the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min were characterized by TEM and SEM, as shown in Figure 2, from which one can see that when the microwave hydrothermal temperature is at 120 oC, the product consists of aggregated porous microspheres with sizes of several hundred nanometers. When the microwave hydrothermal temperature increases to 140, 160 or 180 oC, the product is composed of rod-assembled microspheres with rods as building blocks directing from
8
the center outwards.
Figure 3a-f shows the FTIR spectra of mPEG-PLA, Na2ATP and the samples prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min. According to the previous report [19], the broad peak between 3600 cm–1 and 3100 cm–1 is assigned to the O–H group of adsorbed water; the weak absorption bands at about 2950 and 2890 cm–1 can be attributed to the stretching vibration of –CH2 and –CH3 groups, which originate from mPEG-PLA block copolymer; the absorption peak around 1760 cm–1 may arise from the C=O group in mPEG-PLA; the absorption peak at 1637 cm–1 is related with stretching vibration of C=O, stretching of C=C, stretching of N=C and primary bending of N–H in the –NH2 group; the broad absorption peak located at 1110 cm–1 originates from the stretching of the C–O group in mPEG-PLA; the absorption peak at 1115 cm–1 results from the C–O–C stretching from mPEG-PLA. The absorption peaks at around 1100, 1027, 605 and 560 cm–1 are characteristic bands of PO43- ions in calcium phosphate in accordance with the XRD results in Figure 1. The absorption peak at 915 cm–1 comes from asymmetric P–O–C stretching and P–OH stretching in phosphorus ester of ATP biomolecules. The absorption peak at 915 cm–1 is only observable in the product prepared at 120 oC for 30 min, implying the existence of ATP molecules in the product of ACP. This may be explained by uncomplete hydrolysis of ATP biomolecules in the microwave-assisted hydrothermal conditions at 120 oC for 30 min [17].
9
We also investigated the effect of the concentrations of Na2ATP and CaCl2 on the crystal phase and morphology of the product prepared using mPEG-PLA, Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min. As shown in Figure 1a, e–g, the products prepared using 0.0500 g mPEG-PLA and different amounts of Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min are composed of amorphous calcium phosphate. As shown in Figure 2a and a’, when 0.0550 g of Na2ATP and 0.0555 g of CaCl2 are used, the product consists of ACP microspheres with a porous structure. Similarly, when 0.1100 g of Na2ATP and 0.1110 g of CaCl2 are used, ACP porous microspheres are also obtained (Figure 4a and a’). However, when the amounts of Na2ATP and CaCl2 further increase, ACP porous hollow microspheres are formed (Figure 4b, b’ and 4c, c’). The FTIR spectra of the products prepared using 0.0500 g mPEG-PLA and different amounts of Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min (Figure 3c, g–i) indicate that more ATP biomolecules are adsorbed on ACP porous microspheres with increasing the amount of ATP.
Figure 5a and b shows the size distributions measured by both SEM and dynamic lighting scattering (DLS) of ACP porous hollow microspheres prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min. The average size of ACP porous hollow microspheres measured by SEM is (387 ± 114) nm, as shown in
10
Figure 5a. In addition, the hydrodynamic size distributions of the ACP porous hollow microspheres were characterized by DLS, as shown in Figure 5b, from which one can see that the average hydrodynamic diameter of as-prepared ACP porous hollow microspheres is (947 ± 130) nm. The aggregation of ACP porous hollow microspheres is observed by DLS. Two sets of size distributions of ACP porous hollow microspheres are observed: one is located at approximately 500 nm and the other is around 1300 nm. This result is caused by the aggregation of ACP porous hollow microspheres.
The possible formation process of calcium phosphate porous microspheres prepared using mPEG-PLA, Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at different temperatures for 30 min is schematically shown in Figure 6. At the early stage of the reaction, mPEG-PLA molecules form spherical micelles with the PLA segment as the core and the PEG segment as the shell. And calcium ions adsorb on the PEG segments of micelles as a result of attraction force between the polymer molecules and calcium ions [20–23]. When the reaction solution is microwave-heated, ATP molecules are hydrolyzed and release phosphate ions, and then the reaction between calcium ions and phosphate ions occurs on the surface of mPEG-PLA micelles. The morphology of the final product depends on the microwave hydrothermal temperature and concentrations of the reactants. When the microwave hydrothermal temperature is 120 oC, the remaining ATP molecules can stabilize the amorphous calcium phosphate and prevent the transformation from ACP to
11
hydroxyapatite in aqueous solution, and ss a result, the product is composed of ACP. ACP porous microspheres are formed at lower concentrations of Na2ATP and CaCl2, however, ACP porous hollow microspheres are obtained at higher concentrations of Na2ATP and CaCl2. At higher microwave hydrothermal temperatures (140 oC, 160 oC and 180 oC), ATP molecules can be effectively hydrolyzed and the inhibition effect on the phase transformation from ACP to HAP is weakened, leading to the significant phase transformation from ACP to HAP. The rod-like morphology is common during the crystal growth of HAP. Due to the space limitation and higher concentrations of calcium ions and phosphate ions in the outer environment of the spheres, the HAP rods prefer to grow from the central core outwards, resulting in HAP rods-assembled microspheres.
This research group previously reported highly stable ACP porous spheres with relatively uniform sizes and an average pore diameter of about 10 nm by the microwave-assisted hydrothermal method using Na2ATP as the phosphorus source and stabilizer [17], and the sizes of the as-prepared ACP porous spheres were relatively uniform with the average diameter of (238 ± 35) nm. In this work, the ACP porous hollow microspheres prepared by the microwave-assisted hydrothermal method in the presence of a block copolymer mPEG-PLA at 120 oC for 30 min are comparatively larger with an average size of (387 ± 114) nm. However, the ACP porous microspheres obtained in the presence of mPEG-PLA by the present method are hollow, while the ACP porous spheres obtained in the absence of mPEG-PLA in
12
reference 17 were solid not hollow, indicating that the presence of mPEG-PLA is the key factor for the formation of the hollow structure.
