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Calcium phosphate drug nanocarriers with ultrahigh and adjustable drug-loading capacity: One-step synthesis, in situ drug loading and prolonged drug release
BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 428 – 434
Research Article
nanomedjournal.com
Calcium phosphate drug nanocarriers with ultrahigh and adjustable drug-loading capacity: One-step synthesis, in situ drug loading and prolonged drug release Qi-Li Tang, PhDa,b , Ying-Jie Zhu, PhDa,b,⁎, Jin Wu, PhDa,b , Feng Chen, MSa , Shao-Wen Cao, PhDa,b a
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, P. R. China b Graduate School of Chinese Academy of Sciences, Shanghai, P. R. China Received 22 September 2010; accepted 17 December 2010
Abstract Calcium phosphates (CPs) are regarded as the most biocompatible inorganic biomaterials; however, they are limited in the drug-delivery applications, especially for hydrophobic drugs. Achieving high drug-loading capacity and a controllable drug-release property are two main challenges. In this study we report a strategy for the preparation of novel drug delivery systems based on a concerted process in which the formation of the CP nanocarriers and the drug storage are accomplished in one step in mixed solvents of water and ethanol. The key advantage of this strategy is that the formation of CP nanocarriers and in situ loading of the drug occur simultaneously in the same reaction system, which makes it possible to achieve ultrahigh drug-loading capacity and prolonged drug release due to ultrahigh specific surface area and numerous binding sites of the CP nanocarriers. A series of hydrophobic drug-delivery systems with adjustable drug-loading capacities and drug-release rates have been successfully synthesized. In addition, the drug-release kinetics of the as-prepared drug-delivery systems have been found in which the cumulative amount of drug release has a linear relationship with the natural logarithm of release time. From the Clinical Editor: Calcium phosphates (CPs) are highly biocompatible inorganic biomaterials with thus far limited drug-delivery applications. This study reports the preparation of a novel drug delivery system where the formation of CP nanocarriers and in situ loading of the drug occur simultaneously in the same reaction, enabling ultra-high drug-loading. © 2011 Elsevier Inc. All rights reserved. Key words: Calcium phosphate; Nanocarrier; Drug delivery; Hydrophobic drug; Biomedical application
Calcium phosphates (CPs) are present throughout the body in the form of amorphous calcium phosphate (ACP) as well as hydroxyapatite (HAp); thus, they are considered the most biocompatible inorganic biomaterials and suitable candidates for drug carriers.1-4 CP can precipitate from a supersaturated solution containing ions of calcium and orthophosphate. Among the experimental parameters, an important factor that influences the CP phase and morphology is the pH value of solution.5,6 For instance, ACP forms in the pH range from 5 to 12, and octacalcium phosphate (OCP) is stable in the pH range from 5.5 Conflicts of Interest: The authors declare no conflicts of interests. Grant support: This work was funded by the Science and Technology Commission of Shanghai (1052nm06200), the National Natural Science Foundation of China (50772124, 50821004), and State Key Laboratory of High Performance Ceramics and Superfine Microstructure. ⁎Corresponding author: Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, P. R. China. E-mail address: [email protected] (Y.-J. Zhu).
to 7.0. The structure of ACP is still uncertain; however, the posner's cluster model is widely accepted. The basic structural unit of this model is the Ca9(PO4)6 cluster with a diameter of 9.5 Å. The clusters are presumed to pack randomly with respect to each other, forming the nanospheres with sizes of 30 to 80 nm, and contain 10-20% by weight of tightly bound water molecules in the interstices between clusters.7,8 We propose that the specific surface area of the ACP clusters is extremely large when they form shortly after the reactants are mixed in solution. Similarly, OCP displays the HAp layers separated by hydrated layers, and OCP with large specific surface area exists at the very early stage of the precipitation reaction. If the drug is present in situ during the initial stage of the formation of CP nanomaterials, it is possible for the drug to be loaded in situ into the CP nanocarriers with ultrahigh specific surface area, leading to a ultrahigh drug loading capacity (DLC) and prolonged drug release. Moreover, in comparison with the traditional CP drugdelivery systems (DDSs), these DDSs prepared by the in-situ
1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2010.12.005 Please cite this article as: Q.-L. Tang, Y.-J. Zhu, J. Wu, F. Chen, S.-W. Cao, Calcium phosphate drug nanocarriers with ultrahigh and adjustable drugloading capacity: One-step synthesis, in situ drug loading and prolonged drug release. Nanomedicine: NBM 2011;7:428-434, doi:10.1016/j.nano.2010.12.005
Q.-L. Tang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 428–434
drug-loading process may exhibit different drug-loading and release properties. Many drugs with great medical and commercial significance are hydrophobic, such as ibuprofen (2-[4-(2-methylpropyl) phenyl]propanoic acid, IBU), AC ((3R,5R)-7-[2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-(pheynylcarbamoyl)pyrrol-1-yl]3,5 -dihydroheptanoic acid, calcium salt (2:1) trihydrate, AC), indomethacin (2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2methyl-1H-indol-3-yl}acetic acid). It was reported that the drug carriers are important for loading the drugs with high DLC and for sustained drug release over a prolonged period to enhance therapeutic efficiency.9,10 Zhu et al.11 synthesized HAp porous ellipsoidal hollow capsules constructed with the self-assembly of nanosheets by using CaCO3 hard template; the as-prepared HAp porous ellipsoidal hollow capsules had a specific surface area as high as 220 m2g-1. They were explored as the drug nanocarriers for IBU, and a high DLC of 459.5 mg g-1 was achieved. Although some progress has been made, some challenges regarding the use of CP materials as drug carriers for clinical applications still exist, such as low drug-loading capacity, uncontrollable-burst drug release, and a toxic environment sometimes adopted for the CP preparation and drug-loading process. In this article, we demonstrate a single-step synchronous method for both the synthesis of CP nanocarriers and the loading of the hydrophobic drug into CP nanocarriers in mixed solvents of water and ethanol, the formation of CP nanocarriers and the loading of the drug occur simultaneously in the same reaction system. In this method, large numbers of CP clusters with an ultrahigh specific surface area form at the early stage of the precipitation reaction, and CP clusters provide many binding sites for the drug molecules. The in situ presence of the drug molecules during the formation of CP nanocarriers makes it possible for the drug molecules to adsorb on the surface of the CP clusters. As a result, a superhigh DLC and prolonged drug-release characteristics can be achieved. Furthermore, The DLC can be adjusted by changing the pH value of solution and the concentration of the drug. This method does not need any surfactant or two-step procedures of synthesis of CP nanocarriers and subsequent drug loading; thus, it avoids the post-treatment processes that may inactivate the binding sites for the drug molecules. Additionally, it avoids the use of toxic organic solvents as the media for hydrophobic drug loading. This method has advantages such as ease of handling, low cost and environmental friendliness, which are promising for the application in drug delivery.
