A multifunctional cascade reactive nano-scale ultrasound contrast agent and its preparation method and application

Abstract

The present invention relates to a multifunctional cascade reactive nano - scale ultrasound contrast agent and its preparation method and application. The contrast agent (GHB - NPs) has a core of chitosan loaded with Basigin - siRNA and modified with hyaluronidase, and a shell membrane of phospholipid - polyethylene glycol - 2GSH. Perfluoropentane is encapsulated inside the shell membrane, and the particle size is 106 - 295 nm. For the GHB - NPs prepared by the present invention, DSPE - PEG2k - 2GSH on its surface can react with highly expressed γ - GGT on the surface of tumor cells, resulting in electrostatic repulsion to expose hyaluronidase, enabling GHB - NPs to penetrate deep into tumor tissues, and having targeting and high efficiency for the treatment of tumors. And it can be used in combination with UTMD. By inhibiting the β - catenin / c - Myc pathway, it down - regulates glycolysis - related proteins and reduces the production of lactic acid and ATP, which is beneficial to the treatment of tumor diseases. And the present invention has experimentally proved that GHB - NPs + UTMD has an obvious therapeutic effect on melanoma model mice, and can be used for the preparation of anti - tumor drugs to achieve the integration of tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to a multifunctional cascade reactive nano - scale ultrasound contrast agent and its preparation method and application, belonging to the technical field of ultrasound molecular imaging. Background Art

[0002] The heterogeneity and complexity of tumor metabolism pose a major challenge to tumor treatment. When tumors proliferate at a rate exceeding angiogenesis, tumor cells far from blood vessels become hypoxic, which promotes glycolysis and the secretion of lactate through monocarboxylate transporter 4 (MCT4). Tumor cells near blood vessels can obtain oxygen and absorb the high - concentration lactate in the tumor microenvironment with the help of monocarboxylate transporter 1 (MCT1) to generate ATP. This process is called "lactate shuttle". Monocarboxylate transporters (MCTs), especially MCT1 and MCT4, have essential but different functions in the transport of lactate and protons. A large number of studies have shown that the disruption of the MCT1 / 4 - basigin interaction hinders tumor progression. Therefore, basigin may be a target for disrupting the expression of MCTs on the cell membrane. However, the effect of siRNA treatment is limited. Ultrasound (US) - mediated gene delivery shows important clinical potential by transiently disrupting the vascular endothelial barrier and promoting cell internalization by enhancing cell membrane permeability. In addition, the combination of US and nano - scale ultrasound contrast agents can promote various cellular responses, including immunogenic cell death, epithelial - mesenchymal transition, and anti - tumor drug resistance. So far, there is a lack of research on the impact of US stimulation on metabolic symbiosis.

[0003] γ - Glutamyl transpeptidase (γ - GGT) is overexpressed on the outer surface of endothelial cells and the cell membranes of a variety of metabolically active tumor cells. The GSH group (Boc - γ - Glu(OtBu)-Cys(Trt)-Gly) is the natural and most active substrate of γ - GGT, which can be specifically recognized and cleaved by γ - GGT overexpressed in tumor tissues, resulting in a charge transition. However, the dense extracellular matrix of tumor tissues hinders the penetration of nanomaterials into deeper regions, thus limiting their efficacy. Hyaluronic acid (HA) is ubiquitous in the extracellular matrix of various solid tumors. Moreover, it severely hinders the penetration of nano - drugs. Hyaluronidase (HAase) has received extensive attention due to its ability to degrade hyaluronic acid. The direct injection of HAase has poor effects due to the presence of proteases in the blood and insufficient targeting. To address these challenges, the development of a tumor microenvironment - responsive HAase release system is crucial.

[0004] Chinese Patent Document CN109260480A discloses a chitosan nanoscale ultrasound contrast agent loaded with doxorubicin, its preparation method and application. The nanoscale ultrasound contrast agent uses chitosan as the shell membrane, and doxorubicin and perfluoropropane gas are wrapped inside the chitosan shell membrane, having strong enhanced imaging ability. However, the nanoscale ultrasound contrast agent prepared by this invention is in the form of nano liquid vesicles, with a low production rate of perfluoropropane nano liquid vesicles and poor stability. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a chitosan nanoscale ultrasound contrast agent loaded with Basigin-siRNA, and modifies hyaluronidase and γ-GGT enzyme-responsive lipids on its outer surface to improve the targeting and permeability of tumor tissues. This cascade reaction nanoscale ultrasound contrast agent is applied to block metabolic symbiosis. This contrast agent has good imaging ability, high biosafety, good stability, and strong ability to inhibit tumor growth.

