Preparation of hierarchical mesoporous CaCO3 by a facile binary solvent approach as anticancer drug carrier for etoposide
© Peng et al.; licensee Springer. 2013
Received: 20 April 2013
Accepted: 1 July 2013
Published: 15 July 2013
To develop a nontoxic system for targeting therapy, a new highly ordered hierarchical mesoporous calcium carbonate nanospheres (CCNSs) as small drug carriers has been synthesized by a mild and facile binary solvent approach under the normal temperature and pressure. The hierarchical structure by multistage self-assembled strategy was confirmed by TEM and SEM, and a possible formation process was proposed. Due to the large fraction of voids inside the nanospheres which provides space for physical absorption, the CCNSs can stably encapsulate the anticancer drug etoposide with the drug loading efficiency as high as 39.7 wt.%, and etoposide-loaded CCNS (ECCNS) nanoparticles can dispersed well in the cell culture. Besides, the drug release behavior investigated at three different pH values showed that the release of etoposide from CCNSs was pH-sensitive. MTT assay showed that compared with free etoposide, ECCNSs exhibited a higher cell inhibition ratio against SGC-7901 cells and also decreased the toxicity of etoposide to HEK 293 T cells. The CLSM image showed that ECCNSs exhibited a high efficiency of intracellular delivery, especially in nuclear invasion. The apoptosis test revealed that etoposide entrapped in CCNSs could enhance the delivery efficiencies of drug to achieve an improved inhibition effect on cell growth. These results clearly implied that the CCNSs are a promising drug delivery system for etoposide in cancer therapy.
KeywordsCaCO3 nano-structure Multistage self-assembled pH-sensitive drug delivery Etoposide
Because of drug resistance, low bioavailability, and undesired severe side effects, the therapeutic effect of chemotherapy has been greatly limited for the treatment of cancer [1–5]. The application of many antitumor drugs, such as curcumin, PPT, and etoposide, has been limited because of their disadvantages of poor water solubility, metabolic inactivation, myelosuppression, side effect for normal tissue, and poor targeting .
To overcome these limitations, drug delivery techniques have been intensively investigated and studied to improve the therapeutic effect . Compared with conventional formulations, an ideal anticancer drug delivery system shows numerous advantages compared with conventional formulation, such as improved efficacy, reduced toxicity, and reduced frequency of doses . Besides, the nanocarriers for anticancer drugs can also take advantage of the enhanced permeation and retention (EPR) effect [9–11] in the vicinity of tumor tissues to facilitate the internalization of drugs in tumors. Drug carriers with diameters less than 600 nm may be taken up selectively by tumor tissues because of the higher permeation of tumor vasculature . Multiplicity carrier and functional nanoparticles exhibit greatly enhanced therapeutic effects and can improve the dispersion stability of the particles in water and endow the particles with long circulation property in vivo[8, 12–18]. However, the nanoscale drug delivery systems may also exhibit some disadvantages, such as poor biocompatibility, incompletely release in vivo, and incomplete degradation. Therefore, people are constantly developing delivery systems which are easily prepared, environment-friendly, and biocompatible.
CaCO3, the most common inorganic material of the nature, widely exists in living creatures and even in some human tissues. There are a large number of reports on calcium carbonate in recent years, but not so much attention has been focused on its biological effects. Compared with other inorganic materials, CaCO3 has shown promising potential for the development of smart carriers for anticancer drugs  because of its ideal biocompatibility, biodegradability, and pH-sensitive properties, which enable CaCO3 to be used for controlled degradability both in vitro and in vivo. It has been used as a vector to deliver genes, peptide, proteins, and drug [21–23]. Furthermore, spherical CaCO3 particle might be found in its uses in catalysis, filler, separations technology, coatings, pharmaceuticals and agrochemicals [24, 25].
Etoposide, a derivative of the anticancer drug podophyllotoxin, is an important chemotherapeutic agent for the treatment of cell lung cancer , testicular carcinoma , and lymphomas . Its direct applications had been limited by its poor water solubility, side effect for normal tissue, and poor targeting. Therefore, an efficient drug delivery system is desired to overcome these drawbacks and improve its clinical therapy efficiency.