Methods Materials CaCl2·2H2O (AR), Na2HPO4·12H2O (AR), NaOH (AR), HCl (36–38%) and ethanol (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ibuprofen (99.99%) was purchased from Shanghai (China) Yuanji Chemical Co., Ltd. (Quzhou, China). Atorvastatin calcium (99.5%) was purchased from Quzhou Aifeimu Chemical Co., Ltd. Simulated body fluid (SBF, pH 7.4 at 37°C ) was prepared according to the literature.12 All the chemicals were used as received without further purification.
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Table 1 The main experimental parameters for the preparation of CP-IBU and CP-AC drug delivery systems and estimated drug-loading capacities (DLCs) Samples
IBU [mol L-1]
OH- [mol L-1]
pH value
DLC [g g-1]
CP-IBU1 CP-IBU2 CP-IBU3 CP-IBU4 CP-IBU5 CP-IBU6 CP-IBU7 CP-IBU8 CP-IBU9 CP-IBU10 CP-IBU11 CP-IBU12 CP-IBU13 CP-IBU14 CP-IBU15 CP-IBU16
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.3 0.4 0.5 0.6 0.3 0.3 0.3
0 0.01 0.03 0.05 0.07 0.08 0.09 0.10 0.06 0.09 0.12 0.15 0.18 0.03 0.06 0.12
3.6 4.6 5.6 6.1 6.6 6.8 7.0 7.4 5.8 5.8 5.8 5.7 5.7 4.9 5.5 6.0
0.35 0.63 0.68 0.46 0.47 0.34 0.24 0.14 0.96 1.34 1.62 1.68 1.96 1.37 1.54 1.40
Samples
AC [mol L-1]
OH- [mol L-1]
pH value
DLC [g g-1]
CP-AC17 CP-AC18 CP-AC19 CP-AC20 CP-AC21 CP-AC22 CP-AC23
0.0087 0.0087 0.0087 0.0087 0.0087 0.0087 0.0087
0.008⁎ 0.0056⁎ 0 0.004 0.008 0.011 0.014
5.1 5.4 5.8 6.1 6.4 6.7 7.2
0.33 0.36 0.48 0.62 0.76 0.92 0.93
The concentrations are normalized to those of the final solutions. ⁎ Represents the concentration of HCl in the reaction system.
Preparation of the drug-loaded calcium phosphate nanocarriers All the experiments were conducted at room temperature (about 15−25°C). In a typical experiment for the preparation of IBUloaded DDS sample (CP-IBU3), 2.063 g IBU and 0.368 g CaCl2·2H2O were dissolved in 65 mL ethanol. Then, 3 mL 1 M NaOH aqueous solution and 7 mL deionized water were injected into the ethanol solution. In addition, 25 mL of phosphate aqueous solution containing 0.537 g Na2HPO4·12H2O was poured into the above reaction system under magnetic stirring and reacted for 1 minute. The product was centrifuged, washed with deionized water and dried at 60°C in vacuum. The volume ratio of ethanol to water was 13:7. Other samples were prepared at different IBU concentrations by changing the amount of IBU or at different pH values of solution by changing the volume of NaOH solution (Table 1). The AC concentration was 10 g L-1 in the preparation of AC-loaded DDS samples, and other experimental parameters were similar to the IBU-loaded DDS samples. In vitro drug release The drug-loaded CP DDS samples were compacted into disks (0.20 g each, diameter = 10 mm) at a pressure of 3 MPa. Then the disk was immersed into 200 mL SBF at 37 °C under shaking at a constant rate of 150 rpm. The IBU release medium (2.0 mL) was extracted for UV–vis analysis at the wavelength of 264 nm at given time intervals and replaced with the same volume of fresh SBF, which was preheated to 37°C.
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Figure 1. (A) TG curves and (B) the DLCs versus pH values for the CP-IBU DDS samples prepared at different pH values at a concentration of [IBU] = 0.1 M; (C) TG curves and (D) the DLCs versus IBU concentrations for the CP-IBU DDS samples; (E) TG curves and (F) the DLCs versus pH values for the CP-AC DDS samples prepared at different pH values at a concentration of [AC] = 0.0087 M. All the samples were prepared in mixed solvents of ethanol and water with a volume ratio of 13:7 at a concentration of [Ca2+] = 0.025 M.
Characterization Thermogravimetry (TG) curves were measured with a simultaneous thermal analyzer (STA 409PC, Netzsch, Germany) at a heating rate of 10°C min-1 in flowing air. UV–vis absorption spectra were performed on a spectrophotometer (UV2300, Techcomp, Shanghai, China). X-ray powder diffraction (XRD) patterns were recorded using an X-ray diffractometer (Rigaku D/max 2550V, Tokyo, Japan) with high-intensity Cu Ka
radiation (λ = 1.54178Å). Fourier transform infrared (FTIR) spectra were obtained on a FTIR spectrometer (Nicolet 380).
Results TG and XRD characterization of CP-IBU DDSs IBU and atorvastatin calcium (AC) were chosen as the model hydrophobic drugs in this investigation. It should be
Q.-L. Tang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 428–434
mentioned that both drugs could be dissolved in ethanol and they did not precipitate in the absence of CP in the mixed solvents of ethanol and water with a volume ratio of 13:7. Figures 1, A, 1, C and 1, E, show the TG curves measured in flowing air of CP-IBU and CP-AC DDSs prepared under different conditions. The overall weight loss can be attributed to the loss of adsorbed water and lattice water and the clean burn of drugs. The similar result was also observed for the CP-AC DDSs. On the basis of the TG analysis, we estimated the DLCs of the as-prepared CP nanocarriers, and the results are shown in Figures 1, B, 1, D and 1, F and also in Table 1. Table 1 also shows the main experimental parameters for the preparation of CP-IBU and CP-AC DDSs. By increasing the IBU concentration from 0.1 to 0.6 M (CPIBU3, CP-IBU9 to CP-IBU13), the DLC significantly increased, as shown in Figure 1, D. The DLC of the CP-IBU13 DDS prepared at an IBU concentration of 0.6 M reached a value as high as 1.96 g IBU per gram carrier. The increased IBU concentration can influence the dynamic equilibrium of the drug adsorption, leading to enhanced DLC. It should be noted that a series of DDSs with adjustable DLCs from 0.14∼1.96 g IBU per gram carrier can be prepared by changing the pH value or IBU concentration. The chemical compositions of the as-prepared drug nanocarriers correlate strongly with the pH value of solution. Figure 2, A shows XRD patterns of the DDS samples prepared at different pH values of solution with an IBU concentration of 0.1 M. In addition, CP-IBU1 prepared at pH = 3.6 was identified as singlephase dicalcium phosphate dehydrate (DCPD) (JCPDS No. 110293), and CP-IBU3 synthesized at pH = 5.6 consisted of a mixture of DCPD, OCP and HAp. By increasing the pH value to 6.6 (CP-IBU5), the product was a mixture of OCP and HAp. When the pH value was further increased to 7.4 (CP-IBU8), essentially only HAp was obtained. These results indicate that different drug nanocarriers can be obtained by adjusting the pH value of the solution. TG and XRD characterization of CP-AC DDSs We also investigated the loading of a hydrophobic drug, AC, in the CP nanocarriers. The CP-AC DDSs were prepared at different pH values at a concentration of [AC] = 0.0087 M in mixed solvents of ethanol and water with a volume ratio of 13:7. It has been found that the DLC of CP-AC DDS can also be controlled by adjusting the pH value of the solution, as shown in Figures 1, E and 1, F and Table 1. The DLC increased with increasing pH value, and it varied from 0.33 to 0.93 g AC per gram carrier in the pH range of 5.1−7.2. This variation trend in DLC of AC is totally different from that of the IBU loading, indicating the pH effect on different drugs. Figure 2, B shows XRD patterns of the samples prepared at different pH values of solution at concentrations of [Ca2+] = 0.025 M and [AC] = 0.0087 M in mixed solvents of ethanol and water with a volume ratio of 13:7. The CP-AC17 sample prepared at pH = 5.1 was identified as single-phase DCPD, and the CP-AC20 sample prepared at pH = 6.1 consisted of a mixture of DCPD, OCP and HAp. When the pH value was further increased to 7.2 (CPAC23), essentially only HAp was obtained.