[0006] Term Explanation:

[0007] Basigin-siRNA: It is a small interfering RNA that can degrade the transcribed RNA of basigin, resulting in the silencing of basigin gene expression.

[0008] DSPE-PEG2k-NH2: Phospholipid-polyethylene glycol-amine

[0009] GSH group (GSH): A polypeptide ligand that can bind to γ-GGT highly expressed on the surface of vascular endothelial cells and tumor cell membranes with high affinity, and its amino acid sequence is Boc-γ-Glu(OtBu)-Cys(Trt)-Gly.

[0010] Room temperature: It has the meaning of common knowledge in the art, generally 25±2°C.

[0011] The technical solution of the present invention is as follows:

[0012] A targeting material phospholipid-polyethylene glycol-2GSH (DSPE-PEG2k-2GSH) has the following structure:

[0013]

[0014] The preparation method of the above-mentioned targeting material phospholipid-polyethylene glycol-2GSH includes the following steps:

[0015] Dissolve 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2k-NH2) in chloroform, then add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine, and Nα,Nε-bis-Boc-L-lysine, and react at room temperature for 4 h. Remove the solvent by distillation under reduced pressure to obtain Product 1. Redissolve Product 1 in dichloromethane, add trifluoroacetic acid, and react at room temperature for 0.5 h. Remove the solvent by distillation under reduced pressure to obtain Product 2. Redissolve Product 2 in dimethylformamide, add PyBOP, triethylamine, and GSH group, and react at room temperature for 4 h. Remove the solvent by distillation under reduced pressure to obtain Product 3. Redissolve Product 3 in a mixed solution of acetyl chloride / methanol, react at room temperature for 0.5 h, and remove the solvent by distillation under reduced pressure to obtain Product 4. Adjust the pH to neutral after redissolving Product 4 in methanol, and obtain 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-2GSH (DSPE-PEG2k-2GSH) after dialysis and freeze-drying.

[0016] Preferably according to the present invention, the mass-volume ratio of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] to chloroform is (80 - 120):3, with the unit of mg / mL; more preferably 100:3, with the unit of mg / mL.

[0017] Preferably according to the present invention, the chemical equivalent ratio of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine, and Nα,Nε-bis-Boc-L-lysine is 1:(2.5 - 3.5):(2.5 - 3.5):(1 - 1.1); more preferably 1:3:3:1.1.

[0018] Preferably according to the present invention, the volume ratio of chloroform, dichloromethane, trifluoroacetic acid, dimethylformamide, the mixed solution of acetyl chloride / methanol, and methanol is 3:(1.5 - 2.5):(0.4 - 0.6):(2.5 - 3.5):3; more preferably 3:2:0.5:3:3:3.

[0019] Preferably according to the present invention, the chemical equivalent ratio of the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], PyBOP, triethylamine, and GSH group is 1:(2 - 2.5):(2.5 - 3.5):(2 - 2.5); more preferably 1:2.2:3.0:2.2.

[0020] Preferably according to the present invention, the mixed solution of acetyl chloride / methanol is obtained by mixing acetyl chloride and methanol in a volume ratio of 1:6.

[0021] Preferably according to the present invention, the cut-off molecular weight of the dialysis is 1500 - 2500 Da, more preferably 2500 Da.

[0022] The application of the above-mentioned targeting material phospholipid-polyethylene glycol-2GSH in the preparation of ultrasonic contrast agents.

[0023] A multifunctional cascade reactive nano-scale ultrasonic contrast agent carrying Basigin-siRNA, with chitosan loaded with Basigin-siRNA and modified with hyaluronidase as the core, and phospholipid-polyethylene glycol-2GSH as the shell membrane. Perfluoropentane is encapsulated inside the shell membrane; the particle size of the multifunctional cascade reactive nano-scale ultrasonic contrast agent carrying Basigin-siRNA is 106 - 295 nm.

