Targeted ultrasonic nanobubble as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical nano materials, in particular to a targeted ultrasonic nano bubble as well as a preparation method and application thereof. The targeted ultrasonic nanobubble can carry a drug and / or a gene of a targeted disease, targeted aggregation of the drug and the gene at a prostate cancer part is realized, the nanobubble is exploded when ultrasonic is applied, the ultrasonic developing effect is enhanced, meanwhile, the drug and the gene are more released at a targeted part, the drug effect and the gene transfection efficiency are favorably improved, and the prostate cancer treatment effect is improved. Toxic and side effects of the whole body are reduced. The targeted nanobubble can be combined with chemotherapy and gene therapy to synergistically treat castration-resistant prostate cancer in multiple modes, and a new strategy is provided for overcoming multidrug resistance while the treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical nanomaterials, and in particular to a targeted ultrasonic nanobubble and a preparation method and application thereof. Background Art

[0002] Prostate cancer (PCa) is the second most common cancer in men, causing nearly 360,000 deaths in 2018. Early localized prostate cancer has a good prognosis, but when the disease progresses to the late stage, tumor cell invasion and metastasis lead to increased patient mortality. Currently, androgen deprivation therapy (ADT) has become the first choice for treatment of locally advanced or metastatic prostate cancer. Despite the significant early treatment effect of ADT, almost all prostate cancer patients will progress to castration-resistant prostate cancer (CRPC) after a period of treatment. Bicalutamide is a first-generation anti-androgen drug, and its mechanism of action is mainly competitive inhibition of androgen binding to androgen receptors (ARs). Enzalutamide (ENZ) is a second-generation anti-androgen drug that has been approved for clinical use by the US Food and Drug Administration (FDA). Compared with first-generation anti-androgen drugs, ENZ is a more effective competitive inhibitor with higher binding efficiency than androgen binding to the AR ligand binding domain. Unfortunately, patients usually eventually develop resistance to ENZ. Therefore, if the effectiveness of drugs can be improved, the development of drug resistance can be delayed, thereby increasing the survival rate of patients.

[0003] With the advancement of material technology, nanocarriers can safely and effectively deliver drugs to specific sites, thereby producing greater effects at smaller doses. Currently, there are many types of nanomaterials available, such as liposomes, polymer carriers, inorganic nanocarriers and other types of carriers. Materials can be selected and adjusted according to different types of drugs, thus having greater flexibility in selecting materials. The application of ultrasound molecular imaging technology in targeted tumor therapy has also attracted more and more attention. Ultrasound contrast agents explode under ultrasound irradiation, producing a series of biological effects, such as thermal effects, cavitation effects, mechanical effects and other effects, thereby improving the anti-cancer effect. Under ultrasound irradiation, gaps temporarily appear on the cell membrane, opening to macromolecules so that macromolecules can penetrate the cell membrane and enter the cell. This process is called the sonoporation effect. As the sound energy increases, the propagation of ultrasound in biological tissues increases accordingly, increasing the permeability of vascular endothelium and cell membranes, which can significantly improve the targeted delivery of macromolecules in the body. These effects can make it easier for genes or drugs to pass through the walls of tumor microvessels and enter tumor tissues and cells, increasing the tumor cells' ability to take up genes or drugs. In addition, the range of ultrasound irradiation can limit the effect of genes or drugs on tumor tissues, thereby reducing systemic toxic side effects. In addition, materials commonly used in ultrasound vesicles, such as lipids, are non-immunogenic and non-toxic, and are ideal carrier wall materials for genes or drugs. If nanocarrier technology is combined with ultrasound molecular imaging, it can provide a new therapeutic strategy for targeted tumor therapy. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a targeted ultrasound nanobubble that can be combined with chemotherapy and gene therapy to synergistically treat tumors and has targeting and safety.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In the first aspect, the present invention provides a targeted ultrasound nanobubble, which is a core-shell structure, wherein the outer shell is a PEGylated phospholipid modified with an RGD short peptide, and the inner core includes PFP and an anti-tumor drug; the outer shell adsorbs siRNA targeting the FGFR gene.