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Figure 2. (A) XRD patterns of the CP-IBU DDS samples prepared at different pH values of solution; (B) XRD patterns of the CP-AC DDS samples prepared at different pH values of solution. All the samples were prepared in mixed solvents of ethanol and water with a volume ratio of 13:7, and then washed by ethanol several times to remove the drug for the purpose of avoiding the peaks of the IBU or AC.
In vitro drug release behaviors of CP-IBU DDSs The drug-release profiles of the CP-IBU5 and CP-IBU13 drugdelivery systems in SBF are shown in Figure 3. Both DDSs exhibited a prolonged drug release property, but the drug-release rates were different for the two DDSs. For the CP-IBU5 DDS prepared at a low IBU concentration and pH value of 6.6, 32% of the loaded drug was released in the first 24 hours, and 64% was released in a period of 2 weeks. However, for the CP-IBU13 DDS prepared at a high IBU concentration, in which 1.96 g IBU was loaded per gram carrier, the drug-release percentage was much lower than that of the CP-IBU5. Only 12% of the loaded drug in the CP-IBU13 DDS was released in a period of 24 hours, and 19% was released in 2 weeks; a much prolonged drug release can be achieved in this DDS. Discussion To understand the high DLC of the CP-IBU DDSs, the Brunauer-Emmett-Teller (BET) specific surface area (SBET) measurements were performed. The CP-IBU5 and CP-IBU13 DDS samples were washed by ethanol several times to remove the IBU before the specific surface area measurement. The SBET
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Figure 4. Illustration of the single-step synchronous method for the preparation of CP nanocarriers and the in situ drug loading.
Figure 3. IBU release profiles: (A) CP-IBU5 and (B) CP-IBU13. The insets show the plots for cumulative IBU release percentage versus natural logarithm of release time.
of the CP-IBU5 and CP-IBU13 DDSs was 60.48 and 98.03 m2 g-1, respectively. The ultrahigh DLC of 1.96 g IBU per gram carrier was achieved for the CP-IBU13 DDS, which is even much higher than that (1.40 g g-1) of nonbiocompatible mesoporous material MIL-101, a chromium terephthalate with an ultrahigh SBET of 5510 m2 g-1.13 This extremely high DLC of CP-IBU13 DDS obviously cannot be accounted for only by SBET. ACP was reported to form at pH values of 5∼12, and it was the initial phase of HAp in a neutral or basic environment. Similarly, it could transform to OCP and DCPD in appropriate conditions.5,6 The formation of Ca9(PO4)6 cluster of ACP at the early stage of the precipitation reaction was reported, and the cluster was estimated to be roughly spherical with a diameter of 9.5 Å.7,8 Wang et al14 studied the formation and transformation of ACP particles by stopped-flow spectrophotometry, simultaneous measurements of particle size and pH of solution, and high-resolution transmission electron microscopy, indicating the formation of ion pairs and clusters in the first few seconds, and crystalline domains developed at multiple sites inside the primary particles of the amorphous phase. Suppose that the diameter of ACP clusters is as small as 9.5 Å, ACP clusters have the lattice similar to HAp and the density of HAp is ρ = 3.16 g cm-3. The specific surface area of this material is roughly calculated as high as 2100 m2g-1. We propose that large numbers of CP clusters with an ultrahigh specific surface area form before the bulk precipitation occurs in solution, and CP clusters provide many binding sites for the drug molecules. Ca2+ ions may form
the complex with carboxyl groups of IBU or atorvastatin calcium. In our experiments the formation of the CP nanocarriers and the loading of the drug were conducted simultaneously in the same reaction system. The in situ presence of drug molecules during the formation of CP nanocarriers may lead to the interaction between organic groups of the drug molecules and Ca2+ cations or PO34 anions on the surface of the CP clusters. Thus, the strong interaction between the drug molecules and CP nanocarriers occurred, and more drug molecules were encapsulated in the interior of the CP nanocarriers in addition to the drug molecules adsorbed on the surface of the CP nanocarriers, as shown in Figure 4, which illustrates the proposed strategy for the one-step simultaneous formation of CP nanocarriers and in situ drug loading in the same reaction system. It is different from the conventional DDSs in which the drug molecules merely adsorb on the surface of the carrier. As a result, a superhigh DLC can be achieved. The pH value of solution not only influences the composition, nucleation and crystallization process of the product, but also changes the ionization state of the IBU and AC drug molecules; thus, it has a significant effect on the DLC. The AC molecule is bigger than the IBU molecule, and it has more functional groups that can interact with the CP nanocarriers. A reversed trend in DLC of AC in the CP-AC DDSs compared with the CP-IBU DDSs in a given pH range is found, indicating the pH effects in different ways. We demonstrate a single-step synchronous method for the preparation of CP nanocarriers and the in situ drug loading conducted in mixed solvents of water and ethanol. In comparison with pure IBU without the nanocarriers, which burns at about 150−280°C in air, the CP-IBU DDS shows a much higher burning temperature for the IBU ingredient in the TG measurement, implying that the thermal stability of IBU can be enhanced to some extent when loaded in the CP nanocarriers
Q.-L. Tang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 428–434
Figure 5. FTIR spectra: (A) pure IBU; (B) CP-IBU5; (C) CP-IBU5 washed with ethanol several times to remove IBU; (D) CP-IBU13; (E) CP-IBU13 washed with ethanol several times to remove IBU.
