3.1 Preparation of blank and drug-loaded NPs
The UV spectra of ABE, VES, CaP and HA are shown in Fig. 1. CaP and HA/CaP have no UV absorption in the range of 250–400 nm, indicating that blank carriers will not interfere with the content determination of ABE and VES. The quantitative results showed that the drug loading rates and encapsulation rate of ABE in the CaP/ABE-VES system were 4.02% and 20.11%, respectively, whereas those of VES were 14% and 69.54%, respectively. The drug loading rate and encapsulation rate of ABE in the HA/CaP/ABE-VES system were 3.65% and 18.24%, respectively, whereas those of VES were 16% and 78.79%, respectively. The drug-loading effects of both systems were better on VES than ABE. This outcome may be helpful because VES is harmless to normal cells as a chemical sensitizer; thus, it could reduce the dosage of ABE and increase anti-cancer activity. This conclusion is verified in the results of subsequent cell viability experiments.
3.2 Shape, particle size, and zeta potential
CTAB micelles further formed to to CaP nanospheres with multivesicular structure and a particle size of about 80 nm under the deposition and mineralization of phosphate and calcium salt (Fig. 2A). The particle size of HA/CaP increased to 90 nm because of the presence of a 10 nm HA shell (Fig. 2B). The SEM results show that CaP has a nano-sized spherical structure and uniform overall morphology. HA/CaP is denser under SEM. This is because the negative charge carried by HA neutralizes the positive charge on the surface of CaP, which weakens the repulsive force against agglomeration on the surface of NPs. Particle size distribution curve (Figs. 2C and 2D) shows that the average hydrodynamic diameter of CaP and HA/CaP measured by dynamic light scattering (DLS) is about 100 nm and 110 nm. The particle size of the sample measured by the electron microscope is smaller than the value measured by the DLS due to the shrinkage and the reduction during the external force of air drying. As shown in Fig. 2D, zeta potential measurement shows that the surface charge of CaP is about 9.4 ± 0.80 mV, this indicates the presence of Ca2+ on CaP surface. The surface potential of HA/CaP decreased to -4.3 ± 0.25 mV, indicating that Ca2+ on the surface of CaP changed into carboxyl anion after HA covered on it, which confirmed that HA was successfully coated on the surface of CaP. Above results show that CaP with a spherical hollow structure was successfully prepared and HA coats on the surface of CaP under electrostatic interaction.
3.3 Infrared analysis
Figures 3A and 3B shows the infrared spectra of blank and drug-loaded CaP and HA/CaP. The absorption at 1036 cm− 1 is attributed to the stretching vibration of the P-O bond. The characteristic absorption at 604 cm− 1 and 566 cm− 1 is caused by the bending vibration of O-P-O. This indicates that CaP with a hydroxyapatite structure was successfully prepared[22]. The absorption peaks of HA at 3423 cm− 1, 2923 cm− 1, and 1044 cm− 1 correspond to the stretching vibrations of its carboxyl, methylene and C-O groups, respectively. Compared with CaP, the absorption of HA/CaP at 2923 cm− 1 is enhanced, as a result of the presence of increased methylene groups in the HA molecule. Infrared spectroscopy analysis also confirmed the presence of ABE and VES in drug carriers. The absorption band in the wavenumber range of 1400–1600 cm− 1 is caused by the benzene ring vibration of ABE, and the double peaks in the spectrum of PM at 1715 cm-1 and 1753 cm-1 correspond to the carbonyl absorption of ester and carboxyl groups in VES. Those peaks appears in PM, but disappears in CaP/ABE-VES and HA/CaP/ABE-VES, this phenomenon may be due to the in-situ loading effect of the nanomaterials on the drug, which makes the drug molecules doped inside the material and causes peaks to weaken or disappear. The infrared results confirm that the prepared CaP is a calcium-deficient hydroxyapatite, and the drugs are encapsulated in the nanomaterials because of the in-situ drug-loading effect.