Figure 7a shows the nitrogen adsorption-desorption isotherm of ACP porous hollow microspheres prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min. According to the International Union of Pure and Applied Chemistry (IUPAC), it can be classified as a type IV isotherm which is the characteristics of the porous structure [24]. The BET specific surface area of ACP porous hollow microspheres is very high (232.9 m2g-1), which is higher than many previous reports on calcium phosphate materials. The high specific area of the sample can be explained by the porous and hollow structure and self-assembly of nanoparticles as the building blocks. The average BJH pore size of ACP porous hollow microspheres is 9.9 nm. TG analysis was employed to investigate the docetaxel loading amount of the as-prepared docetaxel-loaded ACP porous hollow microspheres. Figure 7b shows that the weight loss of ACP porous hollow microspheres is 17.97%, and the weight loss of docetaxel-loaded ACP porous hollow microspheres is 47.07%. The weight percentage of docetaxel loaded in ACP porous hollow microspheres is estimated to be ~550 mg drug per gram ACP carrier.
Figure 8a and b displays the docetaxel drug release behaviors of docetaxel-loaded ACP porous hollow microspheres in PBS solutions with different pH values of pH 4.5
13
and pH 7.4 at 37 oC. In both PBS solutions, docetaxel-loaded ACP porous hollow microspheres undergo a fast release of docetaxel in the first 2 h (nearly 82% of loaded docetaxel is released) and show a little drug release in the next 70 h. There is an approximately linear relationship between the cumulative amount of released drug and the square root of release time for the ACP porous hollow microsphere drug delivery system in both PBS solutions with pH 4.5 and pH 7.4 in the first 2 h. It is well acknowledged that the kinetics of drug release from carrier materials can be well described using the Higuchi model with a linear relationship between the cumulative amount of released drug, C, and the square root of time, C = k ·t1/2, where k is a constant, and the drug release is governed by a diffusion process [25,26]. In this study, the drug release behaviors can be approximately described by the Higuchi model in both PBS solutions with pH 4.5 and pH 7.4 in the first 2 h, and the drug release is controlled by a diffusion process. In addition, the ACP porous hollow microspheres exhibit a pH-responsive drug release behavior. In the PBS solution with pH 4.5, nearly 90 % of loaded docetaxel is released in 72 h; in contrast, approximately 60 % of loaded docetaxel is released in 72 h in the PBS solution with pH 7.4. This pH-responsive drug release behavior can be explained by higher solubility of calcium phosphate in PBS with pH 4.5 than that in PBS with pH 7.4 [27].
The drug release behavior of docetaxel-loaded ACP porous hollow microspheres is very different from the result of ACP porous spheres prepared in the absence of the block copolymer in a previous study [17]. In the work of Reference [17], the drug
14
delivery system exhibited a good linear relationship between the cumulative amount of released drug and the natural logarithm of release time. The difference may be explained by the large specific surface area and porous hollow structure of ACP porous hollow microspheres in this study. The XRD patterns and FTIR spectra of docetaxel-loaded ACP porous hollow microspheres after 72 hour drug release in PBS solutions with pH 4.5 and pH 7.4 at 37 oC (Figure 1h and i; Figure 3j and k) indicate that the products are composed of a mixture of hydroxyapatite and monetite, indicating that the phase transformation occurs during the drug release process in PBS solution.
The cytotoxicity tests of the as-prepared ACP porous hollow microspheres with and without docetaxel loading were examined using human gastric carcinoma (MGC-803) cells. The MTT assays show low toxicity when the cells were co-cultured with ACP porous hollow microspheres at the concentrations in the range of 0.1 – 100 μg mL–1, as shown in Figure 9. As an important inorganic component of biological hard tissues, ACP biomaterial is believed to have low toxicity. On the other hand, the cell viability gradually decreases with increasing concentration of docetaxel-loaded ACP porous hollow microspheres. The cell viability decreases to 54 % and 20 % when the concentration of docetaxel-loaded ACP porous microspheres is 0.4 and 2 μg mL–1, respectively. The as-prepared ACP porous hollow microspheres may be used in in-situ drug delivery systems such as bone tissue engineering scaffolds/implants, which can promote bone repair by releasing active substances including peptides, growth factors,
15
hormones, drugs, and so on.
Figure 10 shows the optical images of MGC-803 cells co-cultured with different concentrations of ACP porous hollow microspheres with and without docetaxel loading. The experiments show that the cells, treated with ACP porous hollow microspheres at the concentrations in the range of 0.1－10 μg mL–1, can maintain a spindle morphology, implying that the cells still have a good physiological state. The morphology of the cells obviously changes to be spherical after being treated with the docetaxel-loaded ACP porous hollow microsphere drug delivery system even at a low concentration of 2 μg mL–1, which is significantly different from the control sample without docetaxel loading. As the concentration of the docetaxel-loaded ACP porous hollow microsphere drug delivery system increases, more cells become spherical. The spherical morphology of the cells is caused by the damage or killing of the cells, which is a result of the release of anticancer drug docetaxel from ACP porous hollow microspheres. The high cytotoxicity of the docetaxel-loaded ACP porous hollow microsphere drug delivery system is consistent with the MTT assay results.
Conclusions Amorphous calcium phosphate (ACP) microspheres with a porous and hollow structure have been successfully prepared using an aqueous solution containing CaCl2 as a calcium source, Na2ATP as a phosphorus source in the presence of a block copolymer mPEG-PLA as the template by the microwave-assisted hydrothermal
16
method. The reported preparation strategy herein is facile, rapid, energy-saving, and environment-friendly. When the reaction solution is microwave-heated, ATP molecules are hydrolyzed and release phosphate ions, and then the reaction between calcium ions and phosphate ions occurs on the surface of mPEG-PLA micelles. The microwave hydrothermal temperature and the concentrations of CaCl2 and Na2ATP have significant effects on the crystal phase and morphology of the product. When the microwave hydrothermal temperature is 120
o
C, the product is composed of
amorphous calcium phosphate; and the ACP porous solid microspheres are formed at lower concentrations of Na2ATP and CaCl2, however, ACP porous hollow microspheres are obtained at higher concentrations of Na2ATP and CaCl2. At higher microwave hydrothermal temperatures (140 oC, 160 oC and 180 oC), the as-prepared products consist of hydroxyapatite microspheres formed by self-assembly of rods. The as-prepared ACP porous hollow microspheres have a high specific surface area. The experiments show that the ACP porous hollow microspheres have a high docetaxel drug loading capacity and favorable pH-responsive drug release property, and the ACP porous hollow microsphere drug delivery system shows a high ability to damage tumor cells. It is expected that the as-prepared ACP porous hollow microspheres are promising for the applications in various biomedical fields such as drug delivery.