[0007] The present invention provides a targeted ultrasound nanobubble that can carry drugs and / or genes targeting diseases. The targeted ultrasound nanobubble uses PEGylated phospholipids as nanobubble wall materials and short peptide RGD as a targeting ligand targeting prostate cancer cells. PEGylated phospholipids have excellent biocompatibility and high drug loading capacity, and are easy to surface modify; RDG is a peptide containing an arginine-glycine-aspartic acid (RGD) triplet, and has a strong affinity and selectivity for integrin receptor αVβ3. Integrin receptor αvβ3 mainly exists in the initial stage of angiogenesis, exists in a small amount on normal epithelial cells and endothelial cells, and is overexpressed on endothelial cells under the stimulation of newly formed blood vessels on tumor cells. Therefore, the RDG short peptide modified on the shell of the ultrasound nanobubble obtained by the present invention can increase the enrichment of nano drug carriers in prostate cancer cells, thereby enhancing drug efficacy and gene transfection efficiency and reducing toxic side effects. FGFR gene abnormalities can lead to overactivation of the FGFR signaling pathway and further induce canceration in normal cells. The present invention silences the expression of FGFR by designing siRNA targeting the gene, thereby eliminating one of the causes of cell canceration. The outer shell of the ultrasonic nanobubble adsorbs siRNA, and the anticancer drug ENZ in the inner core is used in synergistic administration to improve the inhibitory effect on the proliferation of prostate cancer cells. The targeted ultrasonic nanobubble can be used in a multi-modal synergistic manner with chemotherapy and gene therapy to treat prostate cancer, and achieve targeted aggregation of drugs and genes at the site of prostate cancer; the perfluoropentane PFP contained in the inner core is an ultrasonic contrast agent. When ultrasound is applied, the nanobubble is exploded, which enhances the ultrasonic imaging effect, and at the same time releases more drugs and genes at the targeted site, which helps to improve the efficacy and gene transfection efficiency, and at the same time reduces systemic toxic side effects. The targeted ultrasonic nanobubble is expected to overcome multidrug resistance, thereby achieving a higher anti-tumor effect in vivo, and has excellent clinical application prospects.

[0008] Preferably, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.2.

[0009] Preferably, the shell of the targeted ultrasound nanobubble is DSPE-PEG-RGD; and the amino acid sequence of the RGD short peptide is cRGD.

[0010] Preferably, the anti-tumor drug includes ENZ.

[0011] In a second aspect, the present invention provides a method for preparing the above-mentioned targeted ultrasound nanobubbles, comprising the following steps:

[0012] (1) mixing DSPE-PEG-MAL and RGD powders in water, stirring, dialyzing, and freeze-drying to obtain DSPE-PEG-RGD freeze-dried powder;

[0013] (2) taking the anti-tumor drug, DSPE-PEG-PEI powder and the DSPE-PEG-RGD lyophilized powder obtained in step (1), adding an organic solvent, ultrasonically dissolving until fully mixed, adding PFP, ultrasonically treating to obtain a mixed solution, dropping it into deionized water, ice bathing, ultrasonicating, and dialyzing to obtain nanobubbles loaded with the anti-tumor drug;

[0014] (3) The nanobubbles loaded with anti-tumor drugs obtained in step (2) are mixed with siRNA targeting the FGFR gene, and incubated to obtain the targeted ultrasound nanobubbles.

[0015] The invention modifies RGD short peptide on PEGylated phospholipid (DPES-PEG) to obtain the shell of the nanobubble, the DPES-PEG end group contains a maleimide group, the RGD short peptide contains a thiol group -SH, and the maleimide group and the thiol group undergo Michael addition reaction to synthesize the shell DSPE-PEG-RGD required by the invention. The PEGylated phospholipid and the content self-assemble to form the nanobubble. As a cationic carrier, DSPE-PEG-RGD has a positive charge on its surface and binds to negatively charged siRNA by electrostatic adsorption.

[0016] Preferably, in step (1), in DSPE-PEG-MAL, the molecular weight of the PEG fragment is 2000-4000 Da.

[0017] Preferably, in step (1), RGD powder is added with water to prepare a 10 mg / mL aqueous solution which is then added dropwise to the reaction, so that the reaction can be better controlled.

[0018] Preferably, in step (1), the mass ratio of DSPE-PEG-MAL to RGD is DSPE-PEG-MAL:RGD=(10-100):(1-10).

[0019] Preferably, in step (1), the stirring time is 4-12 hours and the reaction temperature is 0-10°C.

[0020] Preferably, in step (2), the mass ratio of the anti-tumor drug, DSPE-PEG-PEI, DSPE-PEG-RGD and PFP is anti-tumor drug: DSPE-PEG-PEI: DSPE-PEG-RGD: PFP = (1-50): (2-100): (2-100): (5-250).

[0021] Preferably, in the step (2), after adding the organic solvent, the ultrasonic treatment conditions are: the ultrasonic power is 150W, and the working mode is: work for 3s and stop for 3s.

[0022] Preferably, in step (2), the molecular weight of the PEG fragment in DSPE-PEG-PEI is 2000-4000Da.

[0023] In DSPE-PEG derivatives, the molecular weight of PEG will affect the particle size and structural stability of nanobubbles.

[0024] Preferably, in step (2), the organic solvent is DMSO.

[0025] Preferably, in step (2), PFP is added dropwise into deionized water while being ultrasonicated using a cell crusher.

[0026] Preferably, in the step (2), after adding PFP, the temperature of ultrasonic treatment is 0-10°C.

[0027] Preferably, in step (3), the mass ratio of the nanobubbles loaded with anti-tumor drugs and siRNA is nanobubbles loaded with anti-tumor drugs: siRNA = (8-20): 1.

