Aptamer‐mediated synthesis of multifunctional nano‐hydroxyapatite for active tumour bioimaging and treatment

Abstract Objectives The nano‐hydroxyapatite (nHAp) is widely used to develop imaging probes and drug carriers due to its excellent bioactivity and biocompatibility. However, traditional methods usually need cumbersome and stringent conditions such as high temperature and post‐modification to prepare the functionalized nHAp, which do not benefit the particles to enter cells due to the increased particle size. Herein, a biomimetic synthesis strategy was explored to achieve the AS1411‐targeted tumour dual‐model bioimaging using DNA aptamer AS1411 as a template. Then, the imaging properties and the biocompatibility of the synthesized AS‐nFAp:Gd/Tb were further investigated. Materials and methods The AS‐nFAp:Gd/Tb was prepared under mild conditions through a one‐pot procedure with AS1411 as a template. Besides, the anticancer drug DOX was loaded to AS‐nFAp:Gd/Tb so as to achieve the establishment of a multifunctional nano‐probe that integrated the tumour diagnosis and treatment. The AS‐nFAp:Gd/Tb was characterized by transmission electron microscopy (TEM), energy disperse X‐ray Spectroscopy (EDS) mapping, X‐ray photoelectron spectroscopy (XPS) spectrum, X‐ray diffraction (XRD), fourier‐transformed infrared (FTIR) spectroscopy, capillary electrophoresis analyses, zeta potential and particle sizes. The in vitro magnetic resonance imaging (MRI) and fluorescence imaging were performed on an MRI system and a confocal laser scanning microscope, respectively. The potential of the prepared multifunctional nHAp for a targeted tumour therapy was investigated by a CCK‐8 kit. And the animal experiments were conducted on the basis of the guidelines approved by the Animal Care and Use Committee of Sichuan University, China. Results In the presence of AS1411, the as‐prepared AS‐nFAp:Gd/Tb presented a needle‐like morphology with good monodispersity and improved imaging performance. Furthermore, due to the specific binding between AS1411 and nucleolin up‐expressed in cancer cells, the AS‐nFAp:Gd/Tb possessed excellent AS1411‐targeted fluorescence and MRI imaging properties. Moreover, after loading chemotherapy drug DOX, in vitro and in vivo studies showed that DOX@AS‐nFAp:Gd/Tb could effectively deliver DOX to tumour tissues and exert a highly effective tumour inhibition without systemic toxicity compared with pure DOX. Conclusions The results indicated that the prepared multifunctional nHAp synthesized by a novel biomimetic strategy had outstanding capabilities of recognition and treatment for the tumour and had good biocompatibility; hence, it might have a potential clinical application in the future.