Acknowledgements Financial support from the National Natural Science Foundation of China (51172260, 51302294), the National Basic Research Program of China (973 Program, No.
17
2012CB933600),
and
Science
and
Technology
Commission
of
Shanghai
(12ZR1452100) is gratefully acknowledged.
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Figures:
Figure 1. XRD patterns of the products prepared by the microwave-assisted hydrothermal method for 30 min: (a)-(d) the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 at various temperatures: (a) 120 oC, (b) 140 oC, (c) 160 oC, and (d) 180 oC; (e)-(g) the products prepared using 0.0500 g of mPEG-PLA and different amounts of Na2ATP and CaCl2 at 120 oC: (e) 0.1100 g of Na2ATP and 0.1110 g of CaCl2, (f) 0.2200 g of Na2ATP and 0.2220 g of CaCl2, and (g) 0.3300 g of Na2ATP and 0.3330 g of CaCl2; (h) and (i) the products, prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC, were loaded with docetaxel and put in phosphate buffered saline (PBS) with different pH values at 37 o
C for 72 h: (h) pH 4.5, and (i) pH 7.4. (Δ stands for hydroxyapatite; ◇ stands for 21
mooneetitee).
Figurre 2. SE EM annd TE EM im magees of the pproduccts preepaaredd usin u ng 0.005000 g of o PEG G-P PLA A, 0.05 0 5500 g of Naa2AT ATP and 0.05 0 555 g of CaC Cl2 byy thhe mic m row wavve-aassiistedd mP hyydro otheerm mal meethood at a varioouss tem mpeeratturees for f 30 min n: (a, ( a’) a 1200 oC; C (b, ( bb’) 1400 oC; C (c,, c’) 1660 oC;; (d, d’) 180 oC.. 22 2
Figure 3. FTIR spectra of the samples: (a) mPEG-PLA; (b) Na2ATP; (c)-(f) the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min: (c) 120 oC, (d) 140 oC, (e) 160 oC, (f) 180 oC; (g)-(i) the products prepared using 0.0500 g of mPEG-PLA, various concentrations of Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min: (g) 0.1100 g of Na2ATP and 0.1110 g of CaCl2, (h) 0.2200 g of Na2ATP and 0.2220 g of CaCl2, and (i) 0.3300 g of Na2ATP and 0.3330 g of CaCl2; (j) and (k) the products, prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min, were loaded with docetaxel and then drug was released in PBS solutions with different pH values at 37 23
o
C C: (jj) pH 44.5,, (k) pH H 7.4. 7
Figurre 4. 4 SEM S M annd TE EM imagees of o ACP A P pooroous miccro osphherees prep p pareed using 0.005000 off
mP m EG G-PL LA
a d and
d fereent diff
amo a ountts
off
Naa2AT TP
a and
CaC C Cl2
bby
thhe
owaavee-assistted hydrootheerm mal met m thod aat 1220 oC forr 300 miin: (a and a d a’) 0.1100 g of o miicro Naa2ATP A P annd 0.11 0 110 g of o CaC C Cl2; (b and d b’’) 00.22200 g of o Na N 2AT A P and a 0.222200 g of CaaCl2; (c and d c’) 0.33 0 3000 g of o Na N 2AT TP aand 0.3 3330 g off CaaCl2.
24 4
Figurre 5 (aa) siize disstrib butiion meeasuureed by b SEM S M and a (b)) size dist d tribbution meeasuuredd byy LS in w wateer oof AC ACP por p rouss holl h ow micr m rosppherres prrepaaredd usin u ng 0.005000 g of o DL mP PEG G-P PLA A, 0.33 0 3000 g of Naa2AT ATP and 0.33 0 3300 g of CaC Cl2 byy thhe mic m row wavve-aassiistedd hyydro otheerm mal meethood at a 120 oC forr 30 0 min.
25 5
6 Schhem matiic illu i ustraatioon of thee prep p paraatioon of callciuum ph hospphaate porrouus Figurre 6. u ng mP PEG G-P PLA A, Naa2ATP AT annd CaaCll2 bby thhe hoollow miicroosphherres prrepaaredd usin miicro owaavee-assistted hyd drootheerm mal met m thodd att diifferrennt teempperaaturres forr 300 miin.
26 6
Figurre 7. 7 (a) N2 aadso orpptionn–ddesoorptionn isothherrms an nd BJJH dessorp ptioon porre sizze CP por p rouss hoolloow miicroosphherees pre p eparred usiingg 0.005000 g of o disstributtionn cuurvees of AC mP PEG G-P PLA A, 0.33 0 3000 g of Naa2AT ATP and 0.33 0 3300 g of CaC Cl2 byy thhe mic m row wavve-aassiistedd hyydro otheerm mal meethood at a 120 oC forr 30 0 min; (b)) TG G cuurvves of tthe sam me sam mplle as a inn (aa) wiithoout doccetaaxeel looadiing (A A) annd witth doce d etaxxel loaadin ng (B) ( .
27 7
Figurre 8. 8 (aa) Doc D cetaaxell drrug releease prof p filess inn PB BS solu utioons wiith diff d fereent pH H vaaluees oaded AC CP pporrouss hoolloow miccroosphherees prep p pareed using 0.005000 at 37oC of doccetaaxeel-lo g off mPE m EG G-PL LA,, 0.33 0 3000 g of o Naa2A ATP P and a 0.33 0 330 g of o CaaCl2 by b thhe owaavee-assistted hy ydrootheerm mal meethood at a 11200 oC foor 30 3 min; (b) thee cum c mulaativve miicro am mou unt of relleassed dru ug verrsuss thhe squ s uaree rooot of releeasse tiimee foor the t AC CP porrouus hoollow m miccrossphheree drrug delliveery sysstem m inn both b h PB BS sollutiionss w with pH H 4.5 and a d pH H 7.44 inn thhe fiirstt 2 hou h urs at a 37 3 oC.
28 8
Figure 9. Cytotoxicity tests of the as-prepared ACP porous hollow microspheres prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min with and without anticancer drug docetaxel loading using human gastric carcinoma cells (MGC-803).