due to the interaction between the loaded drug molecules and CP nanocarriers. The interaction between the drug molecules and CP nanocarriers was also investigated by FTIR spectra. Figure 5 shows the FTIR spectra of typical CP-IBU DDSs (CP-IBU5 and CP-IBU13) and corresponding control samples. The samples were washed with ethanol several times to remove the IBU before the FTIR measurements for comparison. In the FTIR spectrum of pure IBU (Figure 4, A), the band at 1724 cm-1 is attributed to the C = O group of IBU molecules, and the peaks at around 2900 cm-1 originate from the –CH3 and –CH2 groups. The FTIR spectra of CP-IBU5 and CP-IBU13 exhibit the presence of the characteristic peaks of both IBU and CP nanocarrier, indicating the combination of IBU and CP nanocarriers in the CP-IBU drug-delivery system. In comparison with the FTIR spectra of pure IBU and the ethanol washed samples, red shift of the C = O band from 1724 to 1550 cm-1 was observed for the CP-IBU5 DDS. Meanwhile, the PO34 vibration peak was split into two bands at 1119 and 1026 cm-1. These changes may originate from the interaction between the loaded IBU molecules and the CP nanocarriers. However, the FTIR spectrum of CP-IBU13 is more complicated. The C=O peaks were observed at 1720 and 1554 cm-1, implying the existence of both strongly bonded and weakly bonded IBU molecules. Moreover, the ethanol washed CP-IBU13 sample exhibited characteristic peaks of both IBU molecules and CP nanocarriers, indicating that some strongly bonded IBU molecules were still left after washing with ethanol due to a relatively strong interaction between the IBU molecules and the CP nanocarriers. We estimated by TG analysis that 0.12 g IBU per gram carrier remained for the ethanol washed CP-IBU13 sample. For the medicinal applications, it is desirable that the drug release is reasonably slow and prolonged to achieve the highly efficient treatment. The ideal DDS should have a high loading capacity of the drug, and it should release the drug with a reasonable rate to reach the concentration required to maintain efficacy within a relatively short time, and then, the drug release rate should maintain at an appropriate level for a prolonged
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period. Our previous studies showed that the release of the pure drug without the nanocarrier in SBF was very rapid, and the drug release completed within several hours. In contrast, the drug release could be well controlled with a reasonable rate and a prolonged release time with the help of the nanocarriers.11,15 In this study, the drug-release time of the CP-IBU5 and CP-IBU13 DDSs significantly increased in SBF, but the drug-release rates were different for different DDSs. For the CP-IBU5 DDS prepared at a low IBU concentration, 32% of the loaded drug was released in the first 24 hours, and 64% in a period of 2 weeks. However, for the CP-IBU13 DDS with a much higher DLC, only 12% of the loaded drug was released in a period of 24 hours and 19% in 2 weeks. The sustained and prolonged drug-release profile can be achieved in the as-prepared DDSs. The drug-release kinetics of the CP-IBU5 and CP-IBU13 DDSs in SBF were investigated. It is well acknowledged that the kinetics of drug release from the carrier materials can be well described using the Higuchi model (C = k·t1/2),16-18 in which there exists a linear relationship between the cumulative amount of released drug and the square root of release time; the drug release is governed by a diffusion process. In this study we have found different drug-release kinetics for the CP-IBU5 and CPIBU13 DDSs in SBF. Our experiments showed that the drugrelease kinetics of the CP-IBU5 and CP-IBU13 DDSs did not follow the Higuchi model. Instead, the cumulative amount of released drug had a linear relationship with the natural logarithm of release time, ln(t), in the time period from 2 hours to 14 days for the CP-IBU5 and CP-IBU13 DDSs (insets of Figure 3, A and B). The drug release kinetics of this type of DDS may be governed by synchronous mechanisms including desorption, diffusion and phase transformation of CP nanocarriers. This unique drug-release kinetics has also been found in the DDS of hierachically nanostructured mesoporous spheres of calcium silicate hydrate.9 For the CP-IBU5 DDS prepared at a low IBU concentration and pH value of 6.6, 32% of the loaded drug was released in the first 24 hours and 64% was released in a period of 2 weeks. However, for the CP-IBU13 DDS prepared at a high IBU concentration, in which 1.96 g IBU was loaded per gram carrier, the drug-release percentage was much lower than that of the CPIBU5. Only 12% of the loaded drug in the CP-IBU13 DDS was released in a period of 24 hours and 19% was released in 2 weeks; a prolonged drug-release can be achieved in this DDS. The FTIR spectra suggest that a quantity of IBU molecules that are strongly bonded with the CP nanocarriers may exist, especially for the CP-IBU13 DDS, which can explain the prolonged drug-release behavior. As for the clinical application, these DDSs representing different drug-release behaviors may be used in various situations. HAp is the most stable phase in SBF at room temperature (about 15−25°C). Thermodynamically metastable phases of CP undergo the processes of dissolution, re-nucleation and crystal growth, and finally transform to HAp. The XRD patterns indicated that the transformation to HAp occurred after the drugrelease process in SBF, and the CP component of the as-prepared DDSs were completely transformed to HAp after the drug release in SBF for 2 weeks. Similar experimental results were also reported.19,20
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In summary, a single-step synchronous method has been developed in which the formation of CP nanocarriers and the in situ loading of the drug occur simultaneously in the same reaction system. It does not need any surfactant or the two-step procedures of synthesis of CP nanocarriers and subsequent drug loading, thus, avoids the complex post-treatment processes. Also, it avoids the use of toxic organic solvents as the media for hydrophobic drug loading. This method has advantages, such as ease of handling, low cost and environmental friendliness, which are promising for the application in drug delivery. The superhigh DLC and prolonged drug-release characteristics have been achieved in the as-prepared DDSs. Furthermore, DLC can be adjusted by changing the pH value of solution and the concentration of the drug. In addition, the drug-release kinetics has been found in which the cumulative amount of drug release has a linear relationship with the natural logarithm of release time. The detailed mechanism of this novel drug-release kinetics needs to be further investigated.