[0024] The preparation method of the above-mentioned multifunctional cascade reactive nano-scale ultrasonic contrast agent carrying Basigin-siRNA includes the following steps:

[0025] (1) Add chitosan to acetic acid, and after fully dissolving, obtain a chitosan solution;

[0026] (2) Disperse perfluorohexane, Tween 20, and lecithin in deionized water, and homogenize to obtain a suspension;

[0027] (3) Sequentially add basigin-siRNA and the chitosan solution to the suspension, and homogenize to obtain an emulsion;

[0028] (4) After allowing the emulsion to stand at room temperature, centrifuge at low speed, collect the middle layer, then centrifuge at high speed, and take the precipitate to obtain chitosan loaded with Basigin-siRNA;

[0029] (5) Disperse the chitosan loaded with Basigin-siRNA in deionized water, add hyaluronidase, incubate under ice bath conditions for 15 - 60 min, then centrifuge at high speed to obtain chitosan loaded with Basigin-siRNA and modified with hyaluronidase;

[0030] (6) Disperse the chitosan loaded with Basigin-siRNA and modified with hyaluronidase in deionized water, add phospholipid-polyethylene glycol-2GSH, incubate under ice bath conditions for 6 - 12 h, then centrifuge at high speed, wash, and obtain the multifunctional cascade reactive nano-scale ultrasonic contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0031] Preferably according to the present invention, in step (1), the concentration of the chitosan solution is 15 - 20 mg / mL, and the concentration of acetic acid is 0.5 - 1%; preferably 20 mg / mL and 1%.

[0032] Preferably according to the present invention, in step (2), the volume-mass ratio of perfluorohexane, Tween 20, lecithin and deionized water is (0.1 - 2):(0.001 - 0.01):(0.001 - 0.01):(2 - 3), with the unit of g / mL; preferably, it is 0.15 mL:0.006 mL:0.004 g:2.7 mL.

[0033] Preferably according to the present invention, in step (2), the homogenization is specifically: under ice bath conditions, using an ultrasonic cell disruptor to process for 5 - 10 min at a power of 100 W, with the process of oscillation and pause alternating, and the oscillation or pause time being 5 - 15 s; preferably, it is processed for 5 - 10 min, with the process of oscillation and pause alternating, and the oscillation or pause time being 10 s.

[0034] Preferably according to the present invention, in step (3), the volume ratio of basigin-siRNA, chitosan and the suspension is 1:(2 - 2.5):(25 - 30); preferably, it is 1:2.25:27.

[0035] Preferably according to the present invention, in step (3), the homogenization is specifically: under ice bath conditions, using an ultrasonic cell disruptor to process for 1 - 5 min at a power of 65 - 100 W, with the process of oscillation and pause alternating, and the oscillation or pause time being 5 - 10 s; preferably, the power is 65 W, processed for 3 min, with the process of oscillation and pause alternating, and the oscillation or pause time being 8 s.

[0036] Preferably according to the present invention, in steps (4) - (6), the speed of low-speed centrifugation is 100 - 300 rpm, the time is 1 - 5 min, the speed of high-speed centrifugation is 12000 - 13000 rpm, and the time is 5 - 10 min; preferably, it is 300 rpm, 3 min and 13000 rpm, 5 min.

[0037] Preferably according to the present invention, in step (5), the mass-volume ratio of chitosan loaded with Basigin-siRNA, deionized water and hyaluronidase is (2 - 5):(1 - 3):(2 - 5), with the unit of mg / mL; preferably, it is 4.5 mg:3 mL:4 mg.

[0038] Preferably according to the present invention, in step (6), the mass-volume ratio of chitosan loaded with Basigin-siRNA and modified with hyaluronidase, deionized water and phospholipid-polyethylene glycol-2GSH is (2 - 5):(1 - 3):(0.1 - 0.3), with the unit of mg / mL; preferably, it is 4.5 mg:3 mL:0.15 mg.

[0039] Use of the multifunctional cascade reactive nano-ultrasound contrast agent carrying Basigin-siRNA in the preparation of anti-tumor drugs.

[0040] Preferably according to the present invention, the anti-tumor drug can effectively block the metabolic symbiosis of tumor cells.

[0041] The experimental steps not detailed in the present invention are carried out according to the conventional operations in the technical field.

[0042] Technical features and beneficial effects of the present invention:

[0043] 1. The multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA prepared by the present invention, the DSPE-PEG2k-2GSH on its surface can react with the highly expressed γ-GGT on the surface of tumor cells, and electrostatic repulsion exposes hyaluronidase, enabling GHB-NPs to penetrate deep into tumor tissues, which has targeting and high efficiency for tumor treatment.

[0044] 2. The multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying basigin-siRNA prepared by the present invention can improve the cell membrane permeability under the action of ultrasound irradiation, significantly improve the gene transfection efficiency, and is conducive to the targeted delivery of the target gene.

