Nano-liposome probe as well as preparation method and application thereof

Abstract

The invention discloses a nano-liposome probe as well as a preparation method and application thereof. Specifically, the nanoliposome probe is targeted to mitochondria, and comprises an antibody targeting to ALDH2 and an antibody targeting to FOLR1. The nano-liposome probe disclosed by the invention can realize noninvasive, accurate and efficient identification and judgment of tumor cells with high invasion and metastasis potentials.

Description

Technical Field

[0001] The invention belongs to the detection field, and specifically relates to a nano liposome probe and a preparation method and application thereof, which can be used for the preparation of tumor in vivo probe experimental materials. Background Art

[0002] Breast cancer is the malignant tumor with the highest incidence rate in women, and its local recurrence and distant metastasis are the main causes of death in patients. If the invasion and metastasis of breast cancer can be accurately diagnosed in vivo and the clinical treatment can be guided accurately, it will be able to effectively improve the quality of life of patients and prolong their survival time. Although previous studies were able to predict the benign and malignant nature of tumors through molecular imaging technology, their metastatic potential could not be determined. This field urgently needs to image tumor metastasis seed cells with metastatic potential in vivo, which will not only enable molecular imaging technology to accurately predict the range and metastasis of the tumor, but also guide precise surgery and prognosis prediction. The prior art urgently needs a probe / method that can efficiently detect tumor cells with high metastatic potential. Summary of the invention

[0003] In view of the lack of probes for efficiently detecting tumor cells with high metastatic potential in the prior art, the present invention provides a nanoliposome probe and its preparation method and application. Specifically, a nanoliposome probe ALDH2-Cy7@LP-FA is designed and synthesized with the high metastatic molecule ALDH2 as the core, which can realize non-invasive, accurate and efficient identification and judgment of tumor cells with high invasion and metastasis potential.

[0004] In order to solve the defects in the prior art, the present invention provides a nanoliposome probe in a first aspect. The nanoliposome probe targets mitochondria and comprises an antibody targeting ALDH2 and an antibody targeting FOLR1.

[0005] In certain embodiments, the antibody targeting ALDH2 is in a liposome, and the antibody targeting FOLR1 is on the cell membrane surface.

[0006] In certain embodiments, the liposomes include the following components: DOTAP, DOPC, Chol and DSPE-PEG2k-folate.

[0007] The second aspect of the present invention provides a method for preparing the nanoliposome probe according to the present invention, the method comprising: mixing ALDH2-Cy7 with the nanoliposome probe LP-FA to prepare the nanoliposome probe ALDH2-Cy7@LP-FA.

[0008] In some embodiments, the method further comprises (a) synthesizing ALDH2-Cy7 and (b) synthesizing nanoliposome probe LP-FA. Preferably, the method (a) comprises: mixing Cy7 and ALDH2 in proportion, adding a catalyst and a solvent to mix, and washing to obtain the probe ALDH2-Cy7.

[0009] In certain embodiments, the (b) comprises: preparing LP-FA by a thin film hydration method.

[0010] Preferably, the step (b) comprises: mixing a chloroform / methanol solution containing DOTAP, DOPC, Chol and DSPE-PEG2k-folate, decompressing the mixture, mixing the mixture with a buffer solution and extruding the mixture through a liposome extruder.

[0011] More preferably, the concentration of DOTAP is 0.23 mg / mL,

[0012] The concentration of DOPC is 3.3 mg / mL,

[0013] The concentration of Chol is 0.775 mg / mL,

[0014] The concentration of the DSPE-PEG2k-folate is 0.215 mg / mL,

[0015] The volume ratio of chloroform to methanol in the chloroform / methanol is 9:1,

[0016] The buffer is Tris buffer with a pH of 9.

[0017] In certain embodiments, the method comprises:

[0018] The ALDH2-Cy7 is dissolved in methanol and mixed with the LP-FA in proportion for encapsulation and ultrafiltration.

[0019] Preferably, the mass volume ratio of the ALDH2-Cy7 solution to the methanol is 1:5.

[0020] The encapsulation uses water bath ultrasound,

[0021] The molecular weight cut-off of the ultrafiltration was 10 kDa.

[0022] The third aspect of the present invention provides use of the nanoliposome probe according to the present invention in the preparation of a diagnostic agent.

[0023] In certain embodiments, the diagnostic agent is a tumor diagnostic agent. Preferably, the tumor is a breast cancer tumor. More preferably, the tumor is a highly metastatic breast cancer tumor.

[0024] A fourth aspect of the present invention provides a method for detecting cancer cells.