29
1 Thhe ooptiicall im mages of hum mann ggastrric carrcinnom ma cell c ls (MG ( GC-8003) treated Figurre 10. A P poorouus hol h low w micr m rosppheeres wiithoout docetaxeel wiith difffereent coonceentrrationss off ACP loaadin ng (thhe first f t roow imaagees) andd with w doocettaxeel load l dingg (tthe seccon nd row r w im magges)). AC CP porrouus hholloow miicroosphheres aare preepaaredd ussingg 0.05000 g of mPE m EG--PL LA, 0.333000 g of o Na N 2AT TP and a d 0.333 30 g oof CaC C Cl2 by b tthe miicroowaave--asssistted hyddro otheerm mal met m thodd at 120 0 oC foor 330 min m n.
30 0
Grap phicall abbstrracct
31 1
Highlights
Amorphous calcium phosphate porous hollow microspheres are prepared. A block copolymer templated microwave-assisted hydrothermal method is reported. The product has high biocompatibility and large specific surface area. The product has high drug loading capacity and pH-responsive drug release.
32
S0021-9797(14)00948-5 http://dx.doi.org/10.1016/j.jcis.2014.12.004 YJCIS 20047
To appear in:
Journal of Colloid and Interface Science
Received Date: Accepted Date:
1 October 2014 1 December 2014
Please cite this article as: 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, Journal of Colloid and Interface Science (2014), doi: http://dx.doi.org/10.1016/j.jcis.2014.12.004
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Porous microspheres of amorphous calcium phosphate: block copolymer templated microwave-assisted hydrothermal synthesis and application in drug delivery
Guan-Jun Ding, Ying-Jie Zhu*, Chao Qi, Bing-Qiang Lu, Jin Wu, Feng Chen
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China
__________________________________________ * Corresponding author. Tel: 0086-21-52412616; Fax: 0086-21-52413122; E-mail address: [email protected] (Y. J. Zhu)
1
Abstract Amorphous calcium phosphate (ACP) microspheres with a porous and hollow structure have been prepared using an aqueous solution containing CaCl2 as a calcium source, adenosine triphosphate disodium salt (Na2ATP) as a phosphorus source in the presence
of
a
block
glycol)-block-poly(D,L-lactide)
copolymer (mPEG-PLA)
methoxyl by
the
poly(ethylene
microwave-assisted
hydrothermal method. The effects of microwave hydrothermal temperature and the concentrations of CaCl2 and Na2ATP on the crystal phase and morphology of the product are investigated. The as-prepared ACP porous hollow microspheres have a relatively high specific surface area of 232.9 m2 g–1 and an average pore size of 9.9 nm. A typical anticancer drug, docetaxel, is used to evaluate the drug loading ability and drug release behavior of ACP porous hollow microspheres in phosphate buffered saline (PBS) with different pH values of 4.5 and 7.4. The experiments reveal that the ACP porous hollow microspheres have a high drug loading capacity and favorable pH-responsive drug release property, and the ACP porous hollow microsphere drug delivery system shows a high ability to damage tumor cells. It is expected that the as-prepared ACP porous hollow microspheres are promising for the applications in various biomedical fields such as drug delivery.
Keyword:
microwave;
amorphous
calcium
nanostructures; drug delivery
2
phosphate;
porous
materials;
Introduction
Calcium phosphate (CP) materials, as the most important inorganic biomaterials with excellent biocompatibility and biodegradability in biological systems, have received much interest in the field of drug and gene delivery [1–5]. Among a variety of CP materials, amorphous calcium phosphate (ACP) is regarded as a metastable phase with a short range order formed from a supersaturated aqueous solution at the early stage of the reaction between calcium ions and phosphate ions. ACP nanostructured materials are promising drug carriers owing to their advantages including large specific surface area, high drug loading capacity and controlled drug release behavior [6,7]. Furthermore, ACP has a good biodegradability and an ability to promote osteoblast adhesion and osteconductivity [8,9].
Block copolymers have been demonstrated to be effective for the template-directed synthesis of CP porous materials. Various block copolymers such as poly(ethylene glycol)-block-polylactide (PEG-PLA), triblock copolymer (P123), and polymer micelles composed of pluronic P123 and Tween-60, were coated with CP nanoparticles or nanosheets for the application in drug loading and release [10–13]. However, to the best of our knowledge, there have been few studies on the synthesis and properties of ACP porous nanostructured materials using a block copolymer as the template by the microwave-assisted hydrothermal method.
3
Adenosine triphosphate (ATP), the most common energy carrier of cells in biology, is recognized as a good organic phosphate source and an effective stabilizer for ACP to inhibit the transformation from ACP to hydroxyapatite (HAP) in aqueous solution via poisoning heterogeneous nucleation sites and/or binding to embryonic HAP nucleus to prevent their growth [14–17]. Qi et al. [17] reported the microwave-assisted hydrothermal preparation of highly stable amorphous calcium phosphate porous nanospheres with a relatively uniform size using ATP as the phosphorus source and stabilizer. The as-prepared ACP porous nanospheres had a high stability in the PBS solution and were efficient for anticancer drug loading and sustained release. The ACP porous nanosphere drug-delivery system showed a high ability to damage tumor cells after loading docetaxel.
In recent years, the microwave-assisted synthesis of nanostructured materials has attracted much interest because of its advantages such as rapidness, high efficiency, low-cost and energy-saving [18]. Herein, we report a facile microwave-assisted hydrothermal synthesis of ACP porous hollow microspheres using calcium chloride as a calcium source and Na2ATP as a phosphorus source in the presence of a block copolymer methoxyl poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PLA) as a soft template. The effects of the microwave hydrothermal temperature and concentrations of CaCl2 and Na2ATP on the crystal phase and morphology of the product are investigated. The prepared ACP porous hollow microspheres have a relatively high specific surface area and are efficient for drug loading and release
4
using docetaxel as a model anticancer drug. The as-prepared ACP porous hollow microspheres loaded with docetaxel show a high ability to damage tumor cells, thus, they are promising for the application in drug delivery.
