CN111658786A - Molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of medical detection, in particular to a molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer and a preparation method thereof. The probe is prepared by coupling the aptamer A10-3.2 with manganese-zinc ferrite nanoparticles. The APT10-3.2-MZF-NPs molecular probe constructed in the present invention has a specific targeting effect on prostate cancer LNCaP cells, can be detected by MR, and is expected to be a new type of targeted contrast agent for MR molecular imaging.

Description

A molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer and its Preparation

technical field

The invention belongs to the field of medical detection, in particular to a molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer and a preparation method thereof.

Background technique

Prostate cancer (PCa) is a urological malignancy with a very high incidence in European and American men. Although the incidence of prostate cancer in my country is lower than that of developed countries in Europe and the United States, in recent years, due to changes in lifestyle and the aging of the population, the incidence of prostate cancer is increasing year by year, and there is a trend of younger age. The incidence of prostate cancer is insidious, and the existing detection methods lack high sensitivity and specificity. Many patients have reached the advanced or advanced stage at the initial treatment, thus missing the optimal treatment time. Androgen deprivation therapy (ADT) is a classic treatment for advanced prostate cancer, but most patients will gradually develop castration-resistant prostate cancer after treatment. Patients at this stage lack effective treatments and have a very poor prognosis. Therefore, it is of great practical significance to explore new methods for the effective diagnosis of prostate cancer.

Magnetic resonance imaging (MRI), as a non-invasive examination, has no ionizing radiation, can perform multi-parameter and multi-sequence imaging, and has good soft tissue resolution. It has now become one of the main examination methods for prostate diseases. Among them, DCE-MRI , DWI, MRS and other inspection sequences are used to evaluate prostate cancer from the aspects of hemodynamics, water molecule diffusion and material metabolism. Existing MRI technology plays an increasingly important role in the diagnosis, grading and follow-up of prostate cancer, but it still lacks high sensitivity for the diagnosis of early-stage prostate cancer, especially low-grade and small-sized prostate cancer. In addition, gadolinium-based T1 relaxation contrast agents (such as Gd-DTPA) are currently the most widely used clinically. Although these contrast agents can enhance the contrast between tissues, they lack diagnostic specificity and circulate in the body. The time is short, thus limiting its application in tumor diagnosis.

The rapid development of nanotechnology has brought new opportunities for the diagnosis and treatment of tumors. Among them, magnetic nanoparticles (MNPs) combine the advantages of magnetic particles and nanoparticles, and their special physicochemical properties, magnetic properties and good biocompatibility make them widely concerned in the field of biomedicine. Because of its low toxicity, small particle size, unique superparamagnetic properties and high magnetic susceptibility, MNPs have better performance as MRI contrast agents (Groult H, Poupard N, Herranz F, et al. Family of Bioactive Heparin-Coated Iron Oxide). Nanoparticles with Positive Contrast in Magnetic Resonance Imaging for Specific Biomedical Applications [J]. Biomacromolecules, 2017, 18: 3156-3167). MNPs are highly modifiable, and the modified nanoparticles not only have greatly improved stability, but are also endowed with more diverse functions. For example, MNPs linked to different ligands can bind to specific molecules on the tumor surface and accumulate in tumor cells or tissues as MRI contrast via RES phagocytosis, EPR effect, and receptor-ligand-specific binding reactions in vivo. The agent can achieve the purpose of diagnosis (Hu Ping. Surface modification of nano Fe3O4 magnetic particles and its application in the fields of medicine and environmental protection [J]. Chinese Journal of Chemical Industry, 2017,68(7):2641-2652).

PSMA is an intrinsic protein of prostate epithelial cell membrane, which is highly expressed in prostate cancer tissue, and its expression increases with tumor invasion, metastasis and recurrence, with high sensitivity (65.9%) and specificity (94.5%). Its expression does not depend on the level of androgen in the body, which is quite meaningful for patients who have transformed into androgen-resistant prostate cancer after endocrine therapy (Kiess AP, Banerjee SR, Mease RC, et al.Prostate-specific membrane antigen as a target for Cancer imaging and therapy[J].Q J Nucl Med Mol Imaging, 2015, 59: 241-268). Therefore, PSMA is considered to be an ideal target for prostate cancer imaging and therapy.

Nucleic acid aptamers (aptamers) are single-stranded oligonucleotide molecules screened by SELEX system that can specifically and efficiently bind to various target molecules. Among them, A10-3.2, the specific sequence is: "5'-GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCU-spacer-NH2-3'with 2'-fluoro pyrimidines(Wu M,Wang Y,Wang Y,etal.Paclitaxel-loaded and A10-3.2 aptamer-targeted poly(lactide-co-glycolicacid) nanobubbles for ultrasound imaging and therapy of prostate cancer[J]. IntJ Nanomedicine, 2017, 12:5313-5330) is an RNA aptamer that can specifically bind to PSMA. Compared with other targeting ligands The aptamer or carrier has many advantages such as no immunogenicity, high targeting specificity, small molecular weight, cheap and easy to obtain, etc. The nucleic acid aptamer is used as a targeted modification ligand to increase the localization of the drug or contrast agent in the tumor. It has excellent application prospects in the targeted diagnosis and targeted therapy of prostate cancer.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a molecular probe of prostate cancer.