Considering that the dimensional and structural characteristics of the materials endow them a wide range of remarkable properties and potential applications, the design and synthesis of materials with specific morphologies have drawn significant attention [29–31]. Among the different morphological nanostructures, the hierarchical particles from nanometer to micrometer dimensions reveal the great desirable properties. They have been attracting considerable attention, owing to their widespread applications in catalysis, chemical reactors, drug delivery, controlled release of various substances, protection of environmentally sensitive biological molecules, and lightweight filler materials [19, 32–37]. Highly orderly hierarchical and pH value-sensitive calcium carbonate can stably preserve drug under physiological conditions and selectively release in the intracellular acid environment . Han et al. reported the mesoporous hollow CaCO3 spheres prepared in guanidinium ionic liquid, but the surface area of those products is very low, even only 17 m2/g . It is still attractive to prepare mesoporous high-surface area carbonates with unique morphology and structure.
Etoposide (≥98%) was a kind gift from the University of Science and Technology of China. Dimethyl sulfoxide (DMSO) and MTT formazan were purchased from Sigma Chemical Co. (St Louis, MO, USA). CaCl2 (analytical reagent (AR)), Na2CO3 (AR), citric acid (AR), HCl (36%–38%), and ethanol (AR) were purchased from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China) and were used without further purification. Dulbecco's modified Eagle medium (DMEM) (high glucose), RPMI-1640, fetal calf serum (FCS), penicillin G, streptomycin, and trypsinase were obtained from GIBCOBRL (Grand Island, New York, NY, USA). Deionized water was decarbonated by boiling before its use in all of the applications.
Synthesis of etoposide-loaded calcium carbonate nanospheres
All the experiments were prepared at room temperature. Etoposide-loaded calcium carbonate nanospheres were synthesized by mixing calcium chloride and sodium carbonate aqueous solution in the presence of ethanol, citric acid, and etoposide. Etoposide (0.2 g) and 10 mL CaCl2 (0.1 M) were dissolved in 60 mL mixed solvent of ethanol and deionized water (volume ratio = 1:2), marked as solution A. Na2CO3 (0.02 g) and 10 mL of Na2CO3 (0.1 M) were dissolved in 60 mL mixed solvent of ethanol and deionized water (volume ratio = 1:2), marked as solution B. Solution B was added dropwise to the vigorously stirred solution A. With the reaction proceeding, the milky white precipitation was obtained after 72 h at room temperature. The precipitation was washed thrice with mixed solvent of ethanol and deionized water (volume ratio = 1:2) and dried using vacuum freeze drier. The blank carrier CCNSs were prepared without the addition of etoposide, and other experimental parameters were similar to the ECCNSs sample.
The morphology of the ECCNSs was viewed by field-emission scanning electron microscopy (Hitachi S4800, Chiyoda-ku, Japan) at an acceleration voltage of 1 to 5 kV and a JEOL 1230 transmission electron micrograph (TEM, Akishima-shi, Japan) at an acceleration voltage of 200 kV. Brunauer-Emmett-Teller (BET) surface area and pore distribution of the CaCO3 products were determined from N2 adsorption-desorption isotherms using a Micromeritics TriStar 3000 system (Norcross, GA, USA). The zeta potential distribution of nanoparticles was analyzed by Nano ZS, Malvern Instruments Ltd., Southborough, MA, USA. Fourier transform infrared measurement was recorded on a Bruker Vector 22 spectrophotometer (Madison, WI, USA) in the range of 4,000 to 500 cm−1 using the standard KBr disk method (sample/KBr = 1/100). UV–vis spectra were measured on a CARY50 spectrophotometer (Varian, Victoria, Australia). The crystallographic structure of the solid samples was recorded using an X-ray diffraction (XRD, Bruker D8) with Cu Kα radiation (λ = 0.154056 nm) (Karlsruhe, Germany), using a voltage of 40 kV, a current of 40 mA, and a scanning rate of 0.02°/s, in 2θ ranges from 10° to 70°. The average particle size (z-average size) and size distribution were measured by photon correlation spectroscopy (LS230 Beckman Coulter, Fullerton, CA, USA) under a fixed angle of 90° in disposable polystyrene cuvettes at 25°C.