[0025] Preferably, the method uses the nanoliposome probe as described in the present invention to detect whether the cancer cells are highly metastatic cancer cells, and the method is for non-diagnostic purposes.

[0026] More preferably, the cancer cells are breast cancer cells.

[0027] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0028] The reagents and raw materials used in the present invention are commercially available.

[0029] The positive progressive effect of the present invention is that the mitochondrial targeted nanoliposome probe ALDH2-Cy7@LP-FA provided by the present invention has a unique structure and tumor recognition potential, can evaluate the expression and distribution of tumor cells, can be used for the diagnosis of highly metastatic breast tumors, and can achieve non-invasive, accurate and efficient identification and judgment of tumor cells with high invasion and metastasis potential, providing a key basis for the formulation of precise treatment plans and individual prognosis prediction. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the synthesis route of nanoliposome probes.

[0031] Figure 2 Characterization of nanoliposome probes.

[0032] Figure 3 The expression of ALDH2 protein in highly invasive breast cancer and adjacent tissues.

[0033] Figure 4 Nanoliposome probes can identify breast cancer cells with high metastatic potential.

[0034] Figure 5 Nanoliposome probes recognize different types of breast cancer cells.

[0035] Figure 6 Nanoliposome probes were used to identify breast cancer cells that down-express ALDH2.

[0036] Figure 7 The sensitivity and specificity of nanoliposome probes in identifying breast cancer cells.

[0037] Figure 8 In vivo recognition of highly metastatic breast cancer cells by nanoliposome probes.

[0038] Fig. 9 This paper presents the application of nanoliposome probes in surgical navigation in nude mice.

[0039] Fig.10 This is the toxicity of the nanoliposome probe to various organs of nude mice. DETAILED DESCRIPTION

[0040] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0041] The supplier of ALDH2 antibody is abcam, model number is 133306; the supplier of FolR1, i.e. FA antibody (hereinafter referred to as FA), is ABclonal, model number is A15672.

[0042] Example 1 Synthesis of ALDH2-Cy7@LP-FA near-infrared fluorescent probe

[0043] (a) Synthesis of ALDH2-Cy7 probe

[0044] First, Cy7 was prepared into a 1 mg / mL solution with DMSO, and then Cy7 and ALDH2 were added into a 2 mL EP tube at a ratio of 20:1, TEA was used as a catalyst, DMSO was used as a solvent, a magnetic stirrer was added and stirred at room temperature for 24 h, and then washed with ether three times (1.5 mL × 3) to obtain a blue solid ALDH2-Cy7 ( Figure 1 A).

[0045] (b) Preparation of Nanoliposome LP-FA

[0046] LP-FA was prepared by thin film hydration method. First, a chloroform / methanol (v / v, 9 / 1) solution (total volume 3 mL) of lipid DOTAP (25 mg / mL, 28 μL), DOPC (25 mg / mL, 396 μL), Chol (25 mg / mL, 93 μL), and DSPE-PEG2k-folate (25 mg / mL, 25.8 μL) was added to a 100 mL pear-shaped flask, and the organic solvent was removed by vacuum distillation to form a lipid film. The formed lipid film was placed under vacuum overnight to remove the residual organic solvent. Next, the lipid film was hydrated with 4 mL Tris buffer (pH = 9) at 30°C for 1 h and vortexed, and then extruded 10 times through a 100 nm polycarbonate membrane to obtain nanoliposome LP-FA.

[0047] (c) Preparation of nanoliposome probes Cy7@LP-FA (as a control) and ALDH2-Cy7@LP-FA

[0048] (c-1) Cy7 solid was dissolved in methanol to form a 30 mg / mL solution. Cy7 and LP-FA were added to a 2 mL EP tube at a volume ratio of 1:5. The solution was encapsulated by ultrasonication in a water bath at room temperature for 20 min. After the ultrasonication, the Cy7@LP-FA liposomes were ultrafiltered three times using an ultrafiltration tube (molecular weight cutoff: 10 kDa) to remove the unencapsulated dye, thereby obtaining the nanoliposome probe Cy7@LP-FA, which was then stored in a refrigerator at 4°C.

[0049] (c-2) The ALDH2-Cy7 solid was dissolved in methanol to form a 30 mg / mL solution, and ALDH2-Cy7 and LP-FA were added to a 2 mL EP tube at a volume ratio of 1:5. The liposomes were encapsulated by ultrasonication in a water bath at room temperature for 20 min. After the ultrasonication, the ALDH2-Cy7@LP-FA liposomes were ultrafiltered three times using an ultrafiltration tube (molecular weight cutoff: 10 kDa) to remove the unencapsulated dye, and then stored in a refrigerator at 4°C. Figure 1 B).