[0045] 3. The multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying basigin-siRNA prepared by the present invention has good ultrasound-enhanced contrast ability, high biological safety, and can specifically react in the tumor microenvironment.

[0046] 4. The multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying basigin-siRNA provided by the present invention can be used in combination with ultrasound-targeted microbubble destruction technology (UTMD). By inhibiting the β-catenin / c-Myc pathway, it down-regulates glycolysis-related proteins, reduces lactic acid and ATP production, which is beneficial to the treatment of tumor diseases. And the present invention has proved through experiments that GHB-NPs + UTMD has an obvious therapeutic effect on melanoma model mice, can be used in the preparation of anti-tumor drugs, and realizes tumor diagnosis and treatment integration. Brief Description of the Drawings

[0047] Figure 1 1H NMR identification of the targeting material phospholipid-polyethylene glycol-2GSH (DSPE-PEG2k-2GSH).

[0048] Figure 2 Synthesis schematic diagram and materials used for the multifunctional cascade reactive nano-ultrasound contrast agent carrying Basigin-siRNA.

[0049] Figure 3 Characterization and detection of a multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA;

[0050] In the figure, A is a transmission electron micrograph, B is a particle size distribution diagram (ordinate is light intensity, abscissa is particle size), C is a surface charge diagram (ordinate is zeta potential), and D is a fluorescence micrograph.

[0051] Figure 4 Entrapment efficiency of a multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA;

[0052] In the figure, A is the entrapment efficiency of basigin-siRNA, and B is the entrapment efficiency of hyaluronidase.

[0053] Figure 5 Charge conversion ability of a multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA;

[0054] In the figure, A is a surface charge change diagram (ordinate is zeta potential), and B is a transmission electron micrograph;

[0055] Figure 6 Cumulative release ability of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0056] Figure 7 Hyaluronic acid degradation ability of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0057] Figure 8 Targeting ability of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0058] In the figure, A and B are the fluorescence results and flow cytometry detection results of γ-GGT-responsive cell binding, respectively, and C and D are the fluorescence results and flow cytometry detection results of hyaluronidase-responsive cell binding, respectively.

[0059] Figure 9 In vivo ultrasound imaging of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0060] Figure 10 Investigation on the inhibition of glycolysis process by ultrasound-targeted microbubble destruction technology using a multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA;

[0061] In the figure, A is the immunoblotting result diagram under different conditions, and B is the columnar analysis corresponding to diagram A;

[0062] Figure 11 It shows the situation of treating in - vivo tumors with a multifunctional cascade - reactive nano - ultrasonic contrast agent carrying Basigin - siRNA under different conditions;

[0063] In the figure, A is the tumor growth situation of each group during the treatment, and B is the weight of the excised tumors of each group after the treatment. Specific implementation manners

[0064] The technical solutions of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods well - known to those skilled in the art.

[0065] For the materials and reagents involved in the embodiments, unless otherwise specified, they are all ordinary commercially available products; for the experimental operations involved in the embodiments, unless otherwise specified, they are all carried out according to the conventional operations in the art.

[0066] The present invention will be further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited only to this.

[0067] Chitosan was purchased from Solarbio, product number CAS 83512 - 85 - 0, with a weight - average molecular weight of 100 - 300 KD; Tween 20 was purchased from Solarbio, product number CAS 9005 - 64 - 5 / T8220; Lecithin was purchased from Macklin, product number CAS8002 - 43 - 5; DSPE - PEG2k - NH2 and GSH groups were purchased from Xi'an Ruixi, and basigin - siRNA was purchased from GenePharma.

[0068] For the drugs and reagents involved in this embodiment, unless otherwise specified, they are all ordinary commercially available products.

[0069] Example 1: Synthesis and identification of the targeting material DSPE - PEG2k - 2GSH

[0070] 1. The synthesis of the targeting material phospholipid - polyethylene glycol - 2GSH (DSPE - PEG2k - 2GSH) is as follows:

[0071] Dissolve 100 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-NH2 (DSPE-PEG2k-NH2) in 3 ml of chloroform, then add 3.0 eq. of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 3.0 eq. of 4-dimethylaminopyridine and 1.1 eq. of Nα,Nε-bis-Boc-L-lysine, and react at room temperature for 4 h. Remove the solvent by distillation under reduced pressure to obtain Product 1; redissolve Product 1 in 2 ml of dichloromethane, add 0.5 ml of trifluoroacetic acid, and react at room temperature for 0.5 h. Remove the solvent by distillation under reduced pressure to obtain Product 2; redissolve Product 2 in 3 ml of N,N-dimethylformamide, add 2.2 eq. of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (PyBOP), 3.0 eq. of triethylamine and 2.2 eq. of glutathione (GSH) group, and react at room temperature for 4 h. Remove the solvent by distillation under reduced pressure to obtain Product 3; redissolve Product 3 in 3 ml of acetyl chloride / methanol mixed solution, react at room temperature for 0.5 h, and remove the solvent by distillation under reduced pressure to obtain Product 4; after redissolving Product 4 in 3 ml of methanol, adjust the pH to neutral with aqueous sodium hydroxide solution, transfer it to a dialysis bag (cut-off molecular weight = 2000 Da) and dialyze in pure water for 24 h. Collect the dialysate and freeze-dry it to obtain 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]-2GSH (DSPE-PEG2k-2GSH).

[0072] 2. Identification of the targeting material DSPE-PEG2k-2GSH:

[0073] The DSPE-PEG2k-2GSH was identified by 1H nuclear magnetic resonance (1H NMR) method, and the results are as Figure 1 shown. It can be seen from Figure 1 that the peaks corresponding to the GSH polypeptide were observed in the final product, indicating that the material was correctly synthesized and DSPE-PEG2k-2GSH was successfully synthesized.

[0074] Example 2. Preparation of a multifunctional cascade reactive nano-ultrasound contrast agent carrying Basigin-siRNA

[0075] As Figure 2 shown, a preparation method of a multifunctional cascade reactive nano-ultrasound contrast agent carrying Basigin-siRNA includes the following steps:

[0076] (1) Add 20 mg of chitosan to 1 mL of acetic acid (concentration: 1%), and after complete dissolution, obtain a chitosan solution;

[0077] (2) Disperse 0.15 ml of perfluorohexane, 0.006 ml of Tween 20, and 0.004 g of lecithin in 2.7 mL of deionized water, and use an ultrasonic cell disruptor to treat it at a power of 100 W for 5 min under ice bath conditions. During the treatment, oscillation and pause are alternated, and the oscillation or pause time is 10 s to obtain a suspension;

[0078] (3) Add 100 μL of basigin-siRNA and 225 μL of the chitosan solution obtained in step (1) successively to 2.7 mL of the suspension obtained in step (2), so that the final concentration of basigin-siRNA in the suspension is 250 μM. Under ice bath conditions, use an ultrasonic cell disruptor to process for 3 min at a power of 65 W. During the process, alternate between shaking and pausing. The shaking or pausing time is 8 s to obtain an emulsion;

[0079] (4) After allowing the emulsion to stand at room temperature, centrifuge at a low speed of 300 rpm for 3 min, collect the middle layer, and then centrifuge at a high speed of 13000 rpm for 5 min. Take the precipitate to obtain chitosan loaded with Basigin-siRNA;

[0080] (5) Disperse 4.5 mg of chitosan loaded with Basigin-siRNA in 3 mL of deionized water, add 4 mg of hyaluronidase, incubate for 30 min using a magnetic stirrer under ice bath conditions, and then centrifuge at a high speed of 13000 rpm for 5 min. Take the precipitate to obtain chitosan loaded with Basigin-siRNA and modified with hyaluronidase;

[0081] (6) Disperse 4.5 mg of chitosan loaded with Basigin-siRNA and modified with hyaluronidase in 3 mL of deionized water, add 0.15 mg of phospholipid-polyethylene glycol-2GSH, incubate for 12 h using a magnetic stirrer under ice bath conditions, and then centrifuge at a high speed of 13000 rpm for 5 min. Wash 3 times with phosphate buffer to remove impurities to obtain a multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0082] Label GHB-NPs with FAM-siRNA instead of basigin-siRNA according to the existing method to obtain FAM-labeled GHB-NPs for standby.

[0083] Example 3

[0084] A method for preparing a multifunctional cascade reactive nano-ultrasound contrast agent carrying Basigin-siRNA, the steps are as described in Example 2, except that in step (3), the volume of basigin-siRNA is 150 μL, and the final concentration of basigin-siRNA in the suspension is 375 μM.

[0085] Example 4

[0086] A method for preparing a multifunctional cascade reactive nano - scale ultrasound contrast agent carrying Basigin - siRNA, the steps are as described in Example 2, except that in step (3), the volume of basigin - siRNA is 75 μL, and the final concentration of basigin - siRNA in the suspension is 187.5 μM.