[0028] Nanobubbles loaded with anti-tumor drugs can be used as cationic carriers to electrostatically adsorb siRNA. When exploring the mass ratio of the carrier to siRNA, the present invention found that the ratio of the two will affect the drug administration effect. Within the ratio range of the present invention, the carrier can completely carry siRNA. If the amount of the carrier is insufficient, siRNA cannot be completely adsorbed. When nanobubbles are used for drug administration, siRNA will separate from the carrier and be degraded by enzymes, thereby failing to achieve the purpose of drug administration.

[0029] More preferably, in step (3), the mass ratio of the nanobubbles loaded with anti-tumor drugs and siRNA is nanobubbles loaded with anti-tumor drugs: siRNA = 20:1.

[0030] Preferably, in step (3), the incubation temperature is 4-10°C.

[0031] Preferably, in step (3), the incubation time is 15 minutes.

[0032] In a third aspect, the present invention provides the use of the above-mentioned targeted ultrasound nanobubbles in the preparation of drugs for treating prostate cancer.

[0033] Preferably, the prostate cancer is castration-resistant prostate cancer.

[0034] The targeted ultrasound nanobubbles of the present invention can down-regulate the expression of P-glycoprotein and FGFR1, improve multidrug resistance, and further treat castration-resistant prostate cancer.

[0035] In a fourth aspect, the present invention provides a drug for treating prostate cancer, comprising the above-mentioned targeted ultrasound nanobubbles.

[0036] The beneficial effects of the present invention are:

[0037] The nanobubble carrier of the present invention is modified with RGD peptide and has a tumor targeting effect. It can carry drugs and / or genes for targeted diseases, improve the bioavailability of drugs and genes, improve the specificity of drugs, realize gene transfection at the targeted site, and achieve precise treatment; and has the advantages of large drug loading capacity and low toxic and side effects.

[0038] The targeted ultrasonic nanobubbles prepared by the present invention have ultrasonic effects, can enhance the ultrasonic contrast effect, and simultaneously locate the ultrasonic blasting nanobubbles. This drug delivery method can improve drug delivery efficiency and gene transfection efficiency.

[0039] The targeted ultrasound nanobubbles prepared by the present invention can be combined with chemotherapy and gene therapy to synergistically treat castration-resistant prostate cancer in a multi-mode manner, which not only improves the treatment effect, but also provides a new strategy for overcoming multidrug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the transmission electron microscopy image of PFP-ENZ@siRNA-NBs-RGD.

[0041] Figure 2 The electrophoresis results of the condensation of PFP-ENZ@NBs-RGD and siRNA in different ratios.

[0042] Figure 3 The results show the effect of PFP-ENZ@siRNA-NBs-RGD on the activity of prostate cancer cells.

[0043] Figure 4 In vivo ultrasound imaging results of PFP-ENZ@siRNA-NBs-RGD in nude mice with prostate cancer model.

[0044] Figure 5 This is the inhibitory effect of PFP-ENZ@siRNA-NBs-RGD on the tumor size of nude mice with prostate cancer model.

[0045] Figure 6 The results show the effect of PFP-ENZ@siRNA-NBs-RGD on the survival rate of nude mice in the prostate cancer model. DETAILED DESCRIPTION

[0046] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0047] The raw materials and reagents used in the present invention are from the following sources: DSPE-PEG-MAL and ENZ were purchased from Aladdin Reagents (Shanghai) Co., Ltd.; DSPE-PEG-PEI, RGD short peptide, and siRNA were purchased from Guangzhou Yuanhe Biological Co., Ltd.; and PFP was purchased from J&K Reagents Company.

[0048] Preparation of 10 mg / mL RGD solution: Weigh RGD short peptide powder and dilute to 10 mg / mL with deionized water.

[0049] Embodiment 1:

[0050] An embodiment of the targeted ultrasound nanobubble of the present invention is a core-shell structure, wherein the outer shell is a nanobubble carrier DSPE-PEG-RGD, and the inner core includes perfluoropentane PFP and anti-tumor drug ENZ; the outer shell adsorbs siRNA targeting the FGFR gene.

[0051] The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2.

[0052] The amino acid sequence of the RGD short peptide is cRGD.

[0053] The method for preparing the targeted ultrasonic nanobubbles comprises the following steps:

[0054] (1) Preparation of DSPE-PEG-RGD:

[0055] Take 100 mg of DSPE-PEG-MAL powder, add 30 mL of deionized water and ultrasonically dissolve it for 10 minutes to make it fully dispersed, add 1 mL of RGD solution (10 mg / mL) dropwise, stir and react for 4 hours after the addition, then transfer to a 3500Da dialysis bag, dialyze with deionized water to remove free RGD, and freeze-dry to obtain DSPE-PEG-RGD lyophilized powder.