Sedimentation study in RPMI-1640 medium
Etoposide (5 mg) was placed in a centrifugal tube of 15 mL and resuspended with 10 mL RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. The tube then stood for over 4 h after vortexing for 2 min. Photographs of the sample were taken at 1 h, 2 h, and 4 h. Other samples were studied in the same way. Based on the drug encapsulation efficiency, the same quantity of etoposide was applied to all formulations for the sedimentation study.
Determination of loading amount and in vitro release test
The etoposide release test was performed in 180 mL PBS at pH 7.4, 5.8, and 3.0. ECCNS (25 mg) was resuspended in 10 mL PBS and loaded in a dialysis bag. The release system was swayed in a bath reciprocal shaker at 100 rpm and at constant temperature of 37°C above for 120 h. Aliquots (2 mL) were extracted at desired time intervals, and another 2 mL fresh PBS was added to the system. The accumulated amount of etoposide released was determined by UV absorption at 285 nm.
Inhibition against SGC-7901 cells
Fluorescence activated cell sorter analysis
The number of the apoptosis cells was determined with the Annexin V-PI detection kit (KeyGEN Biotech). SGC-7901 cells with 1 × 106 were cultured, suspended in RPMI-1640 with 10% pasteurized FCS, and seeded on a 24-well flat-bottomed plate and incubated for 24 h at 37°C. The free etoposide, ECCNSs, and culture medium were only added to each group with the concentration of 30 μg/mL. Based on the drug encapsulation efficiency, the same quantity of etoposide was applied to all formulations for the apoptosis analysis. The incubation continued for 24 h at 37°C. Then, the cells were harvested and washed with PBS, and then PI and Annexin V were added directly to the cell suspended in the binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). The cells were incubated in the dark for 15 min at 37°C and submitted to FACS analysis on a Beckton-Dickinson (Mountain View, CA, USA) spectrophotometer.
Confocal laser scanning microscopy
CLSM images of the ECCNSs and etoposide were obtained using confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with an oil immersion objective (60×, Zeiss, Oberkochen, Germany). A suspension of the particles was placed on a glass slide and dried prior to use. Fluorescence images were obtained at an excitation wavelength of 488 nm (fluorescein isothiocyanate (FITC)) and 405 nm (4',6-diamidino-2-phenylindole (DAPI)).
Results and discussion
The morphology of sphere-shaped nanoparticles was confirmed by TEM and SEM (Additional file 1: Figure S1). As shown in Figure 2, nanoparticles synthesized via binary solvent method exhibited uniform size and good dispersal. It can be observed that the ECCNSs are large spheres with the diameter of about 2 μm. Meanwhile, some small nanocrystals with the size of about 50 to 200 nm (secondary nanoparticles) can also be observed in the PCS images (Additional file 2: Figure S2), which were possibly due to the decomposition of ECCNSs into the secondary nanoparticles when the pH decreased.
CaCO3 shows two characteristic absorption peaks centered at 875 cm−1 (bending vibration of calcite) and 745 cm−1 (bending vibration of vaterite) in its infrared absorption spectrum . In curve b of Additional file 3: Figure S3, CCNSs display three strong absorption bands at 875, 1426, and 712 cm−1, which are characteristic absorption bands of calcite. It indicated that CCNSs are the crystal form of calcite, which agrees with the results from XRD patterns. The spectra of etoposide (c) shows the following bands: 2,923 cm−1 (C-H stretch), 1,770 cm−1 (C=O stretch of ester bond), 1,613 cm−1 (C=O stretch of carboxyl methyl), and 1,056 cm−1 (C-O-C stretch), as well as the bands at 1,487 cm−1 and 1,405 cm−1, corresponding to the C=C stretching in the backbone of the aromatic phenylring. Compared with that of CCNSs (b) and etoposide (c), the spectra of ECCNSs (a) not only display the visibly characteristic bands of CaCO3 (with a small shift) but also show almost all etoposide characteristic vibration, which indicates that that etoposide was successfully packed into CCNSs.