[0050] Example 2 Performance measurement of nanoliposome probe

[0051] 2.1 Determination of liposome encapsulation efficiency

[0052] Cy7 dye encapsulation efficiency determination: The ultraviolet absorption (750 nm) of methanol solution of Cy7 dye at concentrations of 1, 1.5, 2, 2.5, 3, 3.5, and 4 μM was measured to draw a standard curve of Cy7 concentration and ultraviolet absorption intensity (y = 0.2841x + 0.0181, R 2 =0.9955). Take a small amount of prepared Cy7@LP-FA and ALDH2-Cy7@LP-FA, add 9 times the volume of anhydrous methanol to break the membrane, mix thoroughly, and measure the ultraviolet absorption value at 750nm. The concentration of encapsulated Cy7 is calculated according to the standard curve. Finally, according to "encapsulation rate = actual encapsulated dye concentration / total dye concentration", the encapsulation rate of Cy7@LP-FA and ALDH2-Cy7@LP-FA is calculated.

[0053] 2.2 Characterization of basic properties of liposomes

[0054] Take 20 μL of the prepared Cy7@LP-FA and ALDH2-Cy7@LP-FA solution in 1 mL of PBS solution, and measure the particle size by dynamic light scattering (DLS, Malvern ZEN3690). Measure three times and record the particle size results each time ( Figure 2 A and Figure 2C). Then, 20 μL of the prepared Cy7@LP-FA and the ALDH2-Cy7@LP-FA solution were dispersed in a solvent containing 5% D-glucose, and the solution was adjusted to different pH values ​​(pH=6, pH=7, pH=9). The Zeta potential was measured by a nanoparticle size potential analyzer, and the measurement was performed three times and the results were recorded ( Figure 2 The encapsulation efficiencies of Cy7@LP-FA and ALDH2-Cy7@LP-FA were calculated to be 66.7% and 61.2%, respectively ( Figure 2 D).

[0055] 2.3 Spectral performance test

[0056] First, take a small amount of fluorescent probes Cy7, ALDH2-Cy7, and ALDH2-Cy7@LP-FA, and then use PBS, water, and methanol (1xPBS buffer: methanol = 1000: 1) to prepare a working solution with a working concentration of 5 μM. Absorption spectrum measurement (Edinburgh Instruments, FLS1000): Set the scanning wavelength range to 500-900nm, the scanning interval to 1nm, and perform baseline scanning with the solvent of the above solution, then perform sample scanning, read the absorption wavelength and absorbance value, and save the scanning data. Emission spectrum measurement: Set the excitation light source to a xenon lamp, the detector to a NIR PMT, the excitation and emission slits to 2nm, the excitation wavelength to the maximum absorption wavelength, and the scanning wavelength range to (Ex+20)-900. The working solution was placed in a sample pool for scanning, the emission wavelength and fluorescence intensity were read, and the scanning data was saved. The UV absorption spectrum and fluorescence emission spectrum of ALDH2-Cy7@LP-FA were characterized. The results showed that the liposome encapsulation caused the UV absorption and fluorescence emission of the labeled antibody to blue shift by 4nm and 5nm, respectively. The spectral properties showed that Cy7 successfully labeled the ALDH2 antibody, the liposome encapsulation achieved the antibody nano-sizing, and had little effect on the spectral properties of the labeled antibody ( Figure 2 E and Figure 2 F).

[0057] Example 3 Detection of breast cancer cells with high metastatic potential using ALDH2-Cy7@LP-FA near-infrared fluorescent probe

[0058] Immunohistochemistry was used to detect the protein expression level of ALDH2 in highly invasive tumors and adjacent tissues, and it was found that ALDH2 was highly expressed in highly invasive breast cancer tissues ( Figure 3 ). Immunoblotting experiments were performed to detect the expression levels of ALDH2 protein in 6 wild-type breast cancer cell lines. It was found that ALDH2 was highly expressed in BT474 cells (human breast ductal carcinoma cells) and lowly expressed in normal breast epithelial MCF-10A cells (human normal breast epithelial cells). Figure 4A).

