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.

Keywords: AS1411; biomimetic synthesis; drug carriers; dual-model bioimaging; nano-hydroxyapatite.

SCHEME 1

Schematic illustration of AS‐nFAp:Gd/Tb preparation and application

FIGURE 1

Morphology and composition of AS‐nFAp:Gd/Tb. (A), TEM image shows that the synthesized AS‐nFAp: Gd/Tb has a uniform needle‐like morphology; (B), EDS elemental mapping and patterns of AS‐nFAp: Gd/Tb; (C), XPS spectrum of AS‐nFAp:Gd/Tb; (D), XRD spectrum of AS‐nFAp:Gd/Tb and the vertical lines represent the standard diffraction peaks of nHAp (JCPDS 09‐0432)

FIGURE 2

Characterization of AS‐nFAp:Gd/Tb. (A), FTIR spectra of AS‐nFAp:Gd/Tb and AS1411‐free nFAp:Gd/Tb NPs; (B), Electropherograms of AS‐nFAp:Gd/Tb NPs; (C), Zeta potential values and particle sizes of AS‐nFAp:Gd/Tb with different concentration molar ratio of AS1411 to Ca²⁺; (D), The particle sizes and the fluorescence intensity (542 nm) of AS‐nFAp:Gd/Tb NPs under different synthetic times; (E), The fluorescence intensity of AS‐nFAp:Gd/Tb on the 542 nm with different concentration molar ratio of Gd³⁺ ions to Ca²⁺ ions and Tb³⁺ ions to Ca²⁺ ions; F, The fluorescence intensity of AS‐nFAp:Gd/Tb on the 542 nm with different concentration molar ratio of AS1411 to Ca²⁺

FIGURE 3

The fluorescence and MRI properties of the prepared AS‐nFAp:Gd/Tb NPs: (A), Schematic energy level diagram of Tb³⁺ ions; (B), The PL emission spectrum of AS‐nFAp:Gd/Tb NPs excited by 285 nm. Inset: the images of AS‐nFAp:Gd/Tb and AS1411‐free nFAp:Gd/Tb NPs excited by UV‐light at room temperature; (C), The photostability of AS‐nFAp:Gd/Tb NPs, *P < .05, **P < .01, ****P < .0001, *compare with the base time points. Data are shown as mean ± SD (n = 3); (D), The curve of relaxation values and T1‐weighted MRI images (inset) of AS‐nFAp:Gd/Tb at various concentrations in buffer solution.

FIGURE 4

Cellular uptake of AS‐nFAp:Gd/Tb. (A), CLSM images of SCC‐25 and L929 cells after incubated with AS‐nFAp:Gd/Tb at 100 μg/mL for 6 h; (B), CLSM images of SCC‐25 and L929 cells after incubated with AS‐nFAp: Gd/Tb at 100 μg/mL for 12 h; (C), CLSM images of SCC‐25 and L929 cells after incubated with AS1411‐free nFAp:Gd/Tb at 100 μg/mL for 12 h; (D‐F), Statistical analyses of mean optical density in CLSM images. *P < .05, data are shown as mean ± SD (n = 3)

FIGURE 5

Drug loading capacity and cytotoxicity in vitro. (A), The standard dilution curve of DOX at different concentrations, table inserted shows the loading efficiency of DOX in DOX@AS‐nFAp:Gd/Tb; (B), The release profiles of DOX from DOX@AS‐nFAp:Gd/Tb in different pH buffers (n = 3); (C) and (D), The cell viability of L929 and SCC‐25 cells after treated with AS‐nFAp:Gd/Tb, DOX and DOX@AS‐nFAp:Gd/Tb at different concentrations for 24 h (the content of DOX in DOX@AS‐nFAp:Gd/Tb group was equivalent to the pure DOX group). ****P < .0001, ^&&&& P < .0001, * represents different concentrations vs ctrl in DOX group, & represents DOX group vs DOX@AS:nFAp:Gd/Tb at the same concentration, data are shown as mean ± SD (n ≥ 3)

FIGURE 6

The anti‐tumour effect of DOX@AS‐nFAp:Gd/Tb in vivo study. (A), The oral squamous cell carcinoma (OSCC) tumour‐bearing mice model (mice injected with SCC‐25 cells) was constructed to investigate the application of DOX@AS‐nFAp:Gd/Tb in vivo experiments; (B), The photographs of tumour‐bearing mice in different treatment groups (saline, AS‐nFAp:Gd/Tb, DOX and DOX@AS‐nFAp:Gd/Tb) at the first day and the 12th day; (C), Ex vivo tumour images at the 12th day. (D), The curves of tumour volumes during different treatments; (E), The curves of body weights of tumour‐bearing mice during different treatments; (F), The H&E staining of tumour issues and liver, and the TUNEL staining of tumour issues in 4 different groups after 12‐day treatments (The magnification is 20 × 4). *P < .05, ***P < .001, ****P < .0001, ^### P < .001, ^#### P < .0001, ^&& P < .01, * represents DOX group vs saline group at the same day, ^# represents DOX@AS‐nFAp: Gd/Tb group vs saline group at the same day, ^& represents DOX group vs DOX@AS‐nFAp:Gd/Tb group at the same day. Data are shown as mean ± SD (n ≥ 3)