3.4 Crystallinity and element analysis
All of synthesized products showed hydroxyapatite-like characteristics in X-ray diffraction (XRD) analysis. CaP has two characteristic peaks at 26° and 32°. It can be indexed as a calcium-deficient hydroxyapatite structure with poor crystallinity[23]. The poor crystallinity is attributed to the low reaction temperature (40°C), which is consistent with reported literature, that is, CaP particles synthesized at low temperatures often show low crystallinity, high solubility and large specific surface area[24]. This result is conducive because a study on the resorption of biomaterials for hard tissue treatment showed that low-crystalline CaP is more degradable than CaP synthesized by sintering or other methods[25]. Therefore, the synthesized calcium-deficient CaP nanomaterials may have better biocompatibility. Figures 3C and 3D indicates that the drug is not simply mixed after being loaded in situ by CaP and HA/CaP. This result was consistent with the infrared result. The characteristic diffraction peak of HA/CaP/ABE-VES at 26° is weaker than that of blank HA/CaP. This may be due to the hydrogen bond between HA and the two drugs. The hydrogen bonding between the drug and the carrier resulted in lower crystallinity[26]. Energy-dispersive X-ray spectroscopy analysis showed a 1.5 Ca/P ratio, which further indicates that the synthesized CaP is a kind of calcium-deficient hydroxyapatite with poor crystallinity[27]. The C, O, and F elements in HA/CaP/ABE-VES also increased, indicating that the incorporation of HA can increase the drug loading of nanomaterials (Fig. S1).
3.5 Specific surface area and pore size distribution
As shown in Figs. 3E and 3F, the surface area of the CaP nanospheres is 123.90 m2 g− 1. CaP has a large specific surface area, which is beneficial to the adsorption and loading of small molecule drugs. The surface area of CaP decreased to 89.94 m2 g− 1 after coating with HA, because some mesoporous channels were covered by HA, resulting in a decrease in specific surface area. This phenomenon has also been found in previous literature[28]. N2 adsorption curve of CaP shows that it has an obvious mesoporous structure. Figure 3 shows that the pore size of CaP is approximately 31.78 nm. The pore size distribution of HA/CaP is more uniform. After the addition of HA, pore size and pore volume were reduced to 29.74 nm and 0.67 cm3 g− 1, respectively,. This decrease may be because the incorporation of negatively charged HA makes the internal electrostatic force of mesoporous HA/CaP stronger, which results in a more compact structure. This result is consistent with the conclusion of SEM.
3.6 Thermogravimetric (TG) analysis
The thermal decomposition is shown in Fig. 4. The endothermic peak of ABE and VES appeared at 360°C and 306°C, respectively. In the range of 150–350°C, CaP shows a weight loss of 4.04% at 333°C, which is attributed to the decomposition of the CaP framework[29]. The derivative thermogravimetric (DTG) curve of CaP/ABE-VES shows that the endothermic peak at 150–350°C becomes larger and the weight loss is 11.02%. This result is because of the overlap in the endothermic peaks of VES and CaP in this temperature range. A weight loss of 2.26% appears at 406°C, which corresponds to the endothermic peak of ABE in CaP/ABE-VES. The curve of HA/CaP/ABE-VES shows that the mass loss in this range is 26.59%, which is higher than that of CaP/ABE-VES. These results indicate that the HA/CaP system has a higher drug loading capacity for VES than a single CaP system. The higher drug loading capacity of HA/CaP may be due to the COO- group contained in the outer HA, which can provide more binding sites and space for drug molecules. The drug molecule may bind to the C site of CaP through electrostatic force, or be incorporated into the amorphous region of CaP through the Ca bridge action of the P site[30]. The thermal decomposition temperature of loaded drugs is higher compared with free drugs. This result also shows that the prepared HA/CaP can improve the thermal stability of the loaded drug.
3.7 pH Sensitivity of CaP and HA/CaP NPs
The drug release kinetics of CaP may be controlled by acid-assisted dissolution [31]. In pH 7.4, about 30% of ABE was released from CaP/ABE-VES after 8 h, which is higher than the 16% release of a single drug. When the pH dropped to 4.5, the cumulative release of ABE from CaP/ABE-VES can increased to 98% after 8 h but the release of a single drug at pH 4.5 is only 60%. In general, CaP makes the release of ABE more complete at pH 4.5. About 90% of VES was released from CaP/ABE-VES in 8 h at pH 7.4, which is similar to the 80% release of a single drug. The increased release of VES at pH 7.4 is due to the fact that VES is a weakly acidic molecule, and alkaline condition can increase its solubility. When the pH dropped to 4.5, the cumulative release of VES from CaP/ABE-VES can reach 100% after 8 h, whereas only 18% of the single drug was released. Therefore, the pH sensitivity of CaP as a carrier is weak for the controlled release of VES, that is, drug release can be achieved regardless of pH (4.5 or 7.4). Compared with the CaP system, the HA/CaP system released less drugs under acidic conditions. Whereas HA/CaP released only 14% ABE and 32% VES at pH 7.4. This may be because the covering effect of the HA layer prevents the dissolution of the drug. It is surprising that the incorporation of the HA layer greatly reduced the release of ABE and VES at pH 7.4, that is, the HA/CaP/ABE-VES system has stronger pH responsiveness, can release both drugs at pH 4.5, but few released at pH 7.4. Such release characteristics may weaken the damage of the drug to normal human cells, while killing cancer cells.