[0059] Flow cytometry was used to analyze the uptake of the probe in tumor cells. The ALDH2-Cy7@LP-FA probe was co-incubated with BT474 and MDA-MB-231 (human breast cancer cells) cells (37°C 5% CO 2 Incubator), it was found that the binding rate gradually increased with time and reached saturation after 4 hours ( Figure 4 B). BT474 and MDA-MB-231 cells were incubated with different concentrations of ALDH2-Cy7@LP-FA probe for 24 h, and the CCK8 (MCE, HY-K0301) cytotoxicity results showed that ALDH2-Cy7@LP-FA probe had no significant cytotoxicity to both cells ( Figure 4 C). Flow cytometry was used to detect the binding rate of ALDH2-Cy7@LP-FA probe in MCF-10A, MDA-MB-231 and BT474 cells. It was found that the binding rate was highest in BT474 cells with high ALDH2 expression, followed by MDA-MB-231 cells, and lowest in BT474 cells ( Figure 4 D). After knocking down ALDH2 using conventional methods in the art, the cell binding rate of MDA-MB-231 and BT474 decreased ( Figure 4 E).

[0060] MCF-10A, MDA-MB-231 and BT474 cells were incubated with ALDH2-Cy7@LP-FA probe, Mito Tracker mitochondrial dye and Hoechst nuclear stain (37°C, 5% CO). 2 Incubator), it was found that the fluorescence of the ALDH2-Cy7@LP-FA probe overlapped with the mitochondrial luminescence of Mito Tracker, suggesting that ALDH2 protein is mainly located in the mitochondria of the cytoplasm. The probe BT474 showed strong fluorescence staining, and MCF-10A showed very weak fluorescence staining ( Figure 5 ), which is consistent with the results of immunoblotting. The fluorescence of MDA-MB-231 and BT474 cells after knocking down ALDH2 using conventional methods in the art was weakened by co-incubation with ALDH2-Cy7@LP-FA probe ( Figure 6 ), indicating that the ALDH2-Cy7@LP-FA probe can specifically identify the ALDH2 protein expression level in tumor cells. Furthermore, in BT474 cells, we found that compared with the Cy7@LP-FA probe, the ALDH2-Cy7@LP-FA probe had higher sensitivity (82% VS 89%) and specificity (61% VS 93%) ( Figure 7 ).

[0061] Example 4 Tumor cell targeting ability of ALDH2-Cy7@LP-FA near-infrared fluorescent probe in vivo

[0062] 1×10^6 MDA-MB-231-NC (Procell) and MDA-MB-231-shALDH2 (Procell) cells were inoculated into the subcutaneous mammary fat pad of 6-week-old female nude mice. After solid tumors were formed, 5nmol / 200μL of ALDH2-Cy7@LP-FA, ALDH2-Cy7 and Cy7@LP-FA probes were injected into the tail vein of nude mice, respectively, and the IVIS small animal in vivo optical imaging system was used to collect optical imaging information of the probes targeting tumors in tumor-bearing mice. The Cy7 fluorescence signals were collected at different time points of 0h, 2h, 4h, 8h, 24h, and 48h, and it was found that the ALDH2-Cy7@LP-FA probe could accurately locate the tumor cells in the body 4h after tail vein injection ( Figure 8 ). Weak fluorescence signals were observed 2 hours after injection of the three probes, reached saturation after 4 hours, weakened after 24 hours, and disappeared after 48 hours ( Figure 8 By comparing the ALDH2 knockdown group with the NC group (blank control group), it was found that the targeting ability of the ALDH2-Cy7@LP-FA probe to ALDH2 knockdown tumor cells was weakened ( Figure 7 By comparing the fluorescence localization of ALDH2-Cy7@LP-FA, ALDH2-Cy7 and Cy7@LP-FA probes in the MDA-MB-231-NC group, it was found that the ALDH2-Cy7@LP-FA probe had the best tumor binding effect and could accurately locate the tumor boundary ( Figure 8 ).

[0063] Example 5 In vivo surgical navigation in nude mice

[0064] To further explore the clinical application value of the ALDH2-Cy7@LP-FA probe, we constructed an in situ tumor model in the subcutaneous mammary fat pad of BALB / c nude mice: 5×10^6 MDA-MB-231-NC cells in the control group and MDA-MB-231-shALDH2 cells in the experimental group were mixed with 100μg of matrix gel and inoculated into the mammary fat pad of 6-week-old female BALB / c nude mice. When solid tumors were formed subcutaneously, the two groups of nude mice were injected with 5nmol / 200μL of ALDH2-Cy7@LP-FA probe through the tail vein. Six hours after the probe was injected, the tumor was first located by fluorescence in vivo and the fluorescence range was marked; then, the tumor and the surrounding luminous tissue were removed according to the marked range; and fluorescence imaging was continued to observe whether there was fluorescence in the in situ tumor site. If there was no fluorescence, it meant that the tumor cells with high metastatic potential had been completely removed ( Fig. 9 ).