[0056] (2) Preparation of PFP-ENZ@NBs-RGD:

[0057] 50 mg ENZ, 100 mg DSPE-PEG-PEI powder and 100 mg DSPE-PEG-RGD lyophilized powder obtained in step (1) were mixed, 3 mL DMSO was added, and the mixture was ultrasonically dissolved for 10 min. After being fully mixed, 0.15 mL PFP (PFP ​​density is 1.63 g / mL) was added under ice bath conditions, and ultrasonic mixing was uniform. Then, it was added dropwise to 40 mL of deionized water, ice bathed, and ultrasonicated with a cell crusher while dropping. After the dropwise addition, it was ultrasonicated for another 10 min. Then, it was transferred to a 3500 Da dialysis bag and dialyzed at 4° C. for 48 h, during which the water was changed 10 times. After dialyzing to remove free ENZ, PFP and organic solvent DMSO, it was stored at low temperature to obtain a PFP-ENZ@NBs-RGD product solution.

[0058] (3) Preparation of gene-targeted ultrasound nanobubbles PFP-ENZ@siRNA-NBs-RGD by electrostatic adsorption method:

[0059] Take 100 μL of the PFP-ENZ@NBs-RGD solution and siRNA obtained in step (2), mix them in a mass ratio of PFP-ENZ@NBs-RGD solution: siRNA = 20:1, and incubate at room temperature for 15 minutes to obtain the targeted ultrasound nanobubble PFP-ENZ@siRNA-NBs-RGD.

[0060] Embodiment 2:

[0061] An embodiment of the targeted ultrasonic nanobubble of the present invention is different from Example 1 only in that: in step (3) of the preparation method of the targeted ultrasonic nanobubble of this embodiment, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=8:1.

[0062] Embodiment 3:

[0063] An embodiment of the targeted ultrasonic nanobubble of the present invention is different from embodiment 1 only in that in step (3) of the preparation method of the targeted ultrasonic nanobubble of this embodiment, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=10:1.

[0064] Embodiment 4:

[0065] An embodiment of the targeted ultrasonic nanobubble of the present invention is different from Example 1 only in that: in step (3) of the preparation method of the targeted ultrasonic nanobubble of this embodiment, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=12:1.

[0066] Embodiment 5:

[0067] An embodiment of the targeted ultrasonic nanobubble of the present invention is different from embodiment 1 only in that: in step (3) of the preparation method of the targeted ultrasonic nanobubble of this embodiment, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=14:1.

[0068] Embodiment 6:

[0069] An embodiment of the targeted ultrasound nanobubble of the present invention is a core-shell structure, wherein the outer shell is a nanobubble carrier DSPE-PEG-RGD, and the inner core includes perfluoropentane PFP and anti-tumor drug ENZ; the outer shell adsorbs siRNA targeting the FGFR gene.

[0070] The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2.

[0071] The amino acid sequence of the RGD short peptide is cRGD.

[0072] The method for preparing the targeted ultrasonic nanobubbles comprises the following steps:

[0073] (1) Preparation of DSPE-PEG-RGD:

[0074] Take 100 mg of DSPE-PEG-MAL powder, add 30 mL of deionized water and ultrasonically dissolve it for 10 minutes to make it fully dispersed, add 0.1 mL of RGD solution (10 mg / mL) dropwise, stir and react for 4 hours after the addition, then transfer to a 3500Da dialysis bag, dialyze with deionized water to remove free RGD, and freeze-dry to obtain DSPE-PEG-RGD lyophilized powder.

[0075] (2) Preparation of PFP-ENZ@NBs-RGD:

[0076] 25 mg ENZ, 50 mg DSPE-PEG-PEI powder and 50 mg DSPE-PEG-RGD lyophilized powder obtained in step (1) were mixed, 3 mL DMSO was added, and the mixture was ultrasonically dissolved for 10 min. After being fully mixed, 0.046 mL PFP (PFP ​​density is 1.63 g / mL) was added under ice bath conditions, ultrasonically mixed, and then added dropwise to 40 mL deionized water, ice bathed, ultrasonicated with a cell crusher while dropping, and ultrasonicated for another 10 min after the dropwise addition was completed; then, the mixture was transferred to a 3500 Da dialysis bag and dialyzed at 4° C. for 48 h, during which the water was changed 10 times; after dialyzing to remove free ENZ, PFP and organic solvent DMSO, the mixture was stored at low temperature to obtain a PFP-ENZ@NBs-RGD product solution.

[0077] (3) Preparation of gene-targeted ultrasound nanobubbles PFP-ENZ@siRNA-NBs-RGD by electrostatic adsorption method:

[0078] Take 100 μL of the PFP-ENZ@NBs-RGD solution and siRNA obtained in step (2), mix them in a mass ratio of PFP-ENZ@NBs-RGD solution: siRNA = 20:1, and incubate at room temperature for 15 minutes to obtain the targeted ultrasound nanobubble PFP-ENZ@siRNA-NBs-RGD.

[0079] Embodiment 7:

[0080] An embodiment of the targeted ultrasound nanobubble of the present invention is a core-shell structure, wherein the outer shell is a nanobubble carrier DSPE-PEG-RGD, and the inner core includes perfluoropentane PFP and anti-tumor drug ENZ; the outer shell adsorbs siRNA targeting the FGFR gene.

[0081] The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.2.

[0082] The amino acid sequence of the RGD short peptide is cRGD.