The present invention provides a molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer, which is prepared by coupling aptamer A10-3.2 and manganese-zinc ferrite nanoparticles.

The present invention also provides a method for preparing the molecular probe APT10-3.2-MZF-NPs, which is to first prepare manganese-zinc ferrite nanoparticles PEG-MZF-NPs and aptamer A10-3.2, and then use EDC/NHS chemical APT10-3.2 was coupled with PEG-MZF-NPs by cross-linking method to construct APT10-3.2-MZF-NPs molecular probe.

The present invention adopts EDC/NHS chemical cross-linking method to modify the A10-3.2 nucleic acid aptamer that can specifically bind to prostate cancer specific membrane antigen (PSMA) on the prepared PEG-MZF-NPs to obtain targeting molecules probe. According to the DLS measurement results, combined with TEM observation, it can be seen that the hydrodynamic size of the nanoparticles increased slightly before and after APT coupling, while the morphology and dispersion did not change significantly. -Conjugation of NPs and APT, the coupling rate was 89.8%. In order to observe and analyze the cell-specific targeting uptake ability of APT10-3.2-MZF-NPs and explore its potential as a targeted contrast agent for MRI, we used PEG-MZF-NPs and APT10-3.2-MZF-NPs with the same iron concentration. After co-incubating with prostate cancer LNCaP cells (PSMA ⁺ ) and PC-3 cells (PSMA ^- ), Prussian blue staining and MRI T2WI scanning were performed. The results showed that both cells could uptake PEG-MZF-NPs and APT10-3.2 -MZF-NPs, but the uptake of APT10-3.2-MZF-NPs by LNCaP cells was significantly higher than that of PEG-MZF-NPs, which showed that a large number of blue iron particles were deposited in the cells under the microscope; The uptake capacity of MZF-NPs was also significantly stronger than that of PC-3 cells; PC-3 cells had no significant difference in uptake between the two. MRI in vitro imaging experiments showed that the inhibitory effect of APT10-3.2-MZF-NPs on the T2 signal of LNCaP cells was significantly stronger than that of PC-3 cells; the inhibitory effect of APT10-3.2-MZF-NPs on the T2 signal of LNCaP cells was significantly stronger than that of PEG-MZF- NPs, while APT10-3.2-MZF-NPs and PEG-MZF-NPs had no significant difference in the inhibition of T2 signaling in PC-3 cells. Thus, it was confirmed that APT10-3.2-MZF-NPs had certain targeting to prostate cancer LNCaP cells, which could be detected by MRI.

The APT10-3.2-MZF-NPs molecular probe constructed in the present invention has a specific targeting effect on prostate cancer LNCaP cells, can be detected by MR, and is expected to become a new type of targeted contrast agent for MR molecular imaging.

Description of drawings

Figure 1 shows the TEM photo of APT10-3.2-MZF-NPs

Figure 2: a-d are the particle size and Zeta potential analysis of PEG-MZF-NPs and APT10-3.2-MZF-NPs, respectively: a, b are the particle size and Zeta characterization of PEG-MZF-NPs, respectively; c, d are APT10 -3.2 Particle size and Zeta characterization of MZF-NPs

Figure 3: a-d are Prussian blue staining images of PEG-MZF-NPs and APT10-3.2-MZF-NPs co-incubated with PC-3 and LNCaP cells, respectively: a is LNCaP cells co-incubated with PEG-MZF-NPs; b is LNCaP Cells co-incubated with APT10-3.2-MZF-NPs; c is PC-3 cells co-incubated with PEG-MZF-NPs; d is PC-3 cells co-incubated with APT10-3.2-MZF-NPs

Figure 4 shows the magnetic resonance scan results of PEG-MZF-NPs and APT10-3.2-MZF-NPs after co-incubating with PC-3 and LNCaP cells: a. The measured T2-weighted image; b. The degree of T2 signal inhibition

Detailed ways

1. Preparation, characterization and targeting of APT10-3.2-MZF-NPs

1. Preparation and characterization of APT10-3.2-MZF-NPs

1) Preparation of PEG-MZF-NPs

Refer to the literature (Guo Ting. In vitro experimental study of shRNA/DDP magnetic nanoliposomes based on MFH technology for targeted diagnosis and treatment of ovarian cancer [M]: [Master's thesis], Nantong: Nantong University, 2016) High temperature thermal decomposition method , to prepare PEG-MZF-NPs;

2) Preparation of aptamer A10-3.2

According to the literature (Wu M, Wang Y, Wang Y, et al. Paclitaxel-loaded and A10-3.2aptamer-targeted poly(lactide-co-glycolic acid) nanobubbles for ultrasoundimaging and therapy of prostate cancer[J]. Int J Nanomedicine, 2017, 12:5313-5330) in the nucleic acid sequence of A10-3.2, to prepare the aptamer A10-3.2.

