Theranostic targeted ultrasonic blasting nano-bubbles, preparation method therefor, and use thereof

Abstract

Theranostic targeted ultrasonic blasting nano-bubbles, a preparation method therefor, and use thereof, wherein the nano-bubbles are the targeted drug-gene nano-bubbles miRNA / FTY720 / PFP@PEI-T7 NBs. The nano-bubbles can be used for preparing a thyroid ultrasound diagnostic agent or a drug for treating thyroid cancer.

Description

A Therapeutic Targeted Ultrasonic Explosive Nanobubble, Its Preparation Method and Application Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, and in particular to a therapeutically integrated targeted ultrasonic ablation nanobubble, its preparation method, and its application. Background Technology

[0002] Thyroid cancer, caused by changes and overgrowth of the thyroid gland tissue in the neck, is the most common endocrine malignancy. Over the past few decades, the incidence of thyroid cancer has shown a significant upward trend, accounting for approximately 1.7% of all cancer cases. Thyroid cancer can be classified into four types based on the cell types found in the tumor: papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and undifferentiated thyroid carcinoma. The vast majority of thyroid cancers are papillary and follicular thyroid carcinomas, formed from the differentiation of follicular epithelial cells. Although the 5-year prognosis for most thyroid cancers is good, some patients are diagnosed at a locally advanced stage or with distant metastases, and are unresponsive to iodine radiotherapy. These patients have short survival times, and effective treatment methods are lacking clinically. Therefore, early diagnosis is particularly important for improving patient survival rates and quality of life.

[0003] Currently, imaging examinations (such as thyroid ultrasound and fine-needle aspiration biopsy) can be used clinically to make a preliminary assessment of thyroid lesions, but the diagnostic accuracy for thyroid cancers smaller than 10 mm remains very low. Treatment methods for thyroid cancer include surgical resection, radioactive iodine (131I) therapy, thyroid-stimulating hormone (TSH) suppression therapy, chemotherapy / radiotherapy, etc., but survival rates have not improved significantly in the past few decades. Therefore, developing new treatment strategies is particularly urgent.

[0004] Chemotherapy is the most commonly used treatment in clinical practice, but its efficacy is often affected by many physiological obstacles (non-specific distribution, restricted blood flow, endothelial extravasation, tumor cell membrane and tissue delivery barriers, multidrug resistance). To overcome these obstacles, nanomedicine carriers have emerged. Compared with traditional chemotherapy drugs, nanomedicine carriers have advantages such as adjustable drug release rate, increased drug permeability through biological membranes and improved drug retention rate (EPR) at the lesion site, enhanced drug targeting, and reduced drug toxicity.

[0005] However, single-drug chemotherapy is highly prone to inducing multidrug resistance (MDR) in the body. MDR reduces drug accumulation within cells, increases DNA damage, and significantly affects treatment efficacy. Therefore, researchers have been exploring more ideal treatment modalities. Combining two or more treatment strategies with different mechanisms of action is gradually becoming a promising approach, and synergistic treatment with multiple therapies has been proven to enhance therapeutic effects.

[0006] In recent years, with the development of nanomedicine and the promotion of precision medicine, people have raised higher demands for disease diagnosis and treatment. Nanocarriers can not only achieve the responsive release of targeted drugs in the tumor environment, but also tightly bind contrast agents to nanocarriers, thereby achieving integrated diagnosis and treatment. Targeted carriers have attracted widespread attention due to their higher drug delivery efficiency. Under the action of specific targeting ligands, nanocarriers can actively accumulate near tumor blood vessels, allowing drugs to act more directly on the tumor site.

[0007] Transferrin receptor (TfR) is overexpressed on the surface of various cancer cells, including glioma, breast cancer, lung cancer, prostate cancer, and thyroid cancer. The heptapeptide (HAIYPRH, T7), screened using a phage display system, exhibits a strong binding affinity for TfR, suggesting that T7 holds promise for improving drug delivery efficiency and reducing off-target effects in targeted drug delivery systems.

[0008] If a comprehensive treatment plan can be developed that is highly targeted to the tumor, has a high drug absorption rate, few side effects, and is comfortable and effective, based on the biological characteristics of thyroid cancer, it can greatly improve the prognosis of thyroid cancer patients and enhance their sense of well-being and quality of life. Summary of the Invention

[0009] To address the above problems, this invention provides an ultrasonically ablated nanobubble that exhibits strong targeting of thyroid cancer cells, high drug absorption rate, and minimal side effects.