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7. Posner AS, Betts F. Synthetic amorphous calcium phosphate and its relation to bone mineral structure. Acc Chem Res 1975;8:273-81. 8. Tropp J, Blumenthal NC, Waugh JS, Phosphorus NMR. study of solid amorphous calcium phosphate. J Am Chem Soc 1983;105:22-6. 9. Wu J, Zhu YJ, Cao SW, Chen F. Hierachically nanostructured mesoporous spheres of calcium silicate hydrate: Surfactant-free sonochemical synthesis and drug-delivery system with ultrahigh drugloading capacity. Adv Mater 2010;22:749-53. 10. Rosenholm JM, Sahlgren C, Lindén M. Towards multifunctional, targeted drug delivery systems using mesoporous silica nanoparticles Opportunities & challenges. Nanoscale 2010;2:1870-83. 11. Ma MY, Zhu YJ, Li L, Cao SW. Nanostructured porous hollow ellipsoidal capsules of hydroxyapatite and calcium silicate: preparation and application in drug delivery. J Mater Chem 2008;18:2722-7. 12. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006;27:2907-15. 13. Vallet-Regí M, Balas F, Arcos D. Mesoporous materials for drug delivery. Angew Chem Int Ed 2007;46:7548-58. 14. Wang CG, Liao JW, Gou BD, Huang J, Tang RK, Tao JH, et al. Crystallization at multiple sites inside particles of amorphous calcium phosphate. Cryst Growth Des 2009;9:2620-6. 15. Cao SW, Zhu YJ. Surfactant-free preparation and drug release property of magnetic hollow core/shell hierarchical nanostructures. J Phys Chem C 2008;112:12149-56. 16. Higuchi T. Rate of release of medicaments from ointment bases containing drugs in suspension. J Pharm Sci 1961;50:874-5. 17. Higuchi T. Mechanism of sustained-action medication: Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J Pharm Sci 1963;52:1145-9. 18. Andersson J, Rosenholm J, Areva S, Lindén M. Influences of material characteristics on ibuprofen drug loading and release profiles from ordered micro- and mesoporous silica matrices. Chem Mater 2004;16: 4160-7. 19. Tang QL, Zhu YJ, Duan YR, Wang Q, Wang KW, Cao SW, et al. Porous nanocomposites of PEG-PLA/calcium phosphate: room-temperature synthesis and its application in drug delivery. Dalton Trans 2010;39: 4435-9. 20. Horváthová R, Müller L, Helebrant A, Greil P, Müller FA. In vitro transformation of OCP into carbonated HA under physiological conditions. Mater Sci Eng C 2008;28:1414-9.
Research Article
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Calcium phosphate drug nanocarriers with ultrahigh and adjustable drug-loading capacity: One-step synthesis, in situ drug loading and prolonged drug release Qi-Li Tang, PhDa,b , Ying-Jie Zhu, PhDa,b,⁎, Jin Wu, PhDa,b , Feng Chen, MSa , Shao-Wen Cao, PhDa,b a
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, P. R. China b Graduate School of Chinese Academy of Sciences, Shanghai, P. R. China Received 22 September 2010; accepted 17 December 2010
Abstract Calcium phosphates (CPs) are regarded as the most biocompatible inorganic biomaterials; however, they are limited in the drug-delivery applications, especially for hydrophobic drugs. Achieving high drug-loading capacity and a controllable drug-release property are two main challenges. In this study we report a strategy for the preparation of novel drug delivery systems based on a concerted process in which the formation of the CP nanocarriers and the drug storage are accomplished in one step in mixed solvents of water and ethanol. The key advantage of this strategy is that the formation of CP nanocarriers and in situ loading of the drug occur simultaneously in the same reaction system, which makes it possible to achieve ultrahigh drug-loading capacity and prolonged drug release due to ultrahigh specific surface area and numerous binding sites of the CP nanocarriers. A series of hydrophobic drug-delivery systems with adjustable drug-loading capacities and drug-release rates have been successfully synthesized. In addition, the drug-release kinetics of the as-prepared drug-delivery systems have been found in which the cumulative amount of drug release has a linear relationship with the natural logarithm of release time. From the Clinical Editor: Calcium phosphates (CPs) are highly biocompatible inorganic biomaterials with thus far limited drug-delivery applications. This study reports the preparation of a novel drug delivery system where the formation of CP nanocarriers and in situ loading of the drug occur simultaneously in the same reaction, enabling ultra-high drug-loading. © 2011 Elsevier Inc. All rights reserved. Key words: Calcium phosphate; Nanocarrier; Drug delivery; Hydrophobic drug; Biomedical application
Calcium phosphates (CPs) are present throughout the body in the form of amorphous calcium phosphate (ACP) as well as hydroxyapatite (HAp); thus, they are considered the most biocompatible inorganic biomaterials and suitable candidates for drug carriers.1-4 CP can precipitate from a supersaturated solution containing ions of calcium and orthophosphate. Among the experimental parameters, an important factor that influences the CP phase and morphology is the pH value of solution.5,6 For instance, ACP forms in the pH range from 5 to 12, and octacalcium phosphate (OCP) is stable in the pH range from 5.5 Conflicts of Interest: The authors declare no conflicts of interests. Grant support: This work was funded by the Science and Technology Commission of Shanghai (1052nm06200), the National Natural Science Foundation of China (50772124, 50821004), and State Key Laboratory of High Performance Ceramics and Superfine Microstructure. ⁎Corresponding author: Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, P. R. China. E-mail address: [email protected] (Y.-J. Zhu).
to 7.0. The structure of ACP is still uncertain; however, the posner's cluster model is widely accepted. The basic structural unit of this model is the Ca9(PO4)6 cluster with a diameter of 9.5 Å. The clusters are presumed to pack randomly with respect to each other, forming the nanospheres with sizes of 30 to 80 nm, and contain 10-20% by weight of tightly bound water molecules in the interstices between clusters.7,8 We propose that the specific surface area of the ACP clusters is extremely large when they form shortly after the reactants are mixed in solution. Similarly, OCP displays the HAp layers separated by hydrated layers, and OCP with large specific surface area exists at the very early stage of the precipitation reaction. If the drug is present in situ during the initial stage of the formation of CP nanomaterials, it is possible for the drug to be loaded in situ into the CP nanocarriers with ultrahigh specific surface area, leading to a ultrahigh drug loading capacity (DLC) and prolonged drug release. Moreover, in comparison with the traditional CP drugdelivery systems (DDSs), these DDSs prepared by the in-situ
1549-9634/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2010.12.005 Please cite this article as: Q.-L. Tang, Y.-J. Zhu, J. Wu, F. Chen, S.-W. Cao, Calcium phosphate drug nanocarriers with ultrahigh and adjustable drugloading capacity: One-step synthesis, in situ drug loading and prolonged drug release. Nanomedicine: NBM 2011;7:428-434, doi:10.1016/j.nano.2010.12.005
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drug-loading process may exhibit different drug-loading and release properties. Many drugs with great medical and commercial significance are hydrophobic, such as ibuprofen (2-[4-(2-methylpropyl) phenyl]propanoic acid, IBU), AC ((3R,5R)-7-[2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-(pheynylcarbamoyl)pyrrol-1-yl]3,5 -dihydroheptanoic acid, calcium salt (2:1) trihydrate, AC), indomethacin (2-{1-[(4-chlorophenyl)carbonyl]-5-methoxy-2methyl-1H-indol-3-yl}acetic acid). It was reported that the drug carriers are important for loading the drugs with high DLC and for sustained drug release over a prolonged period to enhance therapeutic efficiency.9,10 Zhu et al.11 synthesized HAp porous ellipsoidal hollow capsules constructed with the self-assembly of nanosheets by using CaCO3 hard template; the as-prepared HAp porous ellipsoidal hollow capsules had a specific surface area as high as 220 m2g-1. They were explored as the drug nanocarriers for IBU, and a high DLC of 459.5 mg g-1 was achieved. Although some progress has been made, some challenges regarding the use of CP materials as drug carriers for clinical applications still exist, such as low drug-loading capacity, uncontrollable-burst drug release, and a toxic environment sometimes adopted for the CP preparation and drug-loading process. In this article, we demonstrate a single-step synchronous method for both the synthesis of CP nanocarriers and the loading of the hydrophobic drug into CP nanocarriers in mixed solvents of water and ethanol, the formation of CP nanocarriers and the loading of the drug occur simultaneously in the same reaction system. In this method, large numbers of CP clusters with an ultrahigh specific surface area form at the early stage of the precipitation reaction, and CP clusters provide many binding sites for the drug molecules. The in situ presence of the drug molecules during the formation of CP nanocarriers makes it possible for the drug molecules to adsorb on the surface of the CP clusters. As a result, a superhigh DLC and prolonged drug-release characteristics can be achieved. Furthermore, The DLC can be adjusted by changing the pH value of solution and the concentration of the drug. This method does not need any surfactant or two-step procedures of synthesis of CP nanocarriers and subsequent drug loading; thus, it avoids the post-treatment processes that may inactivate the binding sites for the drug molecules. Additionally, it avoids the use of toxic organic solvents as the media for hydrophobic drug loading. This method has advantages such as ease of handling, low cost and environmental friendliness, which are promising for the application in drug delivery.