Experimental Section Materials Methoxyl poly(ethylene glycol)-block-poly(D,L-lactide) (mPEG-PLA, the molecular weight of PEG and PLA was 5000 and 3000, respectively) was obtained from Jinan Daigang Biomaterials Co. Adenosine triphosphate disodium salt (Na2ATP) was purchased from Sigma-Aldrich. Hydrochloric acid (analytical grade, HCl, 36.0%~38.0%), anhydrous CaCl2 (analytical grade), NaOH (analytical grade) were obtained from Sinopharm Chemical Reagent Co. Ltd. Docetaxel (98%) and powdered phosphate buffer were obtained from Sangon Biotech (Shanghai), Co. Ltd. Microwave-assisted hydrothermal synthesis of samples In a typical experiment, 0.0500 g of mPEG-PLA and 0.0550, 0.1100, 0.2200 or 0.3300 g of Na2ATP was dissolved in 30 mL of deionized water to form a clear solution, which was mixed with 5 mL aqueous solution containing 0.0555, 0.1110, 0.2220 or 0.3330 g of anhydrous CaCl2. Then, the pH value of the resulting solution was adjusted to 5.0 by addition of 2 M NaOH aqueous solution. The final volume of the solution was 40 mL with the extra addition of deionized water. The resulting solution was loaded into a 60 mL autoclave, sealed and heated in a microwave oven (MDS-6, Sineo, China) to a temperature of 120, 140, 160 or 180 oC and maintained at
5
this temperature for 30 min, and then cooled down naturally to room temperature. The product was collected by centrifugation and washed with deionized water three times, and dried by freeze drying. In vitro drug loading and release experiments The typical in vitro drug loading and release experiments were performed as follows: 0.100 g of ACP porous hollow microspheres, prepared using an aqueous solution containing 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of anhydrous CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min, was added into 10 mL of ethanol solution with a docetaxel concentration of 40 mg mL-1. The dispersion was then shaken at a constant rate of 150 rpm for 24 h in a sealed vessel at 37 oC. The drug-loaded ACP porous hollow microspheres were collected by centrifugation and dried in air at 60 oC. Then, 3.5 mg docetaxel-loaded ACP porous microspheres were dispersed in 20 mL phosphate buffered saline (PBS) with a pH value of 4.5 or 7.4 at 37 oC under shaking at a constant rate of 150 rpm. The shaking device was a desk-type constant-temperature oscillator (THI-92A, China). The docetaxel release medium (1.0 mL) was withdrawn for UV-vis absorption analysis at 230 nm at given time intervals and replaced with the same volume of fresh PBS. In vitro cytotoxicity tests The human gastric carcinoma (MGC-803) cells were cultured in a RPMI-1640 medium
supplemented
with
10%
fetal
bovine
serum
(FBS)
and
1
%
penicillin-streptomycin at 37oC for 48 h. Then the cells were seeded in 96-well flat-bottom microassay plates at a concentration of 1×104 cells per milliliter and
6
cultured for 24 h. The sterilized docetaxel-loaded ACP porous hollow microspheres or ACP porous hollow microspheres without drug loading were added into the wells at concentrations ranging from 0.1-100 μg mL-1 and were co-cultured with the cells for 48
h.
The
cell
viability
was
quantified
by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data was representative as the main value of three parallel experiments. All reagents used in cell viability experiments were purchased from Sigma-Aldrich. Cell images of MGC-803 cells treated with different concentrations of ACP porous hollow microspheres or docetaxel-loaded ACP porous hollow microspheres were obtained using an Olympus IX71 optical microscope. Characterization Transmission electron microscopy (TEM) images were obtained with a transmission electron microscope (TEM, HITACHI H-800, Japan). Scanning electron microscopy (SEM) micrographs were obtained with a field-emission scanning electron microscope (SEM, Hitachi S-4800, Japan). The thermogravimetric (TG) analysis was performed with a STA 409/PC simultaneous thermal analyzer (Netzsch, Germany) at a heating rate of 10 oC min-1 in air. UV-vis absorption spectra were taken on a UV 2300 spectrophotometer (Techcomp). Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were measured with a surface area and pore size analyzer (V-Sorb 2800P, Gold APP Instruments); Fourier transform infrared (FTIR) spectra were collected on a FTIR spectrometer (FTIR-7600, Lamdba Scientific, Australia). X-ray powder diffraction (XRD) patterns were recorded using an X-ray diffractometer
7
(Rigaku D/max 2500 V, Cu Kα radiation, λ = 1.54178 Å). Hydrodynamic size distribution and surface zeta potential of the ACP porous hollow microspheres were characterized by dynamic lighting scattering (DLS) and zeta potential analyzer (Brookhaven, Zetaplus).
Results and Discussion
Figure 1a-d shows the XRD patterns of the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min. The product prepared at 120 o
C for 30 min is amorphous calcium phosphate (ACP). The products obtained at 140,
160 and 180 oC for 30 min consist of hydroxyapatite (HAP).
We investigated the effect of microwave hydrothermal temperature on the morphology of the product. The morphologies of the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min were characterized by TEM and SEM, as shown in Figure 2, from which one can see that when the microwave hydrothermal temperature is at 120 oC, the product consists of aggregated porous microspheres with sizes of several hundred nanometers. When the microwave hydrothermal temperature increases to 140, 160 or 180 oC, the product is composed of rod-assembled microspheres with rods as building blocks directing from
8
the center outwards.
Figure 3a-f shows the FTIR spectra of mPEG-PLA, Na2ATP and the samples prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min. According to the previous report [19], the broad peak between 3600 cm–1 and 3100 cm–1 is assigned to the O–H group of adsorbed water; the weak absorption bands at about 2950 and 2890 cm–1 can be attributed to the stretching vibration of –CH2 and –CH3 groups, which originate from mPEG-PLA block copolymer; the absorption peak around 1760 cm–1 may arise from the C=O group in mPEG-PLA; the absorption peak at 1637 cm–1 is related with stretching vibration of C=O, stretching of C=C, stretching of N=C and primary bending of N–H in the –NH2 group; the broad absorption peak located at 1110 cm–1 originates from the stretching of the C–O group in mPEG-PLA; the absorption peak at 1115 cm–1 results from the C–O–C stretching from mPEG-PLA. The absorption peaks at around 1100, 1027, 605 and 560 cm–1 are characteristic bands of PO43- ions in calcium phosphate in accordance with the XRD results in Figure 1. The absorption peak at 915 cm–1 comes from asymmetric P–O–C stretching and P–OH stretching in phosphorus ester of ATP biomolecules. The absorption peak at 915 cm–1 is only observable in the product prepared at 120 oC for 30 min, implying the existence of ATP molecules in the product of ACP. This may be explained by uncomplete hydrolysis of ATP biomolecules in the microwave-assisted hydrothermal conditions at 120 oC for 30 min [17].
