Preparation and properties of magnetic-fluorescent endoglin aptamer nanoprobe

In this study, a kind of magnetic-fluorescent endoglin aptamer nanoprobe, Cy5.5-End-Fe3O4/KCTS, was synthesized with magnetic functions of Fe3O4/KCTS nanoparticles and the fluorescence ability of Cy5.5 through SMCC cross-linking with endoglin aptamer labeled with Cy5.5. The microstructure, fluorescence and magnetic properties of the samples were investigated and discussed by Fourier transform-infrared (FT-IR) spectra, X-ray diffraction (XRD), transmission electron microscopy (TEM), photoluminescence spectra, dynamic light scattering (DLS) and Zeta potential. The magnetic-fluorescent aptamer nanoprobe has an average particle size of about 240 nm, Zeta potential of -8.9 mV, excellent fluorescence and magnetic properties. This study provides a new MRI/fluorescent bimodal molecular probe for early diagnosis of hepatocellular carcinoma.


Introduction
Hepatocellular carcinoma (HCC) is a common and highly malignant tumor, has been the third cause of cancer death and the leading cause of mortality due to its difficult to diagnosis at the onset [1]. Therefore, early diagnosis and timely treatment is a key for improving the prognosis and cure rate of patients. Magnetic resonance imaging (MRI) is non-invasive early diagnosis of HCC and widely used for imaging liver lesions, valuable in tumor molecular imaging for its soft tissue resolution, unlimited image depth, multi-parameter imaging, and lack of radioactivity [2,3]. Magnetic nanoparticles (Fe 3 O 4 NPs) are common MRI contrast agents that significantly reduce T2 relaxation times, thereby darkening the T2-weighted image signal [4][5][6].
To date, increasing attention has been paid to the fabrication of bifunctional nanoprobe consisting of discrete functions [7][8][9]. More importantly, a nanoprobe integrating both fluorescent and magnetic properties can be used in cell separation, biological labeling and MRI/fluorescent bimodal molecular imaging, because they possess both magnetic and fluorescent properties that can be traced and visualized [8,10].
Endoglin (also known as CD105), an auxiliary receptor component of the transforming growth factor beta signalling pathway, is expressed mainly by endothelial cells and has been found to be involved in angiogenesis and vascular remodeling [11,12]. The potential application of Endoglin in the diagnosis and treatment of HCC has become important [13]. Aptamer is a short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) molecules screened by systematic evolution of ligands by exponential enrichment (SELEX) and hold promise for applications in disease diagnosis and targeted therapy fields as a potential candidate for biomolecular recognition [14,15].
In previous studies, we screened for an aptamer that specifically binds to endoglin molecules on murine neovascular endothelial cells. On the basis, a specific MRI/fluorescent imaging endoglin aptamer nanoprobe (Cy5.5-End-Fe 3 O 4 /KCTS) was constructed using endoglin aptamers as recognition molecules in this paper. The physical and chemical properties of Cy5.5-End-Fe 3 O 4 /KCTS were verified. The fluorescence-encoded magnetic nanoparticles, with the magnetic functions of Fe 3 O 4 /KCTS nanoparticles and fluorescence ability of Cy5.5, can simultaneously realize two functions of magnetic resonance imaging and fluorescence labeling. We believe that such a competitive multimodality probe has the potential to challenge some met limitations in biomedical area.

Preparation of Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe
The magnetic Fe 3 O 4 /KCTS nanoparticles were prepared according to our previous reported method with Fe 3 O 4 nanoparticles as the magnetic core and chitosan alpha-ketoglutaric acid (KCTS) as the basic skeleton through carbodiimide activation [16]. The Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe was prepared by sulfo-SMCC cross-linking with endoglin aptamer labeled with Cy5.5 as the target molecule. Firstly, Fe 3 O 4 /KCTS NPs was dissolved in HEPES buffer( pH= 7.2) to form 1.0 mg/mL Fe 3 O 4 /KCTS suspends, and 100 μL sulfo-SMCC (5.0 mg/ml) was added and incubated at 25 °C for 30min. After magnetic separation, 10 μL endoglin aptamer labeled with Cy5.5 (10 μmol/L) were added and mixed, and incubated at 25 °C for 2 h. Then, 100 μL of 1% bovine serum albumin was added for 30 min. Lastly, the Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe was obtained by magnetic separation after washed 2-3 times with HEPES buffers. The prepared nanoprobe was resuspended with 1 mL of 0.01% NaN 3 and 1% bovine serum albumin buffer and stored at 4 °C for further use. The conjugation rate is calculated according to the following equation.
Where I 0 was the fluorescence intensity of Cy5.5-End and I 1 was the fluorescence intensity of Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe.

Optimization of experimental conditions
The properties of the Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe were influenced by various formulation and process variables like the concentration of aptamer, concentration of cross-linking agent, incubation time, temperature and so on. Fig. 2 presented the result of factor effects on the conjugation rate. It could be seen from Fig. 2A that different aptamer concentration had important effects on the conjugation rate. When aptamer concentration was 0.2μmol/L, the conjugation rate was the highest. Moreover, the conjugation rate increased with the increase of the SMCC concentration and reached the highest value (79.6%) when the SMCC concentration was 1.25 mg/mL (Fig. 2B). In Fig. 2C, it could be founded that conjugation rate increased markedly with the increase of incubation time from 0.5 h to 2 h. Over 2 h, conjugation rate decreased sharply. Fig. 2D indicated that conjugation rate increased with the increase of incubation temperature, and reached the peak at 25 °C, and then dropped from 25 to 60 °C. Therefore, the maximum conjugation rate of 84.8% was achieved at 25 °C when the endoglin aptamer concentration was 0.2 μmol/L, the SMCC concentration was 1.25 mg/mL and an incubation time of 2 h was employed. The peak around 3431 cm −1 related to the -OH group and the peak at 588 cm -1 or 586 cm -1 related to Fe-O group. The FT-IR spectrum (Fig.3A, curve b)  TEM images showed that the Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe nearly spherical in shape and the average diameter was 35 nm (Fig. 3C). Size distribution was determined by DLS (Fig. 3D) in aqueous solution, the size of the Cy5.5-End-Fe 3 O 4 /KCTS nanoprobe was in the range from 190 to 295 nm and the mean diameter was 240 nm, which was bigger than determined by TEM image, presumably arising from the dry state of the TEM measurement. The Zeta potential of the Cy5.5-End-Fe 3 O 4 /KCTS probe was approximately -8.93 mV, while the Fe 3 O 4 /KCTS were approximately +17.01mV (Fig. 3E).

Conclusions
In summary, magnetic-fluorescent endoglin aptamer nanoprobe, Cy5.5-End-Fe 3 O 4 /KCTS, was successfully prepared with the fluorescence ability of Cy5.5 and magnetic functions of Fe 3 O 4 /KCTS nanoparticles. The maximum Cy5.5-End aptamer's conjugation rate achieved 84.8% at 25 °C when the aptamer concentration was 0.2 μmol/L, the SMCC concentration was 1.25 mg/mL and incubation time was 2 h. The microstructure, fluorescence and magnetic properties of the samples were characterized by FTIR, XRD, TEM, DLS, Zeta potential and fluorescence. The magnetic-fluorescent aptamer nanoprobe has an average particle size of about 240 nm, Zeat potential of -8.9 mV, excellent