Methods Materials CaCl2·2H2O (AR), Na2HPO4·12H2O (AR), NaOH (AR), HCl (36–38%) and ethanol (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ibuprofen (99.99%) was purchased from Shanghai (China) Yuanji Chemical Co., Ltd. (Quzhou, China). Atorvastatin calcium (99.5%) was purchased from Quzhou Aifeimu Chemical Co., Ltd. Simulated body fluid (SBF, pH 7.4 at 37°C ) was prepared according to the literature.12 All the chemicals were used as received without further purification.
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Table 1 The main experimental parameters for the preparation of CP-IBU and CP-AC drug delivery systems and estimated drug-loading capacities (DLCs) Samples
IBU [mol L-1]
OH- [mol L-1]
pH value
DLC [g g-1]
CP-IBU1 CP-IBU2 CP-IBU3 CP-IBU4 CP-IBU5 CP-IBU6 CP-IBU7 CP-IBU8 CP-IBU9 CP-IBU10 CP-IBU11 CP-IBU12 CP-IBU13 CP-IBU14 CP-IBU15 CP-IBU16
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.3 0.4 0.5 0.6 0.3 0.3 0.3
0 0.01 0.03 0.05 0.07 0.08 0.09 0.10 0.06 0.09 0.12 0.15 0.18 0.03 0.06 0.12
3.6 4.6 5.6 6.1 6.6 6.8 7.0 7.4 5.8 5.8 5.8 5.7 5.7 4.9 5.5 6.0
0.35 0.63 0.68 0.46 0.47 0.34 0.24 0.14 0.96 1.34 1.62 1.68 1.96 1.37 1.54 1.40
Samples
AC [mol L-1]
OH- [mol L-1]
pH value
DLC [g g-1]
CP-AC17 CP-AC18 CP-AC19 CP-AC20 CP-AC21 CP-AC22 CP-AC23
0.0087 0.0087 0.0087 0.0087 0.0087 0.0087 0.0087
0.008⁎ 0.0056⁎ 0 0.004 0.008 0.011 0.014
5.1 5.4 5.8 6.1 6.4 6.7 7.2
0.33 0.36 0.48 0.62 0.76 0.92 0.93
The concentrations are normalized to those of the final solutions. ⁎ Represents the concentration of HCl in the reaction system.
Preparation of the drug-loaded calcium phosphate nanocarriers All the experiments were conducted at room temperature (about 15−25°C). In a typical experiment for the preparation of IBUloaded DDS sample (CP-IBU3), 2.063 g IBU and 0.368 g CaCl2·2H2O were dissolved in 65 mL ethanol. Then, 3 mL 1 M NaOH aqueous solution and 7 mL deionized water were injected into the ethanol solution. In addition, 25 mL of phosphate aqueous solution containing 0.537 g Na2HPO4·12H2O was poured into the above reaction system under magnetic stirring and reacted for 1 minute. The product was centrifuged, washed with deionized water and dried at 60°C in vacuum. The volume ratio of ethanol to water was 13:7. Other samples were prepared at different IBU concentrations by changing the amount of IBU or at different pH values of solution by changing the volume of NaOH solution (Table 1). The AC concentration was 10 g L-1 in the preparation of AC-loaded DDS samples, and other experimental parameters were similar to the IBU-loaded DDS samples. In vitro drug release The drug-loaded CP DDS samples were compacted into disks (0.20 g each, diameter = 10 mm) at a pressure of 3 MPa. Then the disk was immersed into 200 mL SBF at 37 °C under shaking at a constant rate of 150 rpm. The IBU release medium (2.0 mL) was extracted for UV–vis analysis at the wavelength of 264 nm at given time intervals and replaced with the same volume of fresh SBF, which was preheated to 37°C.
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Figure 1. (A) TG curves and (B) the DLCs versus pH values for the CP-IBU DDS samples prepared at different pH values at a concentration of [IBU] = 0.1 M; (C) TG curves and (D) the DLCs versus IBU concentrations for the CP-IBU DDS samples; (E) TG curves and (F) the DLCs versus pH values for the CP-AC DDS samples prepared at different pH values at a concentration of [AC] = 0.0087 M. All the samples were prepared in mixed solvents of ethanol and water with a volume ratio of 13:7 at a concentration of [Ca2+] = 0.025 M.
Characterization Thermogravimetry (TG) curves were measured with a simultaneous thermal analyzer (STA 409PC, Netzsch, Germany) at a heating rate of 10°C min-1 in flowing air. UV–vis absorption spectra were performed on a spectrophotometer (UV2300, Techcomp, Shanghai, China). X-ray powder diffraction (XRD) patterns were recorded using an X-ray diffractometer (Rigaku D/max 2550V, Tokyo, Japan) with high-intensity Cu Ka
radiation (λ = 1.54178Å). Fourier transform infrared (FTIR) spectra were obtained on a FTIR spectrometer (Nicolet 380).