9
We also investigated the effect of the concentrations of Na2ATP and CaCl2 on the crystal phase and morphology of the product prepared using mPEG-PLA, Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min. As shown in Figure 1a, e–g, the products prepared using 0.0500 g mPEG-PLA and different amounts of Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min are composed of amorphous calcium phosphate. As shown in Figure 2a and a’, when 0.0550 g of Na2ATP and 0.0555 g of CaCl2 are used, the product consists of ACP microspheres with a porous structure. Similarly, when 0.1100 g of Na2ATP and 0.1110 g of CaCl2 are used, ACP porous microspheres are also obtained (Figure 4a and a’). However, when the amounts of Na2ATP and CaCl2 further increase, ACP porous hollow microspheres are formed (Figure 4b, b’ and 4c, c’). The FTIR spectra of the products prepared using 0.0500 g mPEG-PLA and different amounts of Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min (Figure 3c, g–i) indicate that more ATP biomolecules are adsorbed on ACP porous microspheres with increasing the amount of ATP.
Figure 5a and b shows the size distributions measured by both SEM and dynamic lighting scattering (DLS) of ACP porous hollow microspheres prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min. The average size of ACP porous hollow microspheres measured by SEM is (387 ± 114) nm, as shown in
10
Figure 5a. In addition, the hydrodynamic size distributions of the ACP porous hollow microspheres were characterized by DLS, as shown in Figure 5b, from which one can see that the average hydrodynamic diameter of as-prepared ACP porous hollow microspheres is (947 ± 130) nm. The aggregation of ACP porous hollow microspheres is observed by DLS. Two sets of size distributions of ACP porous hollow microspheres are observed: one is located at approximately 500 nm and the other is around 1300 nm. This result is caused by the aggregation of ACP porous hollow microspheres.
The possible formation process of calcium phosphate porous microspheres prepared using mPEG-PLA, Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at different temperatures for 30 min is schematically shown in Figure 6. At the early stage of the reaction, mPEG-PLA molecules form spherical micelles with the PLA segment as the core and the PEG segment as the shell. And calcium ions adsorb on the PEG segments of micelles as a result of attraction force between the polymer molecules and calcium ions [20–23]. When the reaction solution is microwave-heated, ATP molecules are hydrolyzed and release phosphate ions, and then the reaction between calcium ions and phosphate ions occurs on the surface of mPEG-PLA micelles. The morphology of the final product depends on the microwave hydrothermal temperature and concentrations of the reactants. When the microwave hydrothermal temperature is 120 oC, the remaining ATP molecules can stabilize the amorphous calcium phosphate and prevent the transformation from ACP to
11
hydroxyapatite in aqueous solution, and ss a result, the product is composed of ACP. ACP porous microspheres are formed at lower concentrations of Na2ATP and CaCl2, however, ACP porous hollow microspheres are obtained at higher concentrations of Na2ATP and CaCl2. At higher microwave hydrothermal temperatures (140 oC, 160 oC and 180 oC), ATP molecules can be effectively hydrolyzed and the inhibition effect on the phase transformation from ACP to HAP is weakened, leading to the significant phase transformation from ACP to HAP. The rod-like morphology is common during the crystal growth of HAP. Due to the space limitation and higher concentrations of calcium ions and phosphate ions in the outer environment of the spheres, the HAP rods prefer to grow from the central core outwards, resulting in HAP rods-assembled microspheres.
This research group previously reported highly stable ACP porous spheres with relatively uniform sizes and an average pore diameter of about 10 nm by the microwave-assisted hydrothermal method using Na2ATP as the phosphorus source and stabilizer [17], and the sizes of the as-prepared ACP porous spheres were relatively uniform with the average diameter of (238 ± 35) nm. In this work, the ACP porous hollow microspheres prepared by the microwave-assisted hydrothermal method in the presence of a block copolymer mPEG-PLA at 120 oC for 30 min are comparatively larger with an average size of (387 ± 114) nm. However, the ACP porous microspheres obtained in the presence of mPEG-PLA by the present method are hollow, while the ACP porous spheres obtained in the absence of mPEG-PLA in
12
reference 17 were solid not hollow, indicating that the presence of mPEG-PLA is the key factor for the formation of the hollow structure.
Figure 7a shows the nitrogen adsorption-desorption isotherm of ACP porous hollow microspheres prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min. According to the International Union of Pure and Applied Chemistry (IUPAC), it can be classified as a type IV isotherm which is the characteristics of the porous structure [24]. The BET specific surface area of ACP porous hollow microspheres is very high (232.9 m2g-1), which is higher than many previous reports on calcium phosphate materials. The high specific area of the sample can be explained by the porous and hollow structure and self-assembly of nanoparticles as the building blocks. The average BJH pore size of ACP porous hollow microspheres is 9.9 nm. TG analysis was employed to investigate the docetaxel loading amount of the as-prepared docetaxel-loaded ACP porous hollow microspheres. Figure 7b shows that the weight loss of ACP porous hollow microspheres is 17.97%, and the weight loss of docetaxel-loaded ACP porous hollow microspheres is 47.07%. The weight percentage of docetaxel loaded in ACP porous hollow microspheres is estimated to be ~550 mg drug per gram ACP carrier.