3) Preparation of APT10-3.2-MZF-NPs

Pipette 5 mL of the prepared PEGylated MZF-NPs (Fe: 1 mg/ml) sample, mix it with EDC (500 μl) and NHS (500 μl), and place it on a shaker to shake for 30 min to ensure the PEGylated MZF-NPs The free -COOH at the surface end is fully activated. Then ultrafiltration centrifugation (10KDa), and deionized water washing (3mL×5 times) to obtain activated magnetic nanocrystals. The RNA aptamer A10-3.2 (10OD) was denatured at 85°C and then renatured in an ice bath for 10min. The activated nanoparticles and the treated aptamers were shaken at room temperature for 10 h. After repeated ultrafiltration centrifugation and deionized water washing, the aptamer-modified magnetic nanocrystal (APT10-3.2-MZF-NPs) solution was finally obtained until no RNA aptamer could be detected in the filtrate.

4) The morphology of APT10-3.2-MZF-NPs was observed by transmission electron microscope (TEM).

An appropriate amount of anhydrous ethanol was added to APT10-3.2-MZF-NPs, and after ultrasonic dispersion, it was added dropwise to the coated copper mesh, and the TecnaiG20 transmission electron microscope was used for observation and analysis.

5) Dynamic light scattering (DLS) analysis of PEG-MZF-NPs hydrodynamic particle size and Zeta potential before and after RNA aptamer modification

Pipette 1 mL each of the prepared PEG-MZF-NPs and APT10-3.2-MZF-NPs nanomaterials, and place them in a quartz dish for particle size measurement and a measuring cup for Zeta potential measurement, and measure the particle size under a dynamic light scattering instrument. and Zeta potential.

6) Detection of MZF nanoparticles (PEG-MZF-NPs and APT10-3.2-MZF-NPs) before and after A10-3.2 aptamer modification by UV-Vis spectrophotometer

Since the conjugated double bonds of base molecules in nucleic acids have the property of absorbing ultraviolet light, their maximum absorption peak is at the wavelength of 260 nm, so in this experiment, a UV-visible spectrophotometer was used to measure the MZF nanoparticles before and after aptamer modification. To determine whether the RNA aptamer A10-3.2 was successfully coupled to the nanomaterial surface. 2. APT10-3.2-MZF-NPs targeting study 1) Prussian blue staining experiment

Cell culture: Prostate cancer LNCaP cells (PSMA ⁺ ) were inoculated in RPMI-1640 medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin), and prostate cancer PC-3 cells (PSMA ^- ) were inoculated In the F-12K medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin), the cells were routinely cultured in an incubator at 37°C and a saturated humidity of 5% CO ₂ , so that the cells were in an adherent state. When the cells are confluent (up to 85%-95%), they are digested with 0.25% trypsin, and passaged every 2 to 3 days, and cells in logarithmic growth phase are selected for the experiment.

Operation steps: Dilute APT10-3.2-MZF-NPs and PEG-MZF-NPs with the corresponding cell culture medium to 70 μg/ml, and incubate with prostate cancer LNCaP and PC-3 cells for 12 h respectively; remove the medium and wash with PBS for 3 1 mL of 4% paraformaldehyde fixative solution was added for 30 min; PBS buffer was added to wash cells for 3 times, and 1 mL of Prussian blue staining solution was added for staining for 10 min (staining magnetic nanomaterials); PBS buffer was added to wash cells 3 times, and nuclei were added. The cells were stained with fast red solution for 5 min (staining nuclei); after color development, they were washed 3 times with PBS and observed under a light microscope.

2) In vitro MRI molecular imaging

PC-3 cells and LNCaP cells in logarithmic growth phase were inoculated in 6-well culture plates (2×10 ⁶ /well), at 37°C, 5% CO ₂ saturated humidity overnight, and after adding equal concentration sterilization The PEG-MZF-NPs and APT-PEG-MZF-NPs of the control group were added with PBS and placed in an incubator. Cells were collected and washed, dispersed and fixed with agarose, and then placed in Siemens 3.0 TMR for imaging. Scanning parameters: field of view (FOV): 160mm×160mm; matrix: 384×384; slice thickness: 1.0mm; TE: 80ms; TR: 2000ms; scanning time: 15min.

