Nano-liposome probe, method for preparing same, and use thereof

Abstract

Disclosed are a nano-liposome probe, a method for preparing same, and use thereof. Specifically, the nano-liposome probe targets the mitochondria, and comprises an antibody targeting ALDH2 and an antibody targeting FOLR1. The nano-liposome probe of the present invention can achieve noninvasive, accurate, and efficient recognition and identification of tumor cells with high invasion and metastasis potential.

Description

A nanoliposome probe and its preparation method and application

[0001] This application claims the benefit of Chinese Patent Application No. 2023116310539, filed on November 30, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention belongs to the field of detection, and in particular 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

[0003] Breast cancer is the most common malignant tumor 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 clinical treatment can be guided, the quality of life of patients will be effectively improved and their survival time will be prolonged. Although previous studies have been able to predict the benign or malignant nature of tumors through molecular imaging technology, their metastatic potential has not been determined. There is an urgent need in this field to image tumor metastatic seed cells with metastatic potential in vivo, which will not only enable molecular imaging technology to accurately predict the extent and metastasis of the tumor, but also guide precise surgery and prognosis prediction. There is an urgent need in the prior art for a probe / method that can efficiently detect tumor cells with high metastatic potential. Summary of the Invention

[0004] Addressing the current lack of probes for efficiently detecting tumor cells with high metastatic potential, this invention provides a nanoliposome probe, its preparation method, and its application. Specifically, a nanoliposome probe, ALDH2-Cy7@LP-FA, was designed and synthesized using the highly metastatic molecule ALDH2 as its core. This probe can noninvasively, accurately, and efficiently identify and detect tumor cells with high invasive and metastatic potential.

[0005] To address the deficiencies 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.

[0006] In certain embodiments, the antibody targeting ALDH2 is within a liposome, and the antibody targeting FOLR1 is on the cell membrane surface.

[0007] In certain embodiments, the liposomes include the following components: DOTAP, DOPC, Chol, and DSPE-PEG2k-folate.

[0008] A 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.

[0009] In certain embodiments, the method further comprises (a) synthesizing ALDH2-Cy7 and (b) synthesizing the nanoliposome probe LP-FA. Preferably, (a) comprises: mixing Cy7 and ALDH2 in appropriate proportions, adding a catalyst and a solvent, mixing thoroughly, and washing to obtain the probe ALDH2-Cy7.

[0010] In certain embodiments, the (b) comprises: preparing LP-FA by a thin film hydration method.

[0011] 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 then extruding the mixture through a liposome extruder.

[0012] More preferably, the concentration of DOTAP is 0.23 mg / mL,

[0013] The concentration of DOPC is 3.3 mg / mL,

[0014] The concentration of Chol is 0.775 mg / mL,

[0015] The concentration of the DSPE-PEG2k-folate is 0.215 mg / mL,

[0016] The volume ratio of chloroform to methanol in the chloroform / methanol is 9:1,

[0017] The buffer solution is Tris buffer solution with a pH of 9.

[0018] In certain embodiments, the method comprises:

[0019] The ALDH2-Cy7 is dissolved in methanol and mixed with the LP-FA in proportion, and then encapsulated and ultrafiltered.

[0020] Preferably, the mass volume ratio of the ALDH2-Cy7 solution to the methanol is 1:5.

[0021] The encapsulation uses water bath ultrasound,

[0022] The molecular weight cut-off of the ultrafiltration was 10 kDa.

[0023] 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.

[0024] 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.

[0025] A fourth aspect of the present invention provides a method for detecting cancer cells.

[0026] 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.

[0027] More preferably, the cancer cells are breast cancer cells.

[0028] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0029] The reagents and raw materials used in the present invention are commercially available.

[0030] The positive progress of the present invention lies in: 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, and can be used for the diagnosis of highly metastatic breast tumors. It 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

[0031] FIG1 is a schematic diagram of the synthesis route of the nanoliposome probe.

[0032] Figure 2 shows the characterization of the nanoliposome probe.