Figure 8a and b displays the docetaxel drug release behaviors of docetaxel-loaded ACP porous hollow microspheres in PBS solutions with different pH values of pH 4.5
13
and pH 7.4 at 37 oC. In both PBS solutions, docetaxel-loaded ACP porous hollow microspheres undergo a fast release of docetaxel in the first 2 h (nearly 82% of loaded docetaxel is released) and show a little drug release in the next 70 h. There is an approximately linear relationship between the cumulative amount of released drug and the square root of release time for the ACP porous hollow microsphere drug delivery system in both PBS solutions with pH 4.5 and pH 7.4 in the first 2 h. It is well acknowledged that the kinetics of drug release from carrier materials can be well described using the Higuchi model with a linear relationship between the cumulative amount of released drug, C, and the square root of time, C = k ·t1/2, where k is a constant, and the drug release is governed by a diffusion process [25,26]. In this study, the drug release behaviors can be approximately described by the Higuchi model in both PBS solutions with pH 4.5 and pH 7.4 in the first 2 h, and the drug release is controlled by a diffusion process. In addition, the ACP porous hollow microspheres exhibit a pH-responsive drug release behavior. In the PBS solution with pH 4.5, nearly 90 % of loaded docetaxel is released in 72 h; in contrast, approximately 60 % of loaded docetaxel is released in 72 h in the PBS solution with pH 7.4. This pH-responsive drug release behavior can be explained by higher solubility of calcium phosphate in PBS with pH 4.5 than that in PBS with pH 7.4 [27].
The drug release behavior of docetaxel-loaded ACP porous hollow microspheres is very different from the result of ACP porous spheres prepared in the absence of the block copolymer in a previous study [17]. In the work of Reference [17], the drug
14
delivery system exhibited a good linear relationship between the cumulative amount of released drug and the natural logarithm of release time. The difference may be explained by the large specific surface area and porous hollow structure of ACP porous hollow microspheres in this study. The XRD patterns and FTIR spectra of docetaxel-loaded ACP porous hollow microspheres after 72 hour drug release in PBS solutions with pH 4.5 and pH 7.4 at 37 oC (Figure 1h and i; Figure 3j and k) indicate that the products are composed of a mixture of hydroxyapatite and monetite, indicating that the phase transformation occurs during the drug release process in PBS solution.
The cytotoxicity tests of the as-prepared ACP porous hollow microspheres with and without docetaxel loading were examined using human gastric carcinoma (MGC-803) cells. The MTT assays show low toxicity when the cells were co-cultured with ACP porous hollow microspheres at the concentrations in the range of 0.1 – 100 μg mL–1, as shown in Figure 9. As an important inorganic component of biological hard tissues, ACP biomaterial is believed to have low toxicity. On the other hand, the cell viability gradually decreases with increasing concentration of docetaxel-loaded ACP porous hollow microspheres. The cell viability decreases to 54 % and 20 % when the concentration of docetaxel-loaded ACP porous microspheres is 0.4 and 2 μg mL–1, respectively. The as-prepared ACP porous hollow microspheres may be used in in-situ drug delivery systems such as bone tissue engineering scaffolds/implants, which can promote bone repair by releasing active substances including peptides, growth factors,
15
hormones, drugs, and so on.
Figure 10 shows the optical images of MGC-803 cells co-cultured with different concentrations of ACP porous hollow microspheres with and without docetaxel loading. The experiments show that the cells, treated with ACP porous hollow microspheres at the concentrations in the range of 0.1－10 μg mL–1, can maintain a spindle morphology, implying that the cells still have a good physiological state. The morphology of the cells obviously changes to be spherical after being treated with the docetaxel-loaded ACP porous hollow microsphere drug delivery system even at a low concentration of 2 μg mL–1, which is significantly different from the control sample without docetaxel loading. As the concentration of the docetaxel-loaded ACP porous hollow microsphere drug delivery system increases, more cells become spherical. The spherical morphology of the cells is caused by the damage or killing of the cells, which is a result of the release of anticancer drug docetaxel from ACP porous hollow microspheres. The high cytotoxicity of the docetaxel-loaded ACP porous hollow microsphere drug delivery system is consistent with the MTT assay results.
Conclusions Amorphous calcium phosphate (ACP) microspheres with a porous and hollow structure have been successfully prepared using an aqueous solution containing CaCl2 as a calcium source, Na2ATP as a phosphorus source in the presence of a block copolymer mPEG-PLA as the template by the microwave-assisted hydrothermal
16
method. The reported preparation strategy herein is facile, rapid, energy-saving, and environment-friendly. When the reaction solution is microwave-heated, ATP molecules are hydrolyzed and release phosphate ions, and then the reaction between calcium ions and phosphate ions occurs on the surface of mPEG-PLA micelles. The microwave hydrothermal temperature and the concentrations of CaCl2 and Na2ATP have significant effects on the crystal phase and morphology of the product. When the microwave hydrothermal temperature is 120
o
C, the product is composed of
amorphous calcium phosphate; and the ACP porous solid microspheres are formed at lower concentrations of Na2ATP and CaCl2, however, ACP porous hollow microspheres are obtained at higher concentrations of Na2ATP and CaCl2. At higher microwave hydrothermal temperatures (140 oC, 160 oC and 180 oC), the as-prepared products consist of hydroxyapatite microspheres formed by self-assembly of rods. The as-prepared ACP porous hollow microspheres have a high specific surface area. The experiments show that the ACP porous hollow microspheres have a high docetaxel drug loading capacity and favorable pH-responsive drug release property, and the ACP porous hollow microsphere drug delivery system shows a high ability to damage tumor cells. It is expected that the as-prepared ACP porous hollow microspheres are promising for the applications in various biomedical fields such as drug delivery.
Acknowledgements Financial support from the National Natural Science Foundation of China (51172260, 51302294), the National Basic Research Program of China (973 Program, No.
17
2012CB933600),
and
Science
and
Technology
Commission
of
Shanghai
(12ZR1452100) is gratefully acknowledged.
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Figures:
Figure 1. XRD patterns of the products prepared by the microwave-assisted hydrothermal method for 30 min: (a)-(d) the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 at various temperatures: (a) 120 oC, (b) 140 oC, (c) 160 oC, and (d) 180 oC; (e)-(g) the products prepared using 0.0500 g of mPEG-PLA and different amounts of Na2ATP and CaCl2 at 120 oC: (e) 0.1100 g of Na2ATP and 0.1110 g of CaCl2, (f) 0.2200 g of Na2ATP and 0.2220 g of CaCl2, and (g) 0.3300 g of Na2ATP and 0.3330 g of CaCl2; (h) and (i) the products, prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC, were loaded with docetaxel and put in phosphate buffered saline (PBS) with different pH values at 37 o
C for 72 h: (h) pH 4.5, and (i) pH 7.4. (Δ stands for hydroxyapatite; ◇ stands for 21
mooneetitee).