Results TG and XRD characterization of CP-IBU DDSs IBU and atorvastatin calcium (AC) were chosen as the model hydrophobic drugs in this investigation. It should be
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mentioned that both drugs could be dissolved in ethanol and they did not precipitate in the absence of CP in the mixed solvents of ethanol and water with a volume ratio of 13:7. Figures 1, A, 1, C and 1, E, show the TG curves measured in flowing air of CP-IBU and CP-AC DDSs prepared under different conditions. The overall weight loss can be attributed to the loss of adsorbed water and lattice water and the clean burn of drugs. The similar result was also observed for the CP-AC DDSs. On the basis of the TG analysis, we estimated the DLCs of the as-prepared CP nanocarriers, and the results are shown in Figures 1, B, 1, D and 1, F and also in Table 1. Table 1 also shows the main experimental parameters for the preparation of CP-IBU and CP-AC DDSs. By increasing the IBU concentration from 0.1 to 0.6 M (CPIBU3, CP-IBU9 to CP-IBU13), the DLC significantly increased, as shown in Figure 1, D. The DLC of the CP-IBU13 DDS prepared at an IBU concentration of 0.6 M reached a value as high as 1.96 g IBU per gram carrier. The increased IBU concentration can influence the dynamic equilibrium of the drug adsorption, leading to enhanced DLC. It should be noted that a series of DDSs with adjustable DLCs from 0.14∼1.96 g IBU per gram carrier can be prepared by changing the pH value or IBU concentration. The chemical compositions of the as-prepared drug nanocarriers correlate strongly with the pH value of solution. Figure 2, A shows XRD patterns of the DDS samples prepared at different pH values of solution with an IBU concentration of 0.1 M. In addition, CP-IBU1 prepared at pH = 3.6 was identified as singlephase dicalcium phosphate dehydrate (DCPD) (JCPDS No. 110293), and CP-IBU3 synthesized at pH = 5.6 consisted of a mixture of DCPD, OCP and HAp. By increasing the pH value to 6.6 (CP-IBU5), the product was a mixture of OCP and HAp. When the pH value was further increased to 7.4 (CP-IBU8), essentially only HAp was obtained. These results indicate that different drug nanocarriers can be obtained by adjusting the pH value of the solution. TG and XRD characterization of CP-AC DDSs We also investigated the loading of a hydrophobic drug, AC, in the CP nanocarriers. The CP-AC DDSs were prepared at different pH values at a concentration of [AC] = 0.0087 M in mixed solvents of ethanol and water with a volume ratio of 13:7. It has been found that the DLC of CP-AC DDS can also be controlled by adjusting the pH value of the solution, as shown in Figures 1, E and 1, F and Table 1. The DLC increased with increasing pH value, and it varied from 0.33 to 0.93 g AC per gram carrier in the pH range of 5.1−7.2. This variation trend in DLC of AC is totally different from that of the IBU loading, indicating the pH effect on different drugs. Figure 2, B shows XRD patterns of the samples prepared at different pH values of solution at concentrations of [Ca2+] = 0.025 M and [AC] = 0.0087 M in mixed solvents of ethanol and water with a volume ratio of 13:7. The CP-AC17 sample prepared at pH = 5.1 was identified as single-phase DCPD, and the CP-AC20 sample prepared at pH = 6.1 consisted of a mixture of DCPD, OCP and HAp. When the pH value was further increased to 7.2 (CPAC23), essentially only HAp was obtained.
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Figure 2. (A) XRD patterns of the CP-IBU DDS samples prepared at different pH values of solution; (B) XRD patterns of the CP-AC DDS samples prepared at different pH values of solution. All the samples were prepared in mixed solvents of ethanol and water with a volume ratio of 13:7, and then washed by ethanol several times to remove the drug for the purpose of avoiding the peaks of the IBU or AC.
In vitro drug release behaviors of CP-IBU DDSs The drug-release profiles of the CP-IBU5 and CP-IBU13 drugdelivery systems in SBF are shown in Figure 3. Both DDSs exhibited a prolonged drug release property, but the drug-release rates were different for the two DDSs. For the CP-IBU5 DDS prepared at a low IBU concentration and pH value of 6.6, 32% of the loaded drug was released in the first 24 hours, and 64% was released in a period of 2 weeks. However, for the CP-IBU13 DDS prepared at a high IBU concentration, in which 1.96 g IBU was loaded per gram carrier, the drug-release percentage was much lower than that of the CP-IBU5. Only 12% of the loaded drug in the CP-IBU13 DDS was released in a period of 24 hours, and 19% was released in 2 weeks; a much prolonged drug release can be achieved in this DDS. Discussion To understand the high DLC of the CP-IBU DDSs, the Brunauer-Emmett-Teller (BET) specific surface area (SBET) measurements were performed. The CP-IBU5 and CP-IBU13 DDS samples were washed by ethanol several times to remove the IBU before the specific surface area measurement. The SBET
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Figure 4. Illustration of the single-step synchronous method for the preparation of CP nanocarriers and the in situ drug loading.
Figure 3. IBU release profiles: (A) CP-IBU5 and (B) CP-IBU13. The insets show the plots for cumulative IBU release percentage versus natural logarithm of release time.
of the CP-IBU5 and CP-IBU13 DDSs was 60.48 and 98.03 m2 g-1, respectively. The ultrahigh DLC of 1.96 g IBU per gram carrier was achieved for the CP-IBU13 DDS, which is even much higher than that (1.40 g g-1) of nonbiocompatible mesoporous material MIL-101, a chromium terephthalate with an ultrahigh SBET of 5510 m2 g-1.13 This extremely high DLC of CP-IBU13 DDS obviously cannot be accounted for only by SBET. ACP was reported to form at pH values of 5∼12, and it was the initial phase of HAp in a neutral or basic environment. Similarly, it could transform to OCP and DCPD in appropriate conditions.5,6 The formation of Ca9(PO4)6 cluster of ACP at the early stage of the precipitation reaction was reported, and the cluster was estimated to be roughly spherical with a diameter of 9.5 Å.7,8 Wang et al14 studied the formation and transformation of ACP particles by stopped-flow spectrophotometry, simultaneous measurements of particle size and pH of solution, and high-resolution transmission electron microscopy, indicating the formation of ion pairs and clusters in the first few seconds, and crystalline domains developed at multiple sites inside the primary particles of the amorphous phase. Suppose that the diameter of ACP clusters is as small as 9.5 Å, ACP clusters have the lattice similar to HAp and the density of HAp is ρ = 3.16 g cm-3. The specific surface area of this material is roughly calculated as high as 2100 m2g-1. We propose that large numbers of CP clusters with an ultrahigh specific surface area form before the bulk precipitation occurs in solution, and CP clusters provide many binding sites for the drug molecules. Ca2+ ions may form
the complex with carboxyl groups of IBU or atorvastatin calcium. In our experiments the formation of the CP nanocarriers and the loading of the drug were conducted simultaneously in the same reaction system. The in situ presence of drug molecules during the formation of CP nanocarriers may lead to the interaction between organic groups of the drug molecules and Ca2+ cations or PO34 anions on the surface of the CP clusters. Thus, the strong interaction between the drug molecules and CP nanocarriers occurred, and more drug molecules were encapsulated in the interior of the CP nanocarriers in addition to the drug molecules adsorbed on the surface of the CP nanocarriers, as shown in Figure 4, which illustrates the proposed strategy for the one-step simultaneous formation of CP nanocarriers and in situ drug loading in the same reaction system. It is different from the conventional DDSs in which the drug molecules merely adsorb on the surface of the carrier. As a result, a superhigh DLC can be achieved. The pH value of solution not only influences the composition, nucleation and crystallization process of the product, but also changes the ionization state of the IBU and AC drug molecules; thus, it has a significant effect on the DLC. The AC molecule is bigger than the IBU molecule, and it has more functional groups that can interact with the CP nanocarriers. A reversed trend in DLC of AC in the CP-AC DDSs compared with the CP-IBU DDSs in a given pH range is found, indicating the pH effects in different ways. We demonstrate a single-step synchronous method for the preparation of CP nanocarriers and the in situ drug loading conducted in mixed solvents of water and ethanol. In comparison with pure IBU without the nanocarriers, which burns at about 150−280°C in air, the CP-IBU DDS shows a much higher burning temperature for the IBU ingredient in the TG measurement, implying that the thermal stability of IBU can be enhanced to some extent when loaded in the CP nanocarriers
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Figure 5. FTIR spectra: (A) pure IBU; (B) CP-IBU5; (C) CP-IBU5 washed with ethanol several times to remove IBU; (D) CP-IBU13; (E) CP-IBU13 washed with ethanol several times to remove IBU.
