Yolk @ cage-Shell Hollow Mesoporous Monodispersion Nanospheres of Amorphous Calcium Phosphate for Drug Delivery with High Loading Capacity
© The Author(s). 2017
Received: 3 January 2017
Accepted: 6 April 2017
Published: 13 April 2017
In this paper, yolk-shell hollow nanospheres of amorphous calcium phosphate (ACP) are prepared, and its loading capacity is investigated by comparing with that of solid-shell hollow structure ACP and cage-shell hollow structure ACP. Results show that the products are yolk @ cage-shell of ACP with large shell’s pores size (15-40 nm) and large cavity volume. Adsorption results show that the loading capacity of yolk @ cage-shell hollow spherical ACP is very high, which is more than twice that of hollow ACP and 1.5 times of cage-like ACP. The main reasons are that the big shell’s pore size contributes the large molecular doxorubicin hydrochloride (DOX · HCl) to enter the inner of hollow spheres easier, and the yolk-shell structure provides larger interior space and more adsorption sites for loading drugs.
KeywordsAmorphous calcium phosphate Yolk @ cage-shell hollow nanospheres DOX · HCl Loading capacity
Over the past decades, many efforts have been devoted to design novel controlled drug-delivery systems, which are superior to commercial administrated drugs in terms of dosage, due to their high delivery efficiency , low side effects , and low toxicity . To date, various polymer , inorganic , and inorganic/organic hybrid materials  with diverse structures and shapes have been employed as vehicles for drug delivery. Particularly, calcium phosphate salts have gained considerable attention in the delivery of different drugs due to their excellent biocompatibility, low toxicity, excellent nonimmunogenicity and osteoconductive properties [7–10]. However, the relatively low surface area and small pore volume may limit their application. Thus, developing a kind of functional hollow calcium phosphate spheres should be highly potential, not only due to their biomedical characteristics but for their large interior space and tunable porous shell, which is suitable for loading more drugs and diffusing the drug molecules through the channels freely.
In order to enhance the loading capacity, various calcium phosphate materials with diverse morphologies and size have been prepared [11–14], such as calcium phosphate composite nanoparticles [15, 16], hydroxyapatite hollow microspheres [17–19], hydroxyapatite microtubes [20, 21], hydroxyapatite assembled hollow fibers , hydroxyapatite nanowires , and flower-like hierarchically nanostructured hydroxyapatite hollow spheres . Among the different morphological nanostructures, yolk-shell hollow spheres with porosity  are more advantageous for applications in biomedical fields such as loading drug, protein or DNA molecules, due to their different specific surface areas and morphologies [26, 27]. However, the reports about yolk-shell calcium phosphate particles are very little, and the particles reported previously could not meet the loading requires because their big yolk size compared with the shell, which results in the smaller interior space and low loading capacity .
On the other hand, the shell’s pore sizes of hollow sphere nanoparticles are very important for the delivery of drugs into cells. Large molecular/volume drugs are difficult to enter the small shell’s pores, and mostly adsorb on the surface of hollow spheres. It is a challenge to synthesize yolk-shell hollow structures of ACP with small particle sizes but simultaneously bigger pore sizes and larger interior space for the delivery of large molecular weight therapeutics.
In this paper, we will prepare a kind of yolk-shell hollow mesoporous nanospheres of calcium phosphate with bigger pore sizes and large interior space, and compare the loading capacity of yolk-shell structure with the solid-shell hollow structure and cage-like hollow structure. At the same time, the effect of yolk-shell structure’s pore sizes and cavity volume on the loading capacity will be investigated.
All chemicals used throughout the experiments were of analytical grade and without further purification. Calcium nitrate [Ca(NO3)2 · 4H2O, 99 wt%] as a source of Ca was purchased from Tianjin Hengxing Chemical Co., Ltd., China. Phosphorus pentoxide (P2O5, 98 wt%) as a source of P was purchased from Tianjin Kermel Chemical Reagent Co., Ltd., China, and an ammonia solution (NH3·H2O, 25–28 wt%) was purchased from Zhuzhou Quartzification Glass Co., Ltd., China. Anhydrous ethanol (CH3CH2OH, 99.7 wt%) was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd., China.
Synthesis of Phenol-Formaldehyde Resin Spheres (PRs)
Monodisperse phenol-formaldehyde resin spheres (PRs) were synthesized by using resorcinol and formaldehyde solution as precursors. Generally, ammonia aqueous solution (NH4OH, 25 wt%, 0.1~0.3 mL) was mixed with a solution containing absolute ethanol (EtOH, 0~28 mL) and deionized water (H2O, 0~28 mL) (with totally amount of 28 mL) to prepare PRs with different sizes. After stirring for more than 1 h, different amounts of formaldehyde solution and resorcinol were added to each of the reaction solutions and stirred at 30 °C for 24 h, and subsequently heated at 100 °C for 24 h under a static condition in a Teflon-lined autoclave. The solid product was recovered by centrifugation and air-dried at 100 °C for 48 h .