[0033] Figure 3 shows the expression of ALDH2 protein in highly invasive breast cancer and adjacent tissues.

[0034] Figure 4 shows that the nanoliposome probe can identify breast cancer cells with high metastatic potential.

[0035] Figure 5 shows the recognition of different types of breast cancer cells by nanoliposome probes.

[0036] FIG6 shows the recognition of down-expressing ALDH2 breast cancer cells by the nanoliposome probe.

[0037] FIG7 shows the sensitivity and specificity of the nanoliposome probe in identifying breast cancer cells.

[0038] FIG8 shows the in vivo recognition of highly metastatic breast cancer cells by nanoliposome probes.

[0039] Figure 9 shows the application of nanoliposome probes in surgical navigation in nude mice.

[0040] FIG10 shows the toxicity of the nanoliposome probe to various organs of nude mice. DETAILED DESCRIPTION

[0041] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0042] The ALDH2 antibody was supplied by abcam, model number 133306; the FolR1, FA antibody (hereinafter referred to as FA), was supplied by ABclonal, model number A15672.

[0043] Example 1 Synthesis of ALDH2-Cy7@LP-FA Near-Infrared Fluorescent Probe

[0044] (a) Synthesis of ALDH2-Cy7 probe

[0045] First, Cy7 was prepared into a 1 mg / mL solution with DMSO. Then, Cy7 and ALDH2 were added to a 2 mL EP tube at a ratio of 20:1. TEA was used as a catalyst and DMSO was used as a solvent. A magnetic stirrer was added and the mixture was stirred at room temperature for 24 h. The mixture was then washed three times with ether (1.5 mL × 3) to obtain a blue solid ALDH2-Cy7 (Figure 1A).

[0046] (b) Preparation of nanoliposome LP-FA

[0047] LP-FA was prepared by thin film hydration. First, a chloroform / methanol (v / v, 9 / 1) solution (total volume 3 mL) of lipids 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 residual organic solvent. Next, the lipid film was hydrated with 4 mL of 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 nanoliposomal LP-FA.

[0048] (c) Preparation of nanoliposome probes Cy7@LP-FA (as a control) and ALDH2-Cy7@LP-FA

[0049] (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 ultrasonicated in a water bath at room temperature for 20 min for entrapment. After the ultrasonication, the Cy7@LP-FA liposomes were ultrafiltered three times using an ultrafiltration tube (molecular weight cutoff: 10 kDa) to remove the unentrapped dye. The nanoliposome probe Cy7@LP-FA was obtained and stored in a refrigerator at 4°C.

[0050] (c-2) The ALDH2-Cy7 solid was dissolved in methanol to a 30 mg / mL solution. ALDH2-Cy7 and LP-FA were added to a 2 mL EP tube at a volume ratio of 1:5. The solution was ultrasonicated in a water bath at room temperature for 20 min for entrapment. After the sonication, the ALDH2-Cy7@LP-FA liposomes were ultrafiltered three times using an ultrafiltration tube (molecular weight cutoff: 10 kDa) to remove unentrapped dye and stored in a refrigerator at 4°C (Figure 1B).

[0051] Example 2 Performance Determination of Nanoliposome Probes

[0052] 2.1 Determination of liposome encapsulation efficiency

[0053] 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, and a standard curve of Cy7 concentration and ultraviolet absorption intensity was drawn (y = 0.2841x + 0.0181, R 2 =0.9955). A small amount of the prepared Cy7@LP-FA and ALDH2-Cy7@LP-FA was added to 9 volumes of anhydrous methanol to permeate the membranes. The membranes were mixed thoroughly, and the UV absorbance at 750 nm was measured. The entrapped Cy7 concentration was calculated using the standard curve. Finally, the encapsulation efficiency of Cy7@LP-FA and ALDH2-Cy7@LP-FA was calculated using the formula "Encapsulation efficiency = actual entrapped dye concentration / total administered dye concentration."