due to the interaction between the loaded drug molecules and CP nanocarriers. The interaction between the drug molecules and CP nanocarriers was also investigated by FTIR spectra. Figure 5 shows the FTIR spectra of typical CP-IBU DDSs (CP-IBU5 and CP-IBU13) and corresponding control samples. The samples were washed with ethanol several times to remove the IBU before the FTIR measurements for comparison. In the FTIR spectrum of pure IBU (Figure 4, A), the band at 1724 cm-1 is attributed to the C = O group of IBU molecules, and the peaks at around 2900 cm-1 originate from the –CH3 and –CH2 groups. The FTIR spectra of CP-IBU5 and CP-IBU13 exhibit the presence of the characteristic peaks of both IBU and CP nanocarrier, indicating the combination of IBU and CP nanocarriers in the CP-IBU drug-delivery system. In comparison with the FTIR spectra of pure IBU and the ethanol washed samples, red shift of the C = O band from 1724 to 1550 cm-1 was observed for the CP-IBU5 DDS. Meanwhile, the PO34 vibration peak was split into two bands at 1119 and 1026 cm-1. These changes may originate from the interaction between the loaded IBU molecules and the CP nanocarriers. However, the FTIR spectrum of CP-IBU13 is more complicated. The C=O peaks were observed at 1720 and 1554 cm-1, implying the existence of both strongly bonded and weakly bonded IBU molecules. Moreover, the ethanol washed CP-IBU13 sample exhibited characteristic peaks of both IBU molecules and CP nanocarriers, indicating that some strongly bonded IBU molecules were still left after washing with ethanol due to a relatively strong interaction between the IBU molecules and the CP nanocarriers. We estimated by TG analysis that 0.12 g IBU per gram carrier remained for the ethanol washed CP-IBU13 sample. For the medicinal applications, it is desirable that the drug release is reasonably slow and prolonged to achieve the highly efficient treatment. The ideal DDS should have a high loading capacity of the drug, and it should release the drug with a reasonable rate to reach the concentration required to maintain efficacy within a relatively short time, and then, the drug release rate should maintain at an appropriate level for a prolonged
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period. Our previous studies showed that the release of the pure drug without the nanocarrier in SBF was very rapid, and the drug release completed within several hours. In contrast, the drug release could be well controlled with a reasonable rate and a prolonged release time with the help of the nanocarriers.11,15 In this study, the drug-release time of the CP-IBU5 and CP-IBU13 DDSs significantly increased in SBF, but the drug-release rates were different for different DDSs. For the CP-IBU5 DDS prepared at a low IBU concentration, 32% of the loaded drug was released in the first 24 hours, and 64% in a period of 2 weeks. However, for the CP-IBU13 DDS with a much higher DLC, only 12% of the loaded drug was released in a period of 24 hours and 19% in 2 weeks. The sustained and prolonged drug-release profile can be achieved in the as-prepared DDSs. The drug-release kinetics of the CP-IBU5 and CP-IBU13 DDSs in SBF were investigated. It is well acknowledged that the kinetics of drug release from the carrier materials can be well described using the Higuchi model (C = k·t1/2),16-18 in which there exists a linear relationship between the cumulative amount of released drug and the square root of release time; the drug release is governed by a diffusion process. In this study we have found different drug-release kinetics for the CP-IBU5 and CPIBU13 DDSs in SBF. Our experiments showed that the drugrelease kinetics of the CP-IBU5 and CP-IBU13 DDSs did not follow the Higuchi model. Instead, the cumulative amount of released drug had a linear relationship with the natural logarithm of release time, ln(t), in the time period from 2 hours to 14 days for the CP-IBU5 and CP-IBU13 DDSs (insets of Figure 3, A and B). The drug release kinetics of this type of DDS may be governed by synchronous mechanisms including desorption, diffusion and phase transformation of CP nanocarriers. This unique drug-release kinetics has also been found in the DDS of hierachically nanostructured mesoporous spheres of calcium silicate hydrate.9 For the CP-IBU5 DDS prepared at a low IBU concentration and pH value of 6.6, 32% of the loaded drug was released in the first 24 hours and 64% was released in a period of 2 weeks. However, for the CP-IBU13 DDS prepared at a high IBU concentration, in which 1.96 g IBU was loaded per gram carrier, the drug-release percentage was much lower than that of the CPIBU5. Only 12% of the loaded drug in the CP-IBU13 DDS was released in a period of 24 hours and 19% was released in 2 weeks; a prolonged drug-release can be achieved in this DDS. The FTIR spectra suggest that a quantity of IBU molecules that are strongly bonded with the CP nanocarriers may exist, especially for the CP-IBU13 DDS, which can explain the prolonged drug-release behavior. As for the clinical application, these DDSs representing different drug-release behaviors may be used in various situations. HAp is the most stable phase in SBF at room temperature (about 15−25°C). Thermodynamically metastable phases of CP undergo the processes of dissolution, re-nucleation and crystal growth, and finally transform to HAp. The XRD patterns indicated that the transformation to HAp occurred after the drugrelease process in SBF, and the CP component of the as-prepared DDSs were completely transformed to HAp after the drug release in SBF for 2 weeks. Similar experimental results were also reported.19,20
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In summary, a single-step synchronous method has been developed in which the formation of CP nanocarriers and the in situ loading of the drug occur simultaneously in the same reaction system. It does not need any surfactant or the two-step procedures of synthesis of CP nanocarriers and subsequent drug loading, thus, avoids the complex post-treatment processes. Also, it avoids the use of toxic organic solvents as the media for hydrophobic drug loading. This method has advantages, such as ease of handling, low cost and environmental friendliness, which are promising for the application in drug delivery. The superhigh DLC and prolonged drug-release characteristics have been achieved in the as-prepared DDSs. Furthermore, DLC can be adjusted by changing the pH value of solution and the concentration of the drug. In addition, the drug-release kinetics has been found in which the cumulative amount of drug release has a linear relationship with the natural logarithm of release time. The detailed mechanism of this novel drug-release kinetics needs to be further investigated.
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