3. Statistical analysis

的形式表示；呈现正态分布特点的计量资料，采用t检验及单因素方差分析，不符合正态分布的采用非参数检验。Statistical analysis was performed using SPSS 22.0 software, with mean ± standard deviation

For the measurement data showing the characteristics of normal distribution, t test and one-way analysis of variance were used, and non-parametric tests were used for those that did not conform to the normal distribution.

1. Preparation and characterization of APT10-3.2-MZF-NPs

1) Transmission electron microscopy (TEM) observation of APT10-3.2-MZF-NPs On the basis of the prepared PEG-MZF-NPs, APT10-3.2-MZF-NPs were prepared by coupling APT by EDC/NHS chemical cross-linking method. TEM The results show that the size is uniform and the dispersion is good, about 10 nm. (see picture 1)

2) Dynamic light scattering (DLS) measurement results

The hydrodynamic average particle size of PEG-MZF-NPs is 56.55nm, the potential is -32.5mV, and the dispersion coefficient (PDI) is 0.188; the hydrodynamic average particle size of APT10-3.2-MZF-NPs is 63.12nm, the potential is -38.3mV, and the dispersion coefficient is 63.12nm. (PDI) was 0.23. Combined with the observation of transmission electron microscope, it can be seen that the size of the material increases slightly before and after the nucleic acid aptamer (APT) coupling. The morphology and dispersibility did not change significantly, showing good monodisperse properties. The Zeta potential changed from -32.5mV to -38.3mV. These experimental results all confirmed the successful modification of the electronegative RNA aptamer. (Fig. 2a-d)

3) 260nm absorbance test results

The material was measured by UV-visible spectrophotometer, the results showed that: A10-3.2 aptamer had an obvious RNA characteristic absorption peak at 260nm, while APT10-3.2-MZF-NPss modified aptamer even after ultrafiltration There is still an absorption peak there, which confirms that the A10-3.2 aptamer has been successfully coupled to the nanoparticle surface, and its activity is not affected, and the coupling rate is 89.8% (Table 1). Coupling rate (%)=(absorption value at 260 nm before coupling-absorption value at 260 nm after coupling)/absorption value at 260 nm before coupling*100%.

Table 1 Absorbance at 260 nm and coupling rate before and after RNA aptamer coupling

2. Targeting study of APT10-3.2-MZF-NPs

1) Prussian blue staining results

Prostate cancer LNCaP and prostate cancer PC-3 cells were sequentially stained with Prussian blue and nuclear fast red according to the experimental procedure of 2.271. The experimental results are shown in Figure 3a-d: manganese-zinc ferrite nanoparticles before and after aptamer modification (PEG-MZF-NPs, APT10-3.2-MZF-NPs) were co-incubated with LNCaP cells and PC-3 cells, respectively, the PEG-MZF-NPs/PC-3, APT10-3.2-MZF-NPs/PC-3 groups There were no obvious iron particles deposition in the cells of APT10-3.2-MZF-NPs/LNCaP group, which was significantly more than that in PEG-MZF-NPs/LNCaP group and PEG-MZF-NPs/LNCaP group. PC-3 group, APT10-3.2-MZF-NPs/PC-3 group. It was suggested that the coupling of APT to PEG-MZF-NPs enhanced the specific uptake of magnetic materials by PSMA-positive LNCaP cells.

2) MRI molecular imaging results

PEG-MZF-NPs and APT10-3.2-MZF-NPs were co-incubated with prostate cancer LNCaP cells and PC-3 cells and collected, and then MR scanning was performed. The T ₂ WI signal of the two groups has a certain degree of inhibition. Observation between groups showed that the inhibitory effect of APT10-3.2-MZF-NPs on the T2 signal of LNCaP cells was significantly stronger than that of PC-3 cells; the observation within the group showed that the inhibitory effect of APT10-3.2-MZF-NPs on the T2 signal of LNCaP cells was significantly stronger Compared with PEG-MZF-NPs, APT10-3.2-MZF-NPs and PEG-MZF-NPs had no significant difference in the inhibitory effect of T2 signal on PC-3 cells, which indicated that APT10-3.2-MZF-NPs could inhibit the high expression of PSMA. Prostate cancer LNCaP cells have a certain targeting effect and can be detected by MR (Fig. 4a-b).

Claims (2)

1. A molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer, characterized in that the probe is prepared by coupling aptamer A10-3.2 with manganese-zinc ferrite nanoparticles. 2. A method for preparing molecular probe APT10-3.2-MZF-NPs for diagnosing prostate cancer, characterized in that, firstly, manganese-zinc ferrite nanoparticles PEG-MZF-NPs and aptamer A10-3.2 are obtained Then, APT10-3.2 was coupled with PEG-MZF-NPs by EDC/NHS chemical cross-linking method to construct APT10-3.2-MZF-NPs molecular probe.

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