[0054] 2.2 Characterization of basic properties of liposomes

[0055] 20 μL of the prepared Cy7@LP-FA and ALDH2-Cy7@LP-FA solutions were dissolved in 1 mL of PBS and their particle sizes were measured three times using dynamic light scattering (DLS, Malvern ZEN3690). The particle size results were recorded for each measurement (Figure 2A and Figure 2C). Another 20 μL of each solution was dispersed in a solvent containing 5% D-glucose. The solutions were adjusted to different pH values ​​(pH = 6, pH = 7, and pH = 9). The zeta potentials were measured three times using a nanoparticle size potentiometer (Figure 2B). The encapsulation efficiencies of Cy7@LP-FA and ALDH2-Cy7@LP-FA were calculated to be 66.7% and 61.2%, respectively (Figure 2D).

[0056] 2.3 Spectral performance testing

[0057] First, take a small amount of the fluorescent probes Cy7, ALDH2-Cy7, or ALDH2-Cy7@LP-FA and prepare a working solution with a working concentration of 5 μM using PBS, water, and methanol (1xPBS buffer:methanol = 1000:1). Absorption spectroscopy (Edinburgh Instruments, FLS1000): Set the scanning wavelength range to 500-900 nm with a scanning interval of 1 nm. Perform a baseline scan using the solvent of each of the above solutions, then perform a sample scan, read the absorption wavelength and absorbance value, and save the scan data. Emission spectroscopy: Set the excitation light source to a xenon lamp, the detector to a NIR PMT, the excitation and emission slits to 2 nm, 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 cell and scanned. The emission wavelength and fluorescence intensity were read and saved. The UV absorption and fluorescence emission spectra of ALDH2-Cy7@LP-FA were characterized. The results showed that liposome encapsulation caused a blue shift of 4 nm in UV absorption and 5 nm in fluorescence emission, respectively, of the labeled antibody. Spectral properties demonstrated successful Cy7 labeling of the ALDH2 antibody, that liposome encapsulation achieved nanoscale-scale antibody size, and that the spectral properties of the labeled antibody were minimally affected (Figures 2E and 2F).

[0058] Example 3 Detection of breast cancer cells with high metastatic potential using ALDH2-Cy7@LP-FA near-infrared fluorescent probe

[0059] Immunohistochemistry assays for ALDH2 protein expression in highly invasive tumors and adjacent adjacent tissues revealed high expression of ALDH2 in highly invasive breast cancer tissues (Figure 3). Western blot analysis of ALDH2 protein expression in six wild-type breast cancer cell lines revealed high expression of ALDH2 in BT474 cells (human breast ductal carcinoma cells) and low expression in MCF-10A cells (human normal mammary epithelial cells) (Figure 4A).

[0060] 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 (37°C, 5% CO2 incubator). Binding rates gradually increased over time, reaching saturation after 4 hours (Figure 4B). BT474 and MDA-MB-231 cells were co-incubated with varying concentrations of the ALDH2-Cy7@LP-FA probe for 24 hours. CCK8 (MCE, HY-K0301) cytotoxicity assays revealed that the ALDH2-Cy7@LP-FA probe had no significant cytotoxicity against either cell type (Figure 4C). Flow cytometry analysis of the ALDH2-Cy7@LP-FA probe binding rate in MCF-10A, MDA-MB-231, and BT474 cells revealed the highest binding rate in BT474 cells, which overexpress ALDH2, followed by MDA-MB-231 cells, and the lowest in BT474 cells (Figure 4D). Knockdown of ALDH2 using conventional methods in the art reduced the binding rate in MDA-MB-231 and BT474 cells (Figure 4E).

[0061] MCF-10A, MDA-MB-231, and BT474 cells were co-incubated with ALDH2-Cy7@LP-FA probe, Mito Tracker mitochondrial dye, and Hoechst nuclear stain (37°C 5% CO2 incubator). It was found that the ALDH2-Cy7@LP-FA probe fluorescence overlapped with the Mito Tracker mitochondrial luminescence, suggesting that ALDH2 protein is mainly located in the cytoplasmic mitochondria. The probe showed strong fluorescence staining in BT474 and very weak fluorescence staining in MCF-10A (Figure 5), which is consistent with the immunoblotting results. After knocking down ALDH2 using conventional methods in the field, MDA-MB-231 and BT474 cells were co-incubated with the ALDH2-Cy7@LP-FA probe, and the fluorescence was weakened (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 the ALDH2-Cy7@LP-FA probe had higher sensitivity (82% vs 89%) and specificity (61% vs 93%) than the Cy7@LP-FA probe ( FIG7 ).