Synthesis of Amorphous Calcium Phosphate Nanospheres (ACPs)
Ca(NO3)2 · 4H2O (0.059 g), P2O5 (0.011 g), and PRs (0.100 g) were dissolved in three portions of anhydrous ethanol for 50 mL, respectively. Then, the solution containing P2O5 was added into the PRs solution. After 30 min of ultrasonic treatment, the Ca(NO3)2 solution was dropped into the mixture. Meanwhile, ammonia solution (NH3 · H2O, 0.001 mol L−1, 10 mL) was added dropwise into the mixture and reacted for 24 or 48 h. The whole process of the reaction was carried out under magnetic stirring. The precipitate was collected and washed alternately with anhydrous ethanol for 3 times by centrifugation (8000 rpm, 5 min), followed by drying at 50 °C for at least 24 h. Finally, the dried powder was calcinated up to 500 °C under air atmosphere with heating rate 2 °C/min and 10 °C/min (named ACP-24-2, ACP-48-2 and ACP-48-10, respectively, the number denotes the process parameter). In the process of calcination, the temperature was to keep heat-preservation at 100, 250, 500 °C for 1, 1, and 4 h respectively.
In the equation, Q (in mg g−1) is the amount of DOX · HCl adsorbed; C 0 and C (in mg mL−1) are the concentrations of the solution containing of DOX before and after adsorption, respectively; V (in mL) is the volume of the solution; and m (in g) is the amount of ACPs.
The phase of powders was analyzed by an X-ray diffraction (XRD, Rigaku D/max-2550) with a monochromatic Cu Kα radiation (λ = 1.5419 Å) using a voltage of 40 kV and a current of 250 mA. The data was recorded by a step size of 0.02° s−1 and a scanning range from 2θ = 10 to 80°. The Fourier transform infrared spectroscopy (FT-IR, Nicolet 6700) analysis was used to identify the chemical and structural compositions in ACP particles, which were detected by mixing with KBr powder with the scanning range from 4000 to 400 cm−1. The morphology and size of ACP were observed by using a field-emission scanning electron microscope (FESEM, Quanta FEG 250), and a JEM-2100 F transmission electron microscope (TEM) at an acceleration voltage of 200 kV. The nitrogen adsorption-desorption isotherms were obtained on a Quantachrome Autosorb automatic analyzer to determine the specific surface area and pore volume of the ACP hollow nanospheres. The amount of DOX · HCl adsorbed on the ACP hollow nanospheres was measured using UV-visible absorption spectrophotometer at 480 nm.
Results and Discussion
The surface area (SBET), pore volume, average pore size, and adsorption for samples synthesized under the different conditions
To demonstrate the potential application of yolk @ cage-shell hollow spherical ACP as delivery carrier/vehicles, doxorubicin hydrochloride (DOX · HCl), an anticancer drug, is chosen as a model drug. The DOX · HCl loading capacities of these three ACP particles with different morphologies are evaluated at pH 7.4 in deionized water. The adsorption amount is calculated by the depletion of DOX · HCl in the solution measured by UV-visible spectrophotometer at 480 nm, as shown in Table 1. The adsorption amounts of ACP-24-2, ACP-48-2, and ACP-48-10 are 350.4, 490.7, and 1181.9 mg g−1 , respectively. The loading capacity of yolk @ cage-shell hollow spherical ACP is twice more than that of ACP-24-2 and ACP-48-2, which indicates that yolk @ cage-shell hollow spherical structure contributed to the drug’s loading.
Compared with other types of core-shell nanospheres reported previously [11–13, 15], the yolk @ cage-shell hollow ACP nanospheres have smaller particle size, bigger shell’s pore size and larger cavity space, which are suitable for more DOX · HCl entering through the channels freely and adsorbing on the yolk surface and shell surface (outer and inner surface). They are the reasons why the loading capacity of the yolk @ cage-shell hollow ACP nanospheres is larger than that of others. On the other hand, the Zeta potentials of DOX · HCl and the yolk @ cage-shell hollow ACP nanospheres in deionized water at pH 7.4 are 12.1 and −19.2 mV, respectively. The strong attractive electrostatic force between ACP nanospheres and DOX · HCl enhances the loading capacity.
Yolk @ cage-shell hollow mesoporous monodispersion nanospheres of amorphous calcium phosphate with big pores (from 15 to 40 nm) and large interior space are prepared. The loading capacity of yolk @ cage-shell hollow spherical ACP is 1181.9 mg g−1 , which is more than twice that of solid-shell hollow spheres 350.4 mg g−1 and cage-like hollow spheres (490.7 mg g−1). The reasons are that the big pore sizes make large DOX · HCl moleculars to enter the hollow spheres easily, the yolk-shell structures with the biggest specific surface area and interior space provide more adsorbing sites for DOX · HCl moleculars, which greatly enhanced the loading capacity.
Amorphous calcium phosphate
Field-emission scanning electron microscope
Fourier transform infrared spectroscopy
Phenol-formaldehyde resin spheres
Transmission electron microscope
This work was financially supported by the National Natural Science Foundation of China (No. 51102285).
QX prepared the phenol-formaldehyde resin nanospheres, SH prepared the amorphous calcium phosphate spheres, and CL investigated the loading capacity. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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