[0083] The method for preparing the targeted ultrasonic nanobubbles comprises the following steps:

[0084] (1) Preparation of DSPE-PEG-RGD:

[0085] Take 10 mg of DSPE-PEG-MAL powder, add 30 mL of deionized water and ultrasonically dissolve it for 10 minutes to make it fully dispersed, add 1 mL of RGD solution (10 mg / mL) dropwise, stir and react for 4 hours after the addition, then transfer to a 3500Da dialysis bag, dialyze with deionized water to remove free RGD, and freeze-dry to obtain DSPE-PEG-RGD lyophilized powder.

[0086] (2) Preparation of PFP-ENZ@NBs-RGD:

[0087] 50 mg ENZ, 100 mg DSPE-PEG-PEI powder and 100 mg DSPE-PEG-RGD lyophilized powder obtained in step (1) were mixed, 3 mL DMSO was added, and the mixture was ultrasonically dissolved for 10 min. After being fully mixed, 0.15 mL PFP (PFP ​​density is 1.63 g / mL) was added under ice bath conditions, and ultrasonic mixing was uniform. Then, it was added dropwise to 40 mL of deionized water, ice bathed, and ultrasonicated with a cell crusher while dropping. After the dropwise addition, it was ultrasonicated for another 10 min. Then, it was transferred to a 3500 Da dialysis bag and dialyzed at 4° C. for 48 h, during which the water was changed 10 times. After dialyzing to remove free ENZ, PFP and organic solvent DMSO, it was stored at low temperature to obtain a PFP-ENZ@NBs-RGD product solution.

[0088] (3) Preparation of gene-targeted ultrasound nanobubbles PFP-ENZ@siRNA-NBs-RGD by electrostatic adsorption method:

[0089] Take 100 μL of the PFP-ENZ@NBs-RGD solution and siRNA obtained in step (2), mix them in a mass ratio of PFP-ENZ@NBs-RGD solution: siRNA = 20:1, and incubate at room temperature for 15 minutes to obtain the targeted ultrasound nanobubble PFP-ENZ@siRNA-NBs-RGD.

[0090] Comparative Example 1:

[0091] A comparative example of the targeted ultrasonic nanobubbles of the present invention, the difference between the targeted ultrasonic nanobubbles and Example 1 is that: in step (3) of the preparation method of the targeted ultrasonic nanobubbles in this comparative example, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=2:1.

[0092] Comparative Example 2:

[0093] A comparative example of the targeted ultrasonic nanobubbles of the present invention, the difference between the targeted ultrasonic nanobubbles and Example 1 is that: in step (3) of the preparation method of the targeted ultrasonic nanobubbles in this comparative example, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=4:1.

[0094] Comparative Example 3:

[0095] A comparative example of the targeted ultrasonic nanobubbles of the present invention, the difference between the targeted ultrasonic nanobubbles and Example 1 is that: in step (3) of the preparation method of the targeted ultrasonic nanobubbles in this comparative example, PFP-ENZ@NBs-RGD and siRNA are mixed in a mass ratio of PFP-ENZ@NBs-RGD:siRNA=6:1.

[0096] Comparative Example 4:

[0097] A comparative example of the targeted ultrasound nanobubble of the present invention, the targeted ultrasound nanobubble (PFP-ENZ@NBs-RGD) is different from Example 1 only in that the targeted ultrasound nanobubble in this comparative example does not adsorb siRNA.

[0098] Comparative Example 5:

[0099] A comparative example of the targeted ultrasound nanobubble of the present invention, the targeted ultrasound nanobubble (PFP-ENZ@siRNA-NBs) is different from Example 1 only in that the targeted ultrasound nanobubble in this comparative example is not modified with RGD short peptide.

[0100] The method for preparing the targeted ultrasonic nanobubbles comprises the following steps:

[0101] (1) Preparation of PFP-ENZ@NBs:

[0102] 50 mg ENZ, 100 mg DSPE-PEG-PEI powder and 100 mg DSPE-PEG lyophilized powder were mixed, 3 mL DMSO was added and ultrasonically dissolved for 10 min, and mixed thoroughly; 0.15 mL PFP was added under ice bath conditions, ultrasonically mixed evenly, and then added dropwise to 40 mL deionized water, ice bath, ultrasonicated with a cell crusher while dropping, and ultrasonicated for another 10 min after the dropwise addition; then, it was transferred to a 3500Da dialysis bag and dialyzed at 4°C for 48 h, during which the water was changed 10 times; after dialyzing to remove free ENZ, PFP and organic solvent DMSO, it was stored at low temperature to obtain the PFP-ENZ@NBs product solution.

[0103] (2) Preparation of gene-carrying ultrasound nanobubbles PFP-ENZ@siRNA-NBs by electrostatic adsorption method:

[0104] Take 100 μL of the PFP-ENZ@NBs solution and siRNA obtained in step (1), mix them in a mass ratio of PFP-ENZ@NBs solution: siRNA = 20:1, and incubate at room temperature for 15 minutes to obtain the ultrasonic nanobubble PFP-ENZ@siRNA-NBs.