Figurre 2. SE EM annd TE EM im magees of the pproduccts preepaaredd usin u ng 0.005000 g of o PEG G-P PLA A, 0.05 0 5500 g of Naa2AT ATP and 0.05 0 555 g of CaC Cl2 byy thhe mic m row wavve-aassiistedd mP hyydro otheerm mal meethood at a varioouss tem mpeeratturees for f 30 min n: (a, ( a’) a 1200 oC; C (b, ( bb’) 1400 oC; C (c,, c’) 1660 oC;; (d, d’) 180 oC.. 22 2
Figure 3. FTIR spectra of the samples: (a) mPEG-PLA; (b) Na2ATP; (c)-(f) the products prepared using 0.0500 g of mPEG-PLA, 0.0550 g of Na2ATP and 0.0555 g of CaCl2 by the microwave-assisted hydrothermal method at various temperatures for 30 min: (c) 120 oC, (d) 140 oC, (e) 160 oC, (f) 180 oC; (g)-(i) the products prepared using 0.0500 g of mPEG-PLA, various concentrations of Na2ATP and CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min: (g) 0.1100 g of Na2ATP and 0.1110 g of CaCl2, (h) 0.2200 g of Na2ATP and 0.2220 g of CaCl2, and (i) 0.3300 g of Na2ATP and 0.3330 g of CaCl2; (j) and (k) the products, prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min, were loaded with docetaxel and then drug was released in PBS solutions with different pH values at 37 23
o
C C: (jj) pH 44.5,, (k) pH H 7.4. 7
Figurre 4. 4 SEM S M annd TE EM imagees of o ACP A P pooroous miccro osphherees prep p pareed using 0.005000 off
mP m EG G-PL LA
a d and
d fereent diff
amo a ountts
off
Naa2AT TP
a and
CaC C Cl2
bby
thhe
owaavee-assistted hydrootheerm mal met m thod aat 1220 oC forr 300 miin: (a and a d a’) 0.1100 g of o miicro Naa2ATP A P annd 0.11 0 110 g of o CaC C Cl2; (b and d b’’) 00.22200 g of o Na N 2AT A P and a 0.222200 g of CaaCl2; (c and d c’) 0.33 0 3000 g of o Na N 2AT TP aand 0.3 3330 g off CaaCl2.
24 4
Figurre 5 (aa) siize disstrib butiion meeasuureed by b SEM S M and a (b)) size dist d tribbution meeasuuredd byy LS in w wateer oof AC ACP por p rouss holl h ow micr m rosppherres prrepaaredd usin u ng 0.005000 g of o DL mP PEG G-P PLA A, 0.33 0 3000 g of Naa2AT ATP and 0.33 0 3300 g of CaC Cl2 byy thhe mic m row wavve-aassiistedd hyydro otheerm mal meethood at a 120 oC forr 30 0 min.
25 5
6 Schhem matiic illu i ustraatioon of thee prep p paraatioon of callciuum ph hospphaate porrouus Figurre 6. u ng mP PEG G-P PLA A, Naa2ATP AT annd CaaCll2 bby thhe hoollow miicroosphherres prrepaaredd usin miicro owaavee-assistted hyd drootheerm mal met m thodd att diifferrennt teempperaaturres forr 300 miin.
26 6
Figurre 7. 7 (a) N2 aadso orpptionn–ddesoorptionn isothherrms an nd BJJH dessorp ptioon porre sizze CP por p rouss hoolloow miicroosphherees pre p eparred usiingg 0.005000 g of o disstributtionn cuurvees of AC mP PEG G-P PLA A, 0.33 0 3000 g of Naa2AT ATP and 0.33 0 3300 g of CaC Cl2 byy thhe mic m row wavve-aassiistedd hyydro otheerm mal meethood at a 120 oC forr 30 0 min; (b)) TG G cuurvves of tthe sam me sam mplle as a inn (aa) wiithoout doccetaaxeel looadiing (A A) annd witth doce d etaxxel loaadin ng (B) ( .
27 7
Figurre 8. 8 (aa) Doc D cetaaxell drrug releease prof p filess inn PB BS solu utioons wiith diff d fereent pH H vaaluees oaded AC CP pporrouss hoolloow miccroosphherees prep p pareed using 0.005000 at 37oC of doccetaaxeel-lo g off mPE m EG G-PL LA,, 0.33 0 3000 g of o Naa2A ATP P and a 0.33 0 330 g of o CaaCl2 by b thhe owaavee-assistted hy ydrootheerm mal meethood at a 11200 oC foor 30 3 min; (b) thee cum c mulaativve miicro am mou unt of relleassed dru ug verrsuss thhe squ s uaree rooot of releeasse tiimee foor the t AC CP porrouus hoollow m miccrossphheree drrug delliveery sysstem m inn both b h PB BS sollutiionss w with pH H 4.5 and a d pH H 7.44 inn thhe fiirstt 2 hou h urs at a 37 3 oC.
28 8
Figure 9. Cytotoxicity tests of the as-prepared ACP porous hollow microspheres prepared using 0.0500 g of mPEG-PLA, 0.3300 g of Na2ATP and 0.3330 g of CaCl2 by the microwave-assisted hydrothermal method at 120 oC for 30 min with and without anticancer drug docetaxel loading using human gastric carcinoma cells (MGC-803).
29
1 Thhe ooptiicall im mages of hum mann ggastrric carrcinnom ma cell c ls (MG ( GC-8003) treated Figurre 10. A P poorouus hol h low w micr m rosppheeres wiithoout docetaxeel wiith difffereent coonceentrrationss off ACP loaadin ng (thhe first f t roow imaagees) andd with w doocettaxeel load l dingg (tthe seccon nd row r w im magges)). AC CP porrouus hholloow miicroosphheres aare preepaaredd ussingg 0.05000 g of mPE m EG--PL LA, 0.333000 g of o Na N 2AT TP and a d 0.333 30 g oof CaC C Cl2 by b tthe miicroowaave--asssistted hyddro otheerm mal met m thodd at 120 0 oC foor 330 min m n.
30 0
Grap phicall abbstrracct
31 1
Highlights
Amorphous calcium phosphate porous hollow microspheres are prepared. A block copolymer templated microwave-assisted hydrothermal method is reported. The product has high biocompatibility and large specific surface area. The product has high drug loading capacity and pH-responsive drug release.
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