| INTRODUC TI ON
The preparation of biodegradable and multifunctional nanoparticles with excellent biocompatibility, superior imaging and therapeutic capabilities is of great significance for improving the diagnosis and treatment of tumours. [1][2][3] In recent years, various nanotechnologies have been designed in the field of biomedicine and have received extensive attention. [4][5][6][7] The hydroxyapatite (HAp, Ca 10 (PO 4 ) 6 (OH) 2 ) is a primary inorganic composition of mammalian hard tissues (bone and teeth), as a biomaterial scaffold with good biocompatibility and biodegradability, the nano-HAp (nHAp) and F-substituted nHAp (nFAp) were widely used in the biomedical field. [8][9][10][11] Recently, a variety of bioimaging systems based on nHAp have been prepared for dual/multi-mode tracking. [12][13][14] In general, the construction of nHAp imaging probes is achieved by post-modifying various imaging contrast agents through physical or chemical interaction such as gold nanoparticles, quantum dots and carbon dots. However, the tedious post-modification is not conductive to the good monodispersity of nanoparticles. 12,15 With the feature of unique hexagonal structure and space group, HAp has the ability to easily replace Ca 2+ in the crystal lattice by lanthanide ions (Ln 3+ ) (eg Eu 3+ , Gd 3+ , Tb 3+ ), thereby endowing HAp with specific imaging performance. [16][17][18] Therefore, the dual/multi-modal bioimaging is easily achieved by co-doping two or more Ln 3+ ions, and the tedious modification process is reduced simultaneously. Stringent conditions such as high temperature and specific pH values are usually required for the synthesis of doped-nHAp, 19,20 which are not beneficial to the internalization of the particles in the cells due to the increased particle size. 16 Hence, there is an urgent need for developing a simple method to synthesize multifunctional nHAp under milder conditions for clinical applications.
Recently, biological macromolecules, such as polysaccharides and natural rubber latex, have been widely used as templates in the biomimetic methods to synthesize nHAp nanoparticles (NPs) under mild conditions, because the nHAp NPs synthesized by these methods have better physical, chemical and biological properties than traditional methods. 21,22 In addition, nHAp can be directly functionalized in the biomimetic methods to avoid the post-modification of nHAp . 23 For example, based on the affinity of hyaluronan (HA) to CD44 overexpressed in tumour cells, a HA-mediated Eu/Ba codoped nFAp was constructed, and in comparison with pure nFAp:Eu/ Ba synthesized by traditional methods, the prepared HA@nFAp:Eu/ Ba possessed the ability to target tumours and improved bioimaging performance. 24 Moreover, a synthetic strategy was developed to synthesize nHAp (tHA) with polydopamine (pDA) as a template, and nHAp was then introduced into polycaprolactone (PCL) to prepare tHA/PCL, demonstrating an enhanced osteogenic ability and biocompatibility compared with traditional nHAp equipped with PCL or pure PCL. 25 Consequently, the biomimetic synthesis strategy is feasible to prepare a functionalized nHAp on one-pot procedure under mild conditions.
The DNA aptamer AS1411 is widely employed as a tumourtargeting agent by binding with nucleolin. [26][27][28][29] Nucleolin is considered to be a tumour biomarker overexpressed on the surface of cancer cells (eg breast cancer and melanoma), [30][31][32] which is believed to be dominant in internalization or transport of nanoparticles from the cell surface to the nucleus. 33 Hence, we explored an AS1411templated strategy to synthesize co-doped nFAp (Gd&Tb) for dualmodal imaging targeted cancers via a one-pot procedure in the present study. Besides, the anticancer drug DOX was loaded to nFAp so as to achieve the establishment of a multifunctional nano-probe that integrated the tumour diagnosis and treatment. A series of characterization techniques were performed to prove the successful synthesis of this functionalized nFAp, and the synthetic conditions were further optimized. The AS-nFAp:Gd/Tb synthesized under the AS1411-templated method has better abilities of imaging and recognizing the tumour compared with AS1411-free nHAp:Gd/Tb. In vitro and in vivo experiments demonstrated that the synthesized AS-nFAp:Gd/Tb could be used as a drug carrier to target tumour imaging and treatment. Meanwhile, the reduced systemic toxicity of the chemotherapy drug indicated its greater potential in clinical applications.
Firstly, AS1411 (100 μmol/L) was dissolved in ultrapure water. Then, of Na 3 PO 4 (0.6 mol/L) solution were put dropwise into the reaction system. The total volume was fixed to 1 mL, and the whole reaction system was required to be stirred at room temperature for different times (2, 6, 12 and 24 hours). Finally, the precipitate was collected after washing with ultrapure water for three times by centrifugation (8000 rpm, 5 minutes) and dispersed in the aqueous solution at the end of the reaction. For comparison, the AS1411-Free nFAp:Gd/Tb was synthesized by the same procedure described above except for the use of AS1411.

| In vitro MRI imaging and photostability
The

| In vitro fluorescence imaging of cells
The SCC-25 (human tongue squamous cell carcinoma cell line) and L929 (mouse fibroblast cell line) cells were cultured in confocal dishes with 1 × 10 5 cells/well at 37°C under 5% CO 2 for 24 hours.
The culture mediums of above cells were DMEM and RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin solution, respectively. After removing the culture medium, SCC-25 and L929 cells were incubated with 100 μg/mL AS-nFAp:Gd/Tb for 2 time periods (6 hours and 12 hours), respectively. Then, the cells were fixed with 4% paraformaldehyde, and the cytoskeletons were stained with phalloidin after washing with phosphate buffer solution (PBS). 34 Finally, fluorescence imaging was obtained by a CLSM. Meanwhile, the cells were also incubated with AS1411-free nFAp:Gd/Tb (100 μg/mL) for 12 hours to observe the fluorescence imaging as a comparison.

| Preparation of DOX@AS-nFAp:Gd/Tb
The AS-nFAp:Gd/Tb was used as a drug carrier to load DOX. In general, an equal volume of DOX (100 μg/mL) was added into the AS-nFAp:Gd/Tb aqueous solution (5 mg/mL), and then stirred for 6 hours under room temperature. Then, the complex (DOX@AS-nFAp:Gd/Tb) was acquired after washing with ultrapure water for three times by centrifugation (8000 rpm, 5 minutes) to remove free chemicals.

| In vitro drug loading and release
The drug loading efficiency and loading content were calculated by a UV-vis spectroscopy. The loading content of DOX in DOX@AS-nFAp:Gd/Tb was obtained from a standard dilution curve of DOX based on the absorbance measured under 485 nm at different concentrations. In order to explore the drug release behaviour of DOX@ AS-nFAp:Gd/Tb, the same amount of which was placed in three dialysis bags and incubated in PBS with diverse pH environments (pH = 5.5, 6.5 and 7.4). The release system was incubated at 37°C with slightly stirring. At several time intervals, equal amounts of liquid were withdrawn from the three release systems and substituted with the corresponding PBS. The amount of DOX released from DOX@AS-nFAp:Gd/Tb was measured on a UV-vis spectroscopy. Cells without any treatment were regarded as a control. Then, cells were fixed with 4% paraformaldehyde and treated by 0.05%

| Cell viability and cell apoptosis analyses
TritonX-100 to penetrate the cell membrane. Cells were further blocked with goat serum, and PBS was required to wash cells at each operation interval. The primary antibodies against Bcl-2, Bax and Caspase-3 were used to cultivate cells overnight at 4°C, and the second antibody was applied the next day. The cell nucleus was stained with DAPI, and the prepared samples were observed on a CLSM to monitor the relative expression of apoptosis-related proteins.