[0105] Test Example 1: Characterization of targeted ultrasound nanobubbles and drug loading rate detection

[0106] Test samples: gene-carrying targeted ultrasound nanobubbles PFP-ENZ@siRNA-NBs-RGD prepared in Examples 1, 6 and 7, and targeted ultrasound nanobubbles PFP-ENZ@NBs-RGD without siRNA adsorption in Comparative Example 4 as a control.

[0107] (1) Dynamic light scattering (DLS) was used to measure the particle size distribution of the sample. In this experiment, a Malvern Zetasizer Nano-ZS 90 particle size analyzer was used to measure the particle size distribution of the nanoparticles contained in the sample. The test temperature was 25 °C.

[0108] (2) PFP-ENZ@siRNA-NBs-RGD was observed using transmission electron microscopy.

[0109] (3) Detect the ENZ content in the test sample by HPLC: First, accurately weigh ENZ, add methanol to dissolve, and prepare a standard solution of 5-80 μg / mL. Use the peak area ratio as the ordinate and the concentration (μg / mL) as the abscissa to perform linear regression to obtain the linear regression equation, and calculate the ENZ content in the sample. Calculate the drug loading according to the following formula.

[0110] Drug loading (%) = the mass of drug (ENZ) contained in the sample / the total mass of the sample × 100%

[0111] The test was repeated three times in parallel and the average value was taken. The particle size and drug loading rate test results are shown in Table 1. The transmission electron microscopy (using the PFP-ENZ@siRNA-NBs-RGD sample prepared in Example 1) observation results are shown in Table 1. Figure 1 shown.

[0112] Table 1 Test results

[0113]

[0114]

[0115] The results show that the particle size distribution of the targeted ultrasonic nanobubbles prepared by the present invention is between 200-260nm, and the dispersibility is good. The drug loading amount of the targeted ultrasonic nanobubbles obtained by adsorbing siRNA is not much different from that of the targeted ultrasonic nanobubbles without adsorbing siRNA, indicating that the electrostatic adsorption of siRNA and the carrier PFP-ENZ@NBs-RGD will not affect its drug loading performance.

[0116] Test Example 2: Study on the condensation ratio of PFP-ENZ@NBs-RGD and siRNA

[0117] Test sample: PFP-ENZ@siRNA-NBs-RGD prepared in Examples 1-5 and Comparative Examples 1-3.

[0118] (1) The ability of targeted ultrasound nanobubbles PFP-ENZ@NBs-RGD to condense siRNA was investigated by agarose gel electrophoresis. 5 μL of the test sample was evenly mixed with an appropriate amount of loading buffer to make the concentration of the loading buffer 1X. The prepared sample was placed in a 4% agarose gel, and 12 μL was loaded into each well. The gel was electrophoresed at 120 mV for 20 min using 1×TAE buffer as the medium. After the electrophoresis, the gel was placed in a 0.5 μg / mL ethidium bromide (EtBr) solution for staining for 30 min, and developed using a gel imager at 365 nm to record the data.

[0119] The results are as follows Figure 2 As shown, when the mass ratio of PFP-ENZ@NBs-RGD to siRNA is greater than 6, PFP-ENZ@NBs-RGD can completely capture siRNA.

[0120] (2) To evaluate the transfection efficiency of PFP-ENZ@siRNA-NBs-RGD, the vector PFP-ENZ@NBs-RGD was incubated with eGFP siRNA (for observing fluorescence signals, purchased from Guangzhou Yuanhe Biotechnology Co., Ltd.) at a mass ratio (w / w) of 8, 10, 20, and 30, and then co-cultured with LNCaP cells. The expression level of eGFP was then evaluated, and fluorescence images were taken using a fluorescence microscope. ImageJ was used to measure the average fluorescence intensity. The greater the average fluorescence intensity, the higher the transfection efficiency of siRNA. The results are shown in Table 2.

[0121] Table 2

[0122] Mass ratio (w / w) 8 10 20 30 Average fluorescence intensity (a,u.) 0.589 1.323 2.268 1.159

[0123] Under the condition of ultrasonic power of 2W, when the mass ratio (w / w) was 20, the transfection efficiency of PFP-ENZ@siRNA-NBs-RGD was the best. Therefore, the targeted ultrasonic nanobubbles prepared in Example 1 (the mass ratio of PFP-ENZ@NBs-RGD to siRNA was 20:1) were used for subsequent experiments. Test Example 3: Effect of targeted ultrasonic nanobubbles on the activity of prostate cancer cells

[0124] The targeted ultrasound nanobubble PFP-ENZ@siRNA-NBs-RGD prepared in Example 1 was used as a sample, and different groups were set for drug administration. The CCK-8 method was used to determine the effect of the sample on the proliferation of prostate cancer cells under different concentration conditions and with or without ultrasound intervention. The cells used were ENZ-resistant LNCaP cells.