[0087] Example 5

[0088] A method for preparing a multifunctional cascade reactive nano - scale ultrasound contrast agent carrying Basigin - siRNA, the steps are as described in Example 2, except that in step (3), the volume of basigin - siRNA is 50 μL, and the final concentration of basigin - siRNA in the suspension is 125 μM.

[0089] Example 6

[0090] A method for preparing a multifunctional cascade reactive nano - scale ultrasound contrast agent carrying Basigin - siRNA, the steps are as described in Example 2, except that in step (5), the mass of hyaluronidase is 2 mg.

[0091] Example 7

[0092] A method for preparing a multifunctional cascade reactive nano - scale ultrasound contrast agent carrying Basigin - siRNA, the steps are as described in Example 2, except that in step (5), the mass of hyaluronidase is 3 mg.

[0093] Example 8

[0094] A method for preparing a multifunctional cascade reactive nano - scale ultrasound contrast agent carrying Basigin - siRNA, the steps are as described in Example 2, except that in step (5), the mass of hyaluronidase is 5 mg.

[0095] Comparative Example 1

[0096] A method for preparing an ultrasound contrast agent, the steps are as described in Example 2, except that in step (6), 0.15 mg of DSPE - PEG2k - 2GSH is replaced with 0.15 mg of DSPE - PEG2k - NH2 to obtain an ultrasound contrast agent (DHB - NPs) using DSPE - PEG2k - NH2 as the coating.

[0097] According to the existing method, FAM - siRNA is used to replace basigin - siRNA to label DHB - NPs, and FAM - labeled DHB - NPs are obtained for standby.

[0098] Comparative Example 2

[0099] A preparation method of an ultrasound contrast agent, the steps are as described in Example 2, the difference is that in step (3), basigin-siRNA is not added, and the obtained ultrasound contrast agent (GH-NPs) does not carry basigin-siRNA.

[0100] Comparative Example 3

[0101] A preparation method of an ultrasound contrast agent, the steps are as described in Example 2, the difference is that in step (5), hyaluronidase is not added, and the obtained ultrasound contrast agent (GB-NPs) is not modified with hyaluronidase.

[0102] Example 9: Characterization of the properties of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA

[0103] 1. Observation of the structure of GHB-NPs

[0104] After diluting the GHB-NPs prepared in Example 2 with PBS buffer and observing, the transmission electron microscope of GHB-NPs is as Figure 3 shown in A.

[0105] As can be seen from Figure 3 A, GHB-NPs are round, with a smooth and translucent surface, having a clear core-shell structure, and a narrow band (red arrow) can be observed outside it.

[0106] After diluting the GHB-NPs prepared in Example 2 with PBS buffer, the particle size and Zeta potential were measured by dynamic light scattering method, and the results are as Figure 3 shown in B and C.

[0107] As can be seen from Figure 3 B and C, the particle size of GHB-NPs is 285.4 ± 10.93 nm, the polydispersity index is 0.212 ± 0.02, and the Zeta potential is -(22.20 ± 1.28) mV.

[0108] 2. After diluting the GHB-NPs prepared in Example 2 with PBS buffer and dropping them onto a glass slide, observation was carried out through a fluorescence microscope, and the results are as Figure 3 shown in D.

[0109] As can be seen from Figure 3 D, FAM-siRNA is green, Dil-labeled DSPE-PEG2k-2GSH is red, and the FAM / Dil image is yellow, indicating the superposition of FAM and Dil, indicating that the contrast agent GHB-NPs was successfully prepared.

[0110] Example 10: Determination of the encapsulation efficiency of multifunctional cascade-responsive nano-ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA

[0111] 1. The RiboGreen RNA quantification kit was used to determine the encapsulation efficiency of basigin-siRNA in the GHB-NPs prepared in Examples 2-5. The results are as Figure 4 shown in A.

[0112] As can be seen from Figure 4 A, when the final concentration of basigin-siRNA was 250 μM, the encapsulation efficiency of basigin-siRNA was the highest.

[0113] 2. The BCA kit and method were used to determine the encapsulation efficiency of hyaluronidase in the GHB-NPs prepared in Examples 2, 6-8. The results are as Figure 4 shown in B.

[0114] As can be seen from Figure 4 B, when the mass of hyaluronidase was 5 mg, the encapsulation efficiency of hyaluronidase was the highest.