| In vivo anti-tumour study
The animal experiments were conducted on the basis of the guide- were measured and recorded every two days throughout the whole treatment period. At the end of the treatment, the mice were sacrificed, and the tumour tissues and vital organs (heart, liver, spleen, lung and kidney) were dissected and stored in 4% paraformaldehyde for TUNEL staining and H&E staining.

| Statistical analysis
All experiments were performed for three times, and multiple group comparisons of data were carried out by one-way analysis of variance (ANOVA) in the software GraphPad Prism 8. It was considered statistically significant when the P value was smaller than .05.

| Characterization of AS-nFAp:Gd/Tb
The simplified co-precipitation method to synthesize the AS- With the increasing concentration of doped AS1411, the zeta potential values of AS-nFAp:Gd/Tb were gradually decreased to negative values ( Figure 2C). The particle size of AS1411-free nFAp was optimized by the doping of AS1411, and the particle size of AS-nFAp:Gd/Tb was smaller and tended to be stable when the molar ratio of AS1411 to Ca 2+ was 1/1000, indicating that AS1411 played a crucial role in mediating the growth and the nucleation of nHAp.
In addition, the particle sizes and fluorescence intensity of the prepared AS-nFAp:Gd/Tb at different reaction times were displayed in Figure 2D. Under the reaction time of 6 hours, the particle size of the NPs was the smallest (68.06 nm), followed by the size of the NPs

| Fluorescence properties of AS-nFAp:Gd/Tb
The fluorescent nHAps doped with lanthanide (Ln 3+ ) ions were reported to have a high-performance optical application. 20,41 Due to similar ionic radius and coordination environment of Tb 3+ ions with Ca 2+ , Tb 3+ could be doped into nHAp by replacing Ca 2+ or inserting Ca 2+ vacancies. 42 The PL emission spectrum ( Figure 3B (Table S1)

| In vitro MRI imaging
The Gd 3+ -based systems have been widely designed and characterized as T1-weighted MRI contrast agents in MRI imaging [44][45][46] . Thus, the potential of AS-nFAp:Gd/Tb for MRI imaging was investigated.
The prepared AS-nFAp:Gd/Tb was dissolved in ultrapure water.
Then, the T1-weighted MRI images were evaluated on an MRI system, and the relaxation values were also measured. With the increasing dose of Gd 3+ , a significant dose-dependent colour change and a good linear correlation change in relaxation values were observed ( Figure 3D), proving the good MRI imaging potential of the prepared AS-nFAp:Gd/Tb.

| In vitro fluorescence imaging of cells
The

| Preparation of DOX@AS-nFAp:Gd/Tb
The chemotherapy drug DOX was loaded to AS-nFAp:Gd/Tb to explore the application of the NPs as a drug delivery system. Firstly, capacity of nHAp to DOX. 47 The superior drug loading capacity and pH-induced drug release of DOX@AS-nFAp:Gd/Tb demonstrated the potential of an attractive nanocarrier for drug delivery.

| Cell viability and cell apoptosis analysis
The potential of DOX@AS-nFAp:Gd/Tb for targeted tumour therapy
This indicated excellent biocompatibility of DOX@AS-nFAp:Gd/Tb, which could reduce side effects of DOX.
In addition, the anti-tumour effect of DOX@AS-nFAp:Gd/Tb was further studied by the H&E and TUNEL staining of tumour tissues in 4 groups after 12-day treatments ( Figure 6F)

| CON CLUS IONS
In this study, a co-doped nFAp was successfully synthesized with AS1411 as a template by a one-pot procedure so as to achieve the AS1411-targeted fluorescence/MRI dual-model imaging. In the presence of AS1411, the prepared AS-nFAp:Gd/Tb possessed good monodispersity and excellent fluorescence/MRI imaging properties.
In addition, the chemotherapy drug DOX was loaded on AS-nFAp:Gd/ Tb to construct a multifunctional nano-probe that integrated diag- Overall, the DOX@AS-nFAp:Gd/Tb prepared by the biomimetic strategy showed the outstanding capabilities of recognition and treatment on tumours and thus had a potential clinical application in the future.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

CO N FLI C T S O F I NTE R E S T
No conflict of interest was declared in this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data, supporting the findings of this work, are available from the corresponding author upon reasonable request.