[0010] The first aspect of this invention provides a therapeutically integrated targeted ultrasound-induced ablation nanobubble, wherein the nanobubble is a targeted drug-gene nanobubble miRNA / FTY720 / PFP@PEI-T7NBs, which uses DMG-PEG2000 / DSPE-PEG2000 liposomes as a shell, encapsulates FTY720 / PFP, and has a T7 short peptide coupled to its surface, and adsorbs gene miRNA-34b-5p. Its raw materials include 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (DMG-PEG2000), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-polyethylene glycol-maleimide (DSPE-PEG2000-MAL), polyethyleneimine (PEI), fingolimod (FTY720), perfluoropentane (PFP), gene miRNA-34b-5p, and a thiol-modified T7 short peptide.

[0011] In the nanobubbles of this invention, DMG-PEG2000 / DSPE-PEG2000 liposomes, after encapsulating PFP, maintain an overall carrier particle size at the nanoscale, exhibiting an EPR effect. Furthermore, the surface of these nanobubbles is coupled with a T7 short peptide, significantly enhancing the binding affinity of the prepared nanobubbles to TfR on the surface of thyroid cancer cells. This improves the nanobubble delivery function and reduces off-target effects, thereby improving the diagnostic accuracy of early-stage thyroid cancer. Moreover, these nanobubbles can be precisely ruptured at the tumor site using UTMD (ultrasound-directed microbubble rupture) technology, releasing therapeutic drugs and / or genes. By increasing local drug concentration, this effectively inhibits the proliferation and metastasis of cancer cells.

[0012] Preferably, the nanobubbles have a particle size of 200-300 nm.

[0013] In a second aspect, the present invention also provides a method for preparing the above-mentioned therapeutically integrated targeted ultrasonic bursting nanobubbles, comprising the steps of:

[0014] S1 uses DMG-PEG2000, DSPE-PEG2000-MAL, PEI, FTY720, and PFP as raw materials to synthesize cationic drug-loaded nanobubbles FTY720 / PFP@PEI NBs by thin film hydration method;

[0015] S2 Take FTY720 / PFP@PEI NBs solution, add T7 short peptide modified with thiol group at room temperature, stir the reaction, and dialyze to obtain targeted cationic drug nanobubbles FTY720 / PFP@PEI-T7NBs;

[0016] Since the synthesized cationic drug-loaded nanobubbles FTY720 / PFP@PEI NBs have maleimide groups on their surface, they can be reacted with thiol-modified T7 short peptides using the Michael addition reaction to obtain targeted cationic drug nanobubbles.

[0017] S3 added miRNA-34b-5p gene solution to the FTY720@PFP@PEI-T7NBs solution obtained in step (2) and incubated to obtain the targeted ultrasonic bursting nanobubble miRNA / FTY720@PFP@PEI-T7NBs;

[0018] In this step, the gene complex is obtained by electrostatic adsorption. The product can be precisely blasted at the tumor site using UTMD technology to release therapeutic drugs and genes, thus achieving precision medicine.

[0019] Further, step S1 includes:

[0020] S11 dissolves DMG-PEG2000, DSPE-PEG2000-MAL, PEI, and FTY720 in an organic solvent and then performs vacuum rotary evaporation; the reagents can be purified by vacuum rotary evaporation; preferably, the mass ratio of each raw material is: DMG-PEG2000:DSPE-PEG2000-MAL:PEI:FTY720 = 30:15:5:10; the organic solvent is any one of tetrahydrofuran, chloroform, dichloromethane, benzene, toluene, and acetone;

[0021] S12 adds PBS buffer to the product after vacuum rotary evaporation and sonicates to obtain a liposome emulsion; preferably, the pH of the PBS solution is 7.4 and the concentration is 0.01M.

[0022] S13 adds PFP to the liposome emulsion to obtain a PFP-containing liposome suspension, and then ultrasonically emulsifies the PFP-containing liposome suspension to obtain the cationic drug-loaded nanobubbles FTY720 / PFP@PEI NBs; preferably, the molar ratio of PFP to FTY720 / PFP@PEI NBs is 0.5.

[0023] Furthermore, in step S2, the concentration of the FTY720 / PFP@PEI NBs solution is 10 mg / mL.

[0024] Further, in step S3, the concentration of the gene solution is 0.5-1 nM, and the amounts of FTY720 / PFP@PEI-T7NBs material and gene miRNA-34b-5p are determined according to the nitrogen-phosphorus ratio (N / P), where N / P is 0, 2, 4, 6, 8, 10, 12, 14, or 16, etc.