[0115] Example 10: Determination of the charge conversion ability of multifunctional cascade-responsive nano-ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA

[0116] The GHB-NPs prepared in Example 2 were respectively dispersed in γ-GGT solutions of 0.05 U / mL and 10 U / mL and co-incubated (0.05 U / mL simulates the normal blood environment, and 10 U / mL simulates the tumor microenvironment). The surface charge was measured by dynamic light scattering method and the morphology was observed by transmission electron microscope. The results are as Figure 5 shown.

[0117] As can be seen from Figure 5 A, in the γ-GGT solution of 10 U / mL, the surface charge of GHB-NPs was converted from negative charge to positive charge. In the γ-GGT solution of 0.05 U / mL, the surface charge of GHB-NPs was stably negative. At the same time, the nano-ultrasound contrast agent without DSPE-PEG2k-2GSH showed a stable positive charge, indicating that the charge conversion was the result of the reaction between DSPE-PEG2k-2GSH and γ-GGT.

[0118] As can be seen from Figure 5 B, the narrow band on the periphery of GHB-NPs disappeared in the γ-GGT solution of 10 U / mL, and the narrow band was still visible in the γ-GGT solution of 0.05 U / mL, proving that DSPE-PEG2k-2GSH can be gradually shed in the tumor microenvironment.

[0119] Example 11: Determination of the drug release ability of multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA

[0120] Place the GHB-NPs prepared in Example 2 in a dialysis bag, and place the dialysis bag in γ-GGT solutions of 0.05 U / mL and 10 U / mL respectively. Then place it in a constant temperature shaking incubator and shake at 37 °C and 200 rpm to determine the drug release ability of GHB-NPs. The specific method is as follows:

[0121] At different time intervals (0, 1, 2, 3, 4, 5, 6, 7, 8 h), take 1 ml of the release medium in the dialysis bag for hyaluronidase concentration determination, and replace it with an equal volume of deionized water at 37 °C to keep the total volume of the release medium unchanged. The released medium taken out is detected by the BCA method and the absorbance is measured at a wavelength of 562 nm to calculate the concentration of hyaluronidase in the release medium. The cumulative drug release amount is calculated by the following formula:

[0122]

[0123] where, R is the cumulative drug release rate; V e is the replacement volume of deionized water; V0 is the total volume of the release medium; Ci is the hyaluronidase concentration in the release medium at the i-th replacement sampling; mdrug: the total mass of the drug carried by the nanoparticles; n is the number of times of replacing PBS. The results are as Figure 6 shown.

[0124] It can be seen from Figure 6 that the cumulative drug release rate of GHB-NPs in the γ-GGT solution of 0.05 U / mL at 8 h (8.99%) is lower than that of GHB-NPs in the γ-GGT solution of 10 U / mL at 8 h (78.19%). This indicates that GHB-NPs can release hyaluronidase at the tumor tissue site.

[0125] Example 12: Evaluation of the ability of multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA to degrade hyaluronic acid

[0126] Seed 2×10 4 B16F10 cells per well in a 24-well plate. Use PBS buffer (Control), hyaluronidase (HAase), FAM-labeled DHB-NPs (Comparative Example 1, DHB) and FAM-labeled GHB-NPs (Example 2, GHB) to co-culture with the cells for 6 h respectively. Wash away the unbound contrast agent with PBS buffer, and stain the cells with DAPI dye. Use a confocal microscope to analyze the degradation ability. The results are asFigure 7 as shown

[0127] As Figure 7 shown, with the hyaluronidase group as the positive control group, the degradation level of hyaluronidase after treatment with GHB-NPs was roughly similar to that of the positive group. This confirmed that GHB-NPs could effectively decompose the hyaluronic acid around tumor cells. In contrast, the hyaluronic acid degradation in the DHB-NPs group was insufficient, indicating that the DSPE-PEG2-2GSH coating could achieve responsive exposure of hyaluronidase at the tumor site.

[0128] Example 13: Analysis of the targeting ability of a multifunctional cascade-responsive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA

[0129] 1. γ-GGT-responsive targeting ability

[0130] 2×10 5 MDA-MB-231 cells per well were seeded in a 24-well plate. When the cells grew to a density of 50 - 60%, FAM-labeled DHB-NPs (Comparative Example 1, DHB) and FAM-labeled GHB-NPs (Example 2, GHB) were co-cultured with the cells for 6 h. At the same time, cells without adding any reagents were used as the blank group (Blank). Then, the unbound contrast agent was washed away with PBS buffer, and the cells were stained with DAPI dye. The binding ability was analyzed by confocal microscopy and flow cytometry, and the results are as Figure 8 shown in A and B.