3.8 In vitro cytotoxicity evaluation of CaP and HA/CaP nanomaterials
The toxicity of the nanocarriers to normal cells and cancer cells were significantly different (Fig. 6(a) and Fig. 6(b)). When the concentration of CaP and HA/CaP was greater than 50 µg/mL, the proliferation of MCF-7 was significantly inhibited. Even the concentration reached up to 200 µg/mL, the viability of 293T cells was above 90% (p < 0.001). The results indicate that the prepared nanomaterials are more damaging to cancer cells, other than normal cells, this is because the increase in calcium ions in the acidic environment of the tumor results in cell death[32].
VES can inhibit the proliferation of a variety of cancer cell, including human neuroblastoma cells, prostate cancer cells, promyelocytic cells, and breast cancer cells but is non-toxic to normal cell lines[33]. The experimental results on the cytotoxicity of VES are consistent with these reports. In vitro cytotoxicity of VES on 293T and MCF-7 cells are shown in Fig. 6(c). VES had no significant inhibitory effect on 293T cells at concentrations up to 200 µM. By contrast, MCF-7 cells was significantly inhibited at the VES concentration of 50 µM (p < 0.05).
The stronger inhibitory effect on MCF-7 may be due to the synergistic effect of the two drugs, which strengthened the anti-tumor effect (Fig. 7(b) and Fig. 7(d)). HA/CaP still retained the Ca2+-mediated inhibition of cancer cell proliferation after the dissolution of CaP in the acidic environment of the tumor. On this basis, the surface coating of the HA layer might have further increased the endocytosis of HA/CaP at low concentration, to enhance the inhibition of tumor cells at low concentration and consequently reduce the damage to normal cells. The results indicated that the prepared HA/CaP/ABE-VES utilizes unique pH responsiveness to effectively control the release of the drugs. This result is consistent with the results of the in vitro release experiment described in the previous section. The incorporation of HA endows HA/CaP/ABE-VES with stronger cytostatic effect on MCF-7 compared with that without HA.
3.9 Synergistic effect
Table 1
Evaluation of combined index (CI) at IC50(ABE) in CaP/ABE-VES and HA/CaP/ABE-VES system
Drug/Combo(µM) | ABE | VES | PM-CaP | Co-CaP | PM-HA | co-HA |
IC50(ABE) | 8.106 | / | 9.811 | 4.322 | 9.002 | 3.673 |
IC50(VES) | / | 484.767 | 34.339 | 15.127 | 40.509 | 16.529 |
CI value | / | | 1.337 | 0.327 | 0.983 | 0.188 |
Combination index (CI) value was calculated using CompuSyn software. The curve of log (CI) value and drug effect level (Fa) can clearly show the characteristics and extent of drug interaction[34]. Log (CI) values of > 0, 0, and < 0 mean antagonism, superimposition, and synergy, respectively[35]. In this study, the CI values of (PM) and CaP/ABE-VES on MCF-7 cell were compared. The CI value of HA/CaP/ABE-VES is 0.188, which has a stronger synergy (++++) and is lower than that of CaP/ABE-VES (CI = 0.327). In the CaP/ABE-VES and HA/CaP/ABE-VES group, all the log (CI) values in each corresponding Fa value are lower than log (CI) = 0. The mixed cocktails almost exceed log (CI) = 0. These results indicate that the synergistic effect of HA/CaP/ABE-VES and CaP/ABE-VES on MCF-7 cells is more remarkable than that of the cocktail mixture. In addition, HA/CaP/ABE-VES presents a more remarkable synergistic anti-tumor effect than CaP/ABE-VES because of HA modification.