[0025] In a third aspect, the present invention also provides the application of the above-mentioned integrated diagnostic and therapeutic targeted ultrasonic bursting nanobubbles in the preparation of thyroid ultrasound diagnostic agents or thyroid cancer treatment drugs.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] Clinically, thyroid nodules are generally diagnosed and their nature determined using high-frequency ultrasound and fine-needle aspiration biopsy. However, for thyroid nodules smaller than 5 mm, the accuracy of high-frequency ultrasound imaging is not high, leading to a lower diagnostic rate. This invention presents a therapeutically integrated targeted ultrasound-guided ablation nanobubble. Using DMG-PEG2000 / DSPE-PEG2000 liposomes as a shell, and after encapsulating PFP, the overall carrier particle size is controlled at the nanoscale, exhibiting an EPR effect. Furthermore, the drug core surface is coupled with a T7 short peptide, which significantly improves the binding affinity of the prepared nanobubble to TfR on the surface of thyroid cancer cells, enhancing the nanobubble delivery function and reducing off-target effects, thereby improving the diagnostic accuracy of early thyroid cancer.

[0028] The nanobubbles of this invention are a therapeutic platform that utilizes nanotechnology to simultaneously encapsulate therapeutic drugs and genes. During treatment, the nanobubbles actively target and accumulate at the tumor site. Tumor location is determined using ultrasound imaging, and UTMD technology is used to precisely rupture the nanobubbles, releasing more therapeutic drugs and genes and reducing drug side effects, thereby achieving precise treatment of thyroid cancer. Under ultrasound conditions, exhibiting cavitation and acoustic aperture effects, the nanobubbles of this invention have higher transfection efficiency than conventional targeted gene vectors.

[0029] The manufacturing process of this invention is simple and can be applied to large-scale production. Attached Figure Description

[0030] Figure 1 is a schematic diagram of the particle size distribution of nanobubbles according to an embodiment of the present invention;

[0031] Figure 2 shows the cytotoxicity results of nanobubbles on cancer cells according to an embodiment of the present invention;

[0032] Figure 3. Animal survival rate results of nanobubbles in the embodiments of the present invention;

[0033] Figure 4 shows the ultrasonic imaging results of the nanobubbles in the embodiment of the present invention. Detailed Implementation

[0034] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0035] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0036] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0038] Unless otherwise specified, all reagents, materials, and equipment used in the embodiments of this invention are commercially available; unless otherwise specified, all test methods are conventional test methods in the field.

[0039] Example 1: Preparation of Nanobubbles

[0040] A therapeutically integrated targeted ultrasonic bursting nanobubble miRNA / FTY720 / PFP@PEI-T7NBs preparation method is as follows:

[0041] S1 synthesized drug-loaded nanobubbles FTY720 / PFP@PEI NBs using a thin-film hydration method.

[0042] S11 added long-cycle DMG-PEG2000 (30 mg), DSPE-PEG2000-MAL (15 mg), PEI (5 mg), and FTY720 (10 mg) to a round-bottom flask and dissolved them in tetrahydrofuran (10 mL). After the powder was completely dissolved, the solvent was evaporated at 40 °C for 2 hours using a vacuum rotary evaporator to remove excess organic solvent.

[0043] S12 added PBS buffer (5 mL, pH 7.4, 0.01 M) to a round-bottom flask and placed the flask in an ultrasonic sonicator to obtain a liposome emulsion.

[0044] S13 transferred the emulsion to a 20 mL test tube and slowly added PFP (100 μL) to obtain a suspension containing PFP liposomes. Under ice bath conditions, the suspension containing PFP liposomes was emulsified for 20 minutes using an ultrasonic homogenizer (200 W, 4 s–4 s). The resulting product is FTY720 / PFP@PEI NBs, where the molar ratio of PFP to FTY720 / PFP@PEI NBs is 0.5. It was stored at 4 °C.

[0045] S2 utilizes the maleimide groups on the surface of FTY720 / PFP@PEI NBs to directly react with the thiol-modified T7 short peptide to prepare FTY720 / PFP@PEI-T7NBs via a Michael addition reaction.

[0046] The specific method is as follows: Take 10 mL of FTY720 / PFP@PEI NBs solution (10 mg / mL), add the thiol-modified T7 short peptide at room temperature, and stir for 4 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag (M... W (CO=3500) was used for low-temperature dialyzing with ultrapure water for 24 hours to obtain the product FTY720 / PFP@PEI-T7NBs, which was then stored at 4°C.