[0125] ENZ-resistant LNCaP cells were prepared by the following method: LNCaP cells were exposed to gradually increasing concentrations of ENZ (initial concentration was 10 μmol / L ENZ, then gradually increased to 30 μmol / L), and cells that could continue to grow in 30 μM ENZ for 6 months were selected to obtain ENZ-tolerant LNCaP cells.

[0126] The groups are as follows (each drug is diluted to a certain concentration using normal saline):

[0127] (1) ENZ treatment group (treatment concentrations were 5, 10, 15, 20, and 25 μM);

[0128] (2) PFP-ENZ@siRNA-NBs-RGD group (ENZ equivalents were 5, 10, 15, 20, and 25 μM);

[0129] (3) PFP-ENZ@siRNA-NBs-RGD (ENZ equivalents were 5, 10, 15, 20, and 25 μM) + ultrasound group (the ultrasound head coated with coupling agent was perpendicular to the bottom of the cell well plate, and ultrasound blasting was applied at a power of 2 W);

[0130] (4) Control group: The same amount of normal saline as the treatment group.

[0131] The specific operation is as follows: 100 μL of cell suspension (2000 cells / well) was inoculated in a 96-well plate; the cells were treated with different concentrations of drug diluent (concentrations were 5, 10, 15, 20, and 25 μM, respectively), the cell culture plate was removed after 24 hours, the culture medium was discarded, and 100 μL of a mixture of CCK8 and serum-free culture medium was added to each well (volume ratio CCK8: serum-free culture medium = 1:10); incubated at 37°C for 1 hour, the OD absorbance was detected at 450 nm using an enzyme reader, and the cell activity was calculated according to the following formula:

[0132] Cell activity (%) = (absorbance value of drug-added well - absorbance value of blank well) / (absorbance value of control well - absorbance value of blank well) × 100%

[0133] The results are as follows Figure 3 As shown, compared with the pure ENZ treatment group, the inhibitory effect of PFP-ENZ@siRNA-NBs-RGD treatment on ENZ-resistant LNCaP cells was more obvious, and the cell activity was further reduced after ultrasound irradiation, which shows that the targeted ultrasound nanobubbles prepared by the present invention can improve the resistance of prostate cancer cells to ENZ, thereby inhibiting tumor growth.

[0134] Test Example 4: In vivo targeting effect of targeted ultrasound nanobubbles in prostate cancer

[0135] Construction of ENZ-resistant nude mouse model: ENZ-resistant LNCaP cells (E-LNCaP, passaged, resuspended, and adjusted to a density of 5×10 7 Cells were inoculated in a clean operating table, the right back of the nude mouse was disinfected with alcohol, and the cell suspension was taken with a 16-gauge needle. The cell suspension was inoculated subcutaneously in the right armpit of the nude mouse (inoculation volume 100 μL). After the injection, a slight pressure was applied to prevent the cell suspension from leaking out. When the tumor volume reached about 100 mm 3 Five days later, the nude mice were randomly divided into 5 groups and injected with different samples through the tail vein. Six hours later, an ultrasound probe coated with a coupling agent was used to image the tumor site.

[0136] The groups are as follows:

[0137] (1) Blank control group (tail vein injection, 100 μL normal saline);

[0138] (2) Enzalutamide ENZ-treated group (tail vein injection, 10 mg / kg);

[0139] (3) PFP-ENZ@siRNA-NBs treatment group (PFP-ENZ@siRNA-NBs prepared in Comparative Example 5 were injected into the tail vein, with the equivalent of ENZ being 10 mg / kg);

[0140] (4) PFP-ENZ@siRNA-NBs-RGD treatment group (PFP-ENZ@siRNA-NBs-RGD prepared in Example 1 was injected into the tail vein, with an ENZ equivalent of 10 mg / kg, and the tumor site was irradiated (2W) with an ultrasound probe coated with a coupling agent for 60 s 6 h after injection);

[0141] Ultrasound imaging results such as Figure 4As shown, the blank control group (normal saline) and the ENZ treatment group showed low echo intensity. However, in the PFP-ENZ@siRNA-NBs treatment group, PFP underwent a liquid-gas phase transition after ultrasound, and the echo intensity increased compared with the previous two groups, indicating that the targeted ultrasound nanobubbles prepared by the present invention have good enhanced ultrasound performance. In addition, in the imaging, the echo intensity signal enhancement of PFP-ENZ@siRNA-NBs-RGD was the most obvious, because more nanobubbles were gathered in the tumor site, which proves that the modification of RGD on the nanovesicles of the present invention makes it have stronger tumor targeting in vivo.

[0142] Test Example 5: Anti-tumor effect of targeted ultrasound nanobubbles in a nude mouse model of castration-resistant prostate cancer

[0143] The targeted ultrasonic nanobubbles prepared in Example 1 were used as samples.