Figure 7c, d shows the effect of etoposide formulation on the inhibition against SGC-7901 cell growth. The results showed the suppression of SGC-7901 cell growth by different nanohybrids was concentration and time dependent. The inhibition rates of ECCNSs and the free etoposide are 72.66% and 41.40% over 48 h, respectively. Obviously, ECCNSs showed higher suppression efficiency than free etoposide against the growth of SGC-7901 cells. Synergistic therapeutic effects occurred when etoposide was entrapped by CCNSs. It is possible that good dispersivity and stability of ECCNSs in culture medium (Figure 5) may lead to a greater cellular uptake than that of free etoposide.
Then, the pH values of culture media for SGC-7901 cells were measured as 8.1 (0 h), 7.82 (24 h), and 6.76 (48 h). Therefore, it can be inferred that the release of etoposide from ECCNSs may increase as the pH value of the culture decreases because of its pH-sensitive controlled release behavior investigated above. The stronger cell inhibition of ECCNSs further confirms that the cell uptake of nanoparticles, the decomposition of ECCNSs as the pH descends, and the passive diffusion of the free etoposide released from the ECCNSs, together helped to achieve the cell inhibition effect.
Controlled delivery of drug using carrier materials is based on two strategies: active and passive targeting. The former is technical sophisticated and suffering from many difficulties. Otherwise, the latter is easier to implement practically . Many formulations have been used in the representative passive-targeting strategies based on the EPR effect . Tumor vessels are often dilated and fenestrated due to rapid formation of vessels that can serve the fast-growing tumor while normal tissues contain capillaries with tight junctions that are less permeable to nanosized particle [11, 48]. The EPR effect is that macromolecules can accumulate in the tumor at concentrations five to ten times higher than in normal tissue within 1 to 2 days . Besides, biomaterials with diameters more than 100 nm tend to migrate toward the cancer vessel walls . Therefore, the EPR effect enables ECCNSs (secondary nanoparticles) to permeate the tumor vasculature through the leaky endothelial tissue and then accumulate in solid tumors. On one hand, the uptake of ECCNSs by tumor cells can lead to the direct release of etoposide into intracellular environment to kill tumor cells. On the other hand, the pH-sensitive drug release behavior for ECCNSs may lead to the low release of etoposide from ECCNSs in pH neutral blood, and the rapid release of the drug in relatively acidic extracellular fluids in the tumor. In this way, the targeted delivery of etoposide to tumor tissues may be possible by ECCNSs.
In summary, we have proposed a facile method to prepare mesoporous CCNSs formed by the multistage self-assembled strategy in a binary solvent reaction system. Further studies on CCNSs as carriers for etoposide (loading capacity 39.7%) demonstrated their pH-sensitive drug release profile and enhanced cytotoxicity by increasing cellular uptake and apoptosis to tumor cell. The cytotoxicity test and apoptosis test showed that the carrier of CCNSs was almost nontoxic and ECCNSs were evidently more efficient than free etoposide in antitumor effect and deliver activity. These results also indicated that the hierarchical mesoporous CaCO3 nanospheres (CCNSs) hold great promise to overcome the drawbacks of water-insoluble drugs such as etoposide and thereby enhance their therapeutic effect.
DS and RZ are assistant professors. SW is a professor, and HP, KL, TW, JW, and JW are graduate students from the School of Life Science and Technology, Tongji University.
Calcium carbonate nanospheres
Confocal laser scanning microscopy
- EPR effect:
Enhanced permeation and retention effect
Fluorescence-activated cell sorter
Scanning electron microscope
Transmission electron micrograph.
This work was financially supported by the 973 project of the Ministry of Science and Technology (grant no. 2010CB912604, 2010CB933901), International S&T Cooperation Program of China, (grant no. 0102011DFA32980), Science and Technology Commission of Shanghai Municipality (grant no. 11411951500, 12 nm0502200) and the Fundamental Research Funds for the Central Universities.
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