[0062] Example 4 Tumor cell targeting ability of ALDH2-Cy7@LP-FA near-infrared fluorescent probe in vivo

[0063] 1×10^6 MDA-MB-231-NC (Procell) and MDA-MB-231-shALDH2 (Procell) cells were inoculated into the subcutaneous mammary fat pads of 6-week-old female nude mice. After solid tumors were established, 5 nmol / 200 μL of ALDH2-Cy7@LP-FA, ALDH2-Cy7, and Cy7@LP-FA probes were injected into the tail vein of the nude mice. An IVIS small animal in vivo optical imaging system was used to capture optical images of the probes targeting the tumors in the tumor-bearing mice. Cy7 fluorescence signals were collected at 0, 2, 4, 8, 24, and 48 hours after injection. The ALDH2-Cy7@LP-FA probe accurately localized to tumor cells in vivo 4 hours after tail vein injection (Figure 8). A faint fluorescence signal was observed 2 hours after injection for all three probes, which reached saturation 4 hours later, weakened after 24 hours, and disappeared after 48 hours (Figure 8). Comparison of the ALDH2 knockdown group with the NC group (blank control group) revealed that the ALDH2-Cy7@LP-FA probe had a weakened targeting ability against ALDH2-knockdown tumor cells (Figure 7). Comparison of the fluorescence localization of the ALDH2-Cy7@LP-FA, ALDH2-Cy7, and Cy7@LP-FA probes in the MDA-MB-231-NC group revealed that the ALDH2-Cy7@LP-FA probe had the best tumor binding effect, accurately locating the tumor boundary (Figure 8).

[0064] Example 5 In vivo surgical navigation in nude mice

[0065] To further explore the clinical application of the ALDH2-Cy7@LP-FA probe, we established an orthotopic subcutaneous mammary fat pad tumor model in BALB / c nude mice. 5×10^6 MDA-MB-231-NC cells (control group) and MDA-MB-231-shALDH2 cells (experimental group) were mixed with 100 μg of Matrigel and inoculated into the mammary fat pad of 6-week-old female BALB / c nude mice. After solid subcutaneous tumors had formed, both groups of mice were injected via the tail vein with 5 nmol / 200 μL of the ALDH2-Cy7@LP-FA probe. Six hours after probe injection, in vivo tumor fluorescence localization was performed and the fluorescent area was marked. The tumor and surrounding luminescent tissue were then removed based on the marked area. Fluorescence imaging was then performed to monitor the presence of fluorescence within the orthotopic tumor site. Absence of fluorescence indicates complete excision of highly metastatic tumor cells (Figure 9).

[0066] Example 6 Safety Assessment

[0067] Two groups of BALB / c nude mice were injected with the 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 in the normal saline injection group, there was no statistically significant difference in the body weight of the 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 (Figure 10). The biosafety assessment consistently showed that the probe ALDH2-Cy7@LP-FA has good biocompatibility and can be further used in vivo.

[0068] These experimental results demonstrate that the ALDH2-Cy7@LP-FA near-infrared fluorescent probe or a kit containing it can be used for in vivo detection of highly metastatic breast cancer cells during tumor diagnosis. The test results are specific, can identify ALDH2 protein expression levels in tumor cells, accurately locate tumor boundaries, and exhibit good biocompatibility. ALDH2 expression results can be used to formulate precise treatment plans and predict prognosis.

[0069] 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 precise treatment plans and the prediction of patient prognosis.

[0070] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A nanoliposome probe, 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, 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, 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, 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, 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, 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, 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, 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, characterized in that; 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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