[0144] Construction of ENZ-resistant tumor-bearing nude mouse model: The construction method refers to Test Example 4. When the tumor volume reaches about 100 mm 3 At 14:00 p.m., castration surgery (testicle removal) was performed. Five days later, the nude mice were randomly divided into groups. The group that needed ultrasound was irradiated with an ultrasound probe coated with a coupling agent (2W) at the tumor site for 60 seconds 6 hours after injection. The mice were injected with drugs through the tail vein every other day. The body weight and tumor volume (V = 0.5 × a × b 2 ) and recorded the survival data of mice during the treatment period.

[0145] The groups are as follows:

[0146] (1) Blank control group (tail vein injection, 100 μL normal saline);

[0147] (2) Enzalutamide ENZ treatment group (tail vein injection, 10 mg / kg);

[0148] (3) PFP-ENZ@siRNA-NBs-RGD treatment group (PFP-ENZ@siRNA-NBs-RGD prepared in Example 1 was injected into the tail vein, and the equivalent of ENZ was 10 mg / kg);

[0149] (4) PFP-ENZ@siRNA-NBs-RGD+ultrasound treatment group (PFP-ENZ@siRNA-NBs-RGD prepared in Example 1 was injected into the tail vein, with the equivalent of ENZ being 10 mg / kg, and the tumor site was irradiated (2W) with an ultrasound probe coated with a coupling agent for 60 s 6 h after injection).

[0150] The results are as follows Figure 5As shown, compared with the blank control group (normal saline) and the ENZ treatment group, the PFP-ENZ@siRNA-NBs-RGD treatment group and the PFP-ENZ@siRNA-NBs-RGD+ultrasound treatment group showed anti-tumor effects (small tumor volume growth, that is, tumor growth was inhibited). Compared with the PFP-ENZ@siRNA-NBs-RGD treatment group, the PFP-ENZ@siRNA-NBs-RGD+ultrasound treatment group had a stronger inhibitory effect on tumor growth. This is because the targeted ultrasound nanobubbles respond to the release of more drugs and genes at the tumor site, and the ultrasound effect can improve the gene transfection efficiency, thereby improving the tumor inhibition effect. In addition, PFP-ENZ@siRNA-NBs-RGD+ultrasound treatment can significantly improve the survival rate of tumor-bearing nude mice ( Figure 6 ). Therefore, PFP-ENZ@siRNA-NBs-RGD administration combined with ultrasound multimodal synergistic therapy can enhance the therapeutic effect on castration-resistant prostate cancer.

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A targeted ultrasound nanobubble, characterized in that: The targeted ultrasound nanobubble is a core-shell structure, the outer shell is a PEGylated phospholipid modified with an RGD short peptide, and the inner core includes PFP and anti-tumor drugs; the outer shell adsorbs siRNA targeting the FGFR gene.

2. The targeted ultrasound nanobubble according to claim 1, characterized in that: The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

2.

3. The targeted ultrasound nanobubble according to claim 1, characterized in that: The shell of the targeted ultrasound nanobubble is DSPE-PEG-RGD; the amino acid sequence of the RGD short peptide is cRGD.

4. A method for preparing targeted ultrasound nanobubbles according to any one of claims 1 to 3, characterized in that: The steps include: (1) mixing DSPE-PEG-MAL and RGD powders in water, stirring, dialyzing, and freeze-drying to obtain DSPE-PEG-RGD freeze-dried powder; (2) taking the anti-tumor drug, DSPE-PEG-PEI powder and the DSPE-PEG-RGD lyophilized powder obtained in step (1), adding an organic solvent, ultrasonically dissolving until fully mixed, adding PFP, ultrasonically treating to obtain a mixed solution, dropping it into deionized water, ice bathing, ultrasonicating, and dialyzing to obtain nanobubbles loaded with the anti-tumor drug; (3) The nanobubbles loaded with anti-tumor drugs obtained in step (2) are mixed with siRNA targeting the FGFR gene, and incubated to obtain the targeted ultrasound nanobubbles.

5. The method for preparing targeted ultrasonic nanobubbles according to claim 4, characterized in that: In the step (1), the mass ratio of DSPE-PEG-MAL to RGD is DSPE-PEG-MAL:RGD=(10-100):(1-10).

6. The method for preparing targeted ultrasonic nanobubbles according to claim 4, characterized in that: In the step (2), the mass ratio of the anti-tumor drug, DSPE-PEG-PEI, DSPE-PEG-RGD and PFP is anti-tumor drug: DSPE-PEG-PEI: DSPE-PEG-RGD: PFP = (1-50): (2-100): (2-100): (5-250).

7. The method for preparing targeted ultrasonic nanobubbles according to claim 4, characterized in that: In the step (3), the mass ratio of the nanobubbles loaded with anti-tumor drugs and siRNA is nanobubbles loaded with anti-tumor drugs: siRNA = (8-20):

1.

8. Use of the targeted ultrasound nanobubble according to any one of claims 1 to 3 in the preparation of a drug for treating prostate cancer.

9. The use according to claim 8, characterized in that The prostate cancer is castration-resistant prostate cancer.

10. A drug for treating prostate cancer, characterized in that: It comprises the targeted ultrasound nanobubble as described in any one of claims 1 to 3.

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