[0065] Example 6 Safety Assessment

[0066] Two groups of BALB / c nude mice were injected with ALDH2-Cy7@LP-FA probe and an equal volume of normal saline through the tail vein, respectively. The physiological condition of the nude mice injected through the tail vein was good, and there was no difference compared with the control group injected with normal saline. The nude mice were killed 24 hours after the probe injection, and the tumors and heart, liver, spleen, lung, and kidney organs were collected for H&E staining conventional in the field. Compared with the mice injected with normal saline, there was no statistically significant difference in the body weight of nude mice in the probe group. None of the nude mice in the probe group died, and no obvious side effects occurred. H&E staining showed that the probe ALDH2-Cy7@LP-FA did not cause pathological damage in the heart, liver, spleen, lung, and kidney organs ( Fig.10 ). The biosafety assessment consistently showed that the probe ALDH2-Cy7@LP-FA has good biocompatibility and can be further applied in vivo.

[0067] The above experimental results show that during tumor diagnosis, the ALDH2-Cy7@LP-FA near-infrared fluorescent probe or a kit containing it can be used to detect highly metastatic breast cancer cells in vivo. The detection results are specific, can identify the ALDH2 protein expression level in tumor cells, can accurately locate the tumor boundary, and have good biocompatibility. According to the expression results of ALDH2, accurate treatment plans can be formulated and prognosis predictions can be made.

[0068] The probe provided by the present invention can realize non-invasive, accurate and efficient identification and judgment of tumor cells with high invasion and metastasis potential, providing a key basis for the formulation of accurate treatment plans and the prediction of patient prognosis.

Claims

1. A nanoliposome probe, It is characterized in that The nanoliposome probe targets mitochondria and comprises an antibody targeting ALDH2 and an antibody targeting FOLR1.

2. The nanoliposome probe according to claim 1, It is characterized in that The antibody targeting ALDH2 is in the liposome, and the antibody targeting FOLR1 is on the cell membrane surface.

3. The nanoliposome probe according to claim 1, It is characterized in that The liposomes include the following components: DOTAP, DOPC, Chol and DSPE-PEG2k-folate.

4. A method for preparing the nanoliposome probe according to any one of claims 1 to 3, It is characterized in that The method comprises: mixing ALDH2-Cy7 with a nanoliposome probe LP-FA to prepare the nanoliposome probe ALDH2-Cy7@LP-FA.

5. The method according to claim 4, It is characterized in that It also includes (a) synthesizing ALDH2-Cy7 and (b) synthesizing the nanoliposome probe LP-FA; preferably, the (a) includes: mixing Cy7 and ALDH2 in proportion, adding a catalyst and a solvent to mix, and washing to obtain the probe ALDH2-Cy7.

6. The method according to claim 5, It is characterized in that The method (b) comprises: preparing LP-FA by a thin film hydration method; Preferably, the (b) comprises: mixing a chloroform / methanol solution containing DOTAP, DOPC, Chol and DSPE-PEG2k-folate, decompressing the mixture, mixing the mixture with a buffer solution and extruding the mixture through a liposome extruder; More preferably, the concentration of DOTAP is 0.23 mg / mL, The concentration of DOPC is 3.3 mg / mL, The concentration of Chol is 0.775 mg / mL, The concentration of the DSPE-PEG2k-folate is 0.215 mg / mL, The volume ratio of chloroform to methanol in the chloroform / methanol is 9:1, and / or, The buffer is Tris buffer with a pH of 9.

7. The method according to claim 4, It is characterized in that The method comprises: The ALDH2-Cy7 is dissolved in methanol and mixed with the LP-FA in proportion for encapsulation and ultrafiltration; Preferably, the mass volume ratio of the ALDH2-Cy7 to the methanol is 1:

5. The encapsulation uses water bath sonication, and / or, The molecular weight cut-off of the ultrafiltration was 10 kDa.

8. Use of the nanoliposome probe according to any one of claims 1 to 3 in the preparation of a diagnostic agent.

9. The use according to claim 8, It is characterized in that The diagnostic agent is a tumor diagnostic agent; preferably, the tumor is a breast cancer tumor; more preferably, the tumor is a highly metastatic breast cancer tumor.

10. A method for detecting cancer cells, It is characterized by: Preferably, the nanoliposome probe as described in any one of claims 1 to 3 is used to detect whether the cancer cells are highly metastatic cancer cells, and the method is for non-diagnostic purposes; more preferably, the cancer cells are breast cancer cells.

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