[0047] S3 uses an electrostatic adsorption method to prepare gene complexes.

[0048] The specific method is as follows: Take the FTY720 / PFP@PEI-T7NBs solution, add the miRNA-34b-5p solution, and incubate for 30 min to obtain the product miRNA / FTY720 / PFP@PEI-T7NBs. The nitrogen-phosphorus ratio (N / P) of the FTY720 / PFP@PEI-T7NBs material and the miRNA-34b-5p gene is 8, and the concentration of the miRNA-34b-5p solution is 0.5 nM.

[0049] The nanobubbles prepared according to Example 1 have a narrow particle size distribution (as shown in Figure 1), with an average particle size of 291 ± 1.3 nm and a PDI of 0.231.

[0050] Example 2: Drug Loading Rate Determination

[0051] Sample: Therapeutic targeted ultrasonic bursting nanobubble miRNA / FTY720 / PFP@PEI-T7NBs (hereinafter referred to as NBs) prepared in Example 1.

[0052] Test methods: The loading rate of FTY720 in NBs was measured by UV-Vis spectrophotometer at a wavelength of 220 nm. The content of FTY720 was determined by a standard curve according to Beer-Lambert's law. The loading rate of miRNA was measured by quantitative fluorescence method to determine the content of miRNA in the gene vector.

[0053] Test results:

[0054] As shown in Table 1, the drug FTY720 had a drug loading rate of approximately 15.03% and an encapsulation rate of approximately 90.17%; the miRNA-34b-5p had a drug loading rate of approximately 10.21% and an encapsulation rate of approximately 91.90%.

[0055] Table 1. Results of Drug Loading Rate Test

[0056] Example 3 K1 Cytotoxicity Test

[0057] Sample: Therapeutic targeted ultrasonic bursting nanobubble miRNA / FTY720 / PFP@PEI-T7NBs (hereinafter referred to as NBs) prepared in Example 1.

[0058] Test methods: The cytotoxicity of the prepared NBs to K1 cells (human papillary thyroid carcinoma cells) under different concentrations and with and without ultrasound irradiation was determined using the CCK-8 assay. Cells were seeded in 96-well culture plates at a density of 5 × 10⁶ cells / well. 3 / well, co-cultured with cells in PBS or sample solutions of different concentrations at 37°C. After 24 hours, each group of cells was incubated with CCK-8 reagent at 37°C for 2 hours. Cells co-cultured with PBS served as a blank control group. The absorbance of each sample was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula:

[0059] Test results: As shown in Figure 2, it can be seen that NBs combined with the UTMD administration regimen have an inhibitory effect on the cell activity of K1 cells, indicating that the NBs combined with UTMD administration strategy has a good anti-K1 cell effect in vitro.

[0060] Example 4 Animal survival rate

[0061] Sample: Therapeutic targeted ultrasonic bursting nanobubble miRNA / FTY720@PFP@PEI-T7NBs (hereinafter referred to as NBs) prepared in Example 1.

[0062] Testing method: The treatment period was 40 days. Every other day, the experimental animals were injected with a sample at a dose of 5 mg / kg. The tumor volume of the nude mice was monitored daily, and the survival rate of the experimental animals was recorded.

[0063] Test Results: The results of Example 4 are shown in Figure 3. It can be seen that the tumor volume is reduced by the NBs combined with UTMD administration regimen, indicating that nanobubbles can more effectively inhibit tumor growth under ultrasound-mediated guidance. Statistical analysis of the survival rate of experimental animals showed that the NBs combined with UTMD administration regimen had a higher survival rate.

[0064] Example 5 Ultrasonic Imaging Experiment

[0065] Sample: Therapeutic targeted ultrasonic bursting nanobubble miRNA / FTY720 / PFP@PEI-T7NBs (hereinafter referred to as NBs) prepared in Example 1.

[0066] Test method: Nanobubble solutions of different concentrations were prepared and filled into agarose gel modules. The ultrasound probe was uniformly coated with coupling agent and placed perpendicular to the side of the module. It was confirmed that there were no air bubbles between the module and the ultrasound probe. The ultrasound imaging of each group of samples was recorded using the contrast-enhanced ultrasound (CEUS) mode.

[0067] Test results: As shown in Figure 4, the results of Example 5 show that the ultrasound imaging signal of NBs becomes stronger and clearer from 62.5mM to 250mM, indicating that NBs have the ability to enhance ultrasound imaging.

[0068] The above embodiments illustrate that, targeting the biological characteristics of thyroid cancer, this invention integrates chemotherapy and gene therapy to combat MDR. It provides a therapeutically integrated targeted ultrasound-guided ablation nanobubble and its preparation method. The manufacturing process is simple and can be applied to large-scale production. The product possesses thyroid cancer targeting and ultrasound imaging properties, good biocompatibility, and can be used to prepare thyroid ultrasound diagnostic agents or thyroid cancer therapeutic drugs. It can be combined with UTMD technology to precisely ablate at the tumor site, releasing therapeutic drugs and / or genes. By increasing local drug concentration, it effectively inhibits the proliferation and metastasis of cancer cells, enabling precision medicine to be applied in real-world scenarios and providing a novel and highly effective strategy for the combined use of chemotherapy and gene therapy in the diagnosis and treatment of thyroid cancer.

[0069] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0070] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A diagnosis and treatment integrated targeted ultrasonic burst nanobubble, characterized in that, The nanobubble is a targeted drug-gene nanobubble miRNA / FTY720 / PFP@PEI-T7 NBs, which has a DMG-PEG2000 / DSPE-PEG2000 liposome as a shell, encapsulates FTY720 / PFP, and is coupled with a T7 short peptide on the surface and adsorbed with a gene miRNA-34b-5p, and raw materials thereof include DMG-PEG2000, DSPE-PEG2000-MAL, PEI, FTY720, PFP, and a thiol-modified T7 short peptide.

2. The diagnosis and treatment integrated targeted ultrasonic burst nanobubble of claim 1, wherein, The nanobubble has a particle size of 200-300 nm.

3. The preparation method of the diagnosis and treatment integrated targeted ultrasonic burst nanobubbles according to any one of claims 1-2, characterized in that, The method comprises the following steps: S1, using DMG-PEG, DSPE-PEG2000-MAL, PEI, FTY720, and PFP as raw materials, cationic drug-loaded nanobubbles FTY720 / PFP@PEI NBs are synthesized by a thin film hydration method; S2, a thiol-modified T7 short peptide is added to a FTY720 / PFP@PEI NBs solution under room temperature, and the reaction is stirred and dialyzed to obtain a targeted cationic drug nanobubble FTY720 / PFP@PEI-T7 NBs; S3, a miRNA-34b-5p gene solution is added to the FTY720 / PFP@PEI-T7 NBs solution obtained in step (2) to obtain the diagnosis and treatment integrated targeted ultrasonic blasting nanobubble miRNA / FTY720 / PFP@PEI-T7 NBs.

4. The production method according to claim 3, characterized by, The step S1 comprises: S11, DMG-PEG2000, DSPE-PEG2000-MAL, PEI, and FTY720 are dissolved with an organic solvent, and vacuum rotary evaporation is performed; S12, PBS buffer is added to the product after vacuum rotary evaporation, and ultrasonic oscillation is performed to obtain a liposome emulsion; S13, PFP is added to the liposome emulsion to obtain a PFP-containing liposome suspension, and the PFP-containing liposome suspension is ultrasonic emulsified to obtain the cationic drug-loaded nanobubble FTY720 / PFP@PEI NBs.

5. The preparation method according to claim 4, characterized in that, In the step S11, the mass ratio of each raw material is: DMG-PEG2000: DSPE-PEG2000-MAL: PEI: FTY720 = 30:15:5:10; and the organic solvent is any one of tetrahydrofuran, chloroform, dichloromethane, benzene, toluene, and acetone.

6. The preparation method according to claim 4, characterized in that, In the step S12, the pH of the PBS solution is 7.4, and the concentration is 0.01M.

7. The preparation method according to claim 4, characterized in that, In the step S13, the molar ratio of PFP to FTY720 / PFP@PEI NBs is 0.

5.

8. The preparation method according to claim 3, characterized in that, In the step S2, the concentration of the FTY720 / PFP@PEI NBs solution is 10 mg / mL.

9. The preparation method according to claim 3, characterized in that, In the step S3, the concentration of the gene solution is 0.5-1 nM, and the amount of the FTY720 / PFP@PEI-T7 NBs material and the gene miRNA-34b-5p is determined according to the nitrogen-phosphorus ratio N / P, and the N / P is: 0, 2, 4, 6, 8, 10, 12, 14, or 16.

10. The use of the diagnosis and treatment integrated targeted ultrasonic burst nanobubbles according to any one of claims 1-2 in the preparation of a drug for ultrasonic diagnosis of thyroid or a drug for treatment of thyroid cancer.

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