[0131] As Figure 8 shown in A and B, the internalization of GHB-NPs by B16F10 cells was significantly higher than that of DHB-NPs. This phenomenon indicates that DSPE-PEG2k-2GSH is triggered to shed in the tumor microenvironment by γ-GGT, exposing the positively charged NPs, which bind to tumor cells through electrostatic attraction, thus enhancing the absorption of siRNA by tumor cells.

[0132] 2. Hyaluronidase-responsive targeting ability

[0133] 2×10 5 MDA-MB-231 cells per well were seeded in a 24-well plate. When the cells grew to a density of 50 - 60%, FAM-labeled DHB-NPs (Comparative Example 1) and FAM-labeled GHB-NPs (Example 2) were co-cultured with the cells for 6 h. At the same time, cells without adding any reagents were used as the blank group (Blank). The unbound contrast agent was washed away with PBS buffer, and the cells were stained with DAPI dye. The binding ability was analyzed by confocal microscopy and flow cytometry, and the results are as Figure 8 shown in C and D.

[0134] As can be seen from Figure 8 C and D, the internalization of B16F10 cells to GHB-NPs is significantly higher than that to GB-NPs. This indicates that hyaluronidase can enhance the absorption of siRNA by decomposing hyaluronic acid.

[0135] Example 14: Evaluation of the in vivo ultrasound contrast ability of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA

[0136] Place the GHB-NPs prepared in Example 2 in a round hole made of agar gel, and use the contrast mode of a GE logiq E9 ultrasound diagnostic instrument, with a frequency of 9.0 MHz and a mechanical index (MI) of 0.5. Observe synchronously in the two-dimensional and ultrasound contrast modes, adjust the parameter settings, store the image data using the internal workstation of the ultrasound instrument, and use PBS buffer as the control group. The results are as Figure 9 shown.

[0137] As can be seen from Figure 9 it that enhanced imaging appears for GHB-NPs under ultrasound, while no enhanced imaging appears in the control group.

[0138] Example 14: Inhibiting tumor cell glycolysis by a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA combined with ultrasound-targeted microbubble destruction technology

[0139] Seed 5×10 5 B16F10 cells per well in a 6-well plate. When the cells grow to a density of 50-70%, give three different treatments as follows:

[0140] Control group: negative control group, add PBS buffer;

[0141] GH-NPs group: add an ultrasound contrast agent without carrying basigin-siRNA (Comparative Example 2);

[0142] GH-NPs + US group: add an ultrasound contrast agent without carrying basigin-siRNA (Comparative Example 2) and perform ultrasound irradiation;

[0143] Among them, the ultrasound irradiation intensity is 1 W / cm 2 and the time is 30 s.

[0144] After the treatment, the results are as Figure 10 shown.

[0145] As can be seen from Figure 10It can be seen that, compared with the control group, the levels of related proteins in the GH-NPs group were comparable to those in the control group, indicating that GH-NPs had no effect on the glycolysis process. However, after ultrasound irradiation, it was found that the levels of related proteins were significantly downregulated. In summary, the ultrasound-targeted microbubble destruction technology downregulates the expression of glycolysis-related proteins by inhibiting the β-catenin / c-Myc pathway.

[0146] Example 14: Evaluation of the in vivo tumor growth inhibitory ability of multifunctional cascade reactive nano-ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA

[0147] Healthy C57BL / 6 mice (5 mice per group) were injected with B16F10 cell suspension (1×10 6 ) into the right back to construct melanoma model mice. When the tumor volume reached an appropriate size, different treatments were given on days 0, 3, 6, and 9 respectively. The treatment groups were as follows:

[0148] Control group: negative control group, added with PBS buffer;

[0149] GH-NPs group: added with ultrasound contrast agent without carrying basigin-siRNA (Comparative Example 2), without ultrasound irradiation;

[0150] GH-NPs+US group: added with ultrasound contrast agent without carrying basigin-siRNA (Comparative Example 2), with ultrasound irradiation;

[0151] GB-NPs group: ultrasound contrast agent without modified hyaluronidase (Comparative Example 3), without ultrasound irradiation;

[0152] GHB-NPs group: added with GHB-NPs, without ultrasound irradiation;

[0153] GHB-NPs+US group: added with GHB-NPs, with ultrasound irradiation;

[0154] Among them, the ultrasound irradiation intensity was 1.5W / cm 2 , and the time was 60s. The body weight and tumor volume were recorded every two days (tumor volume calculation formula = 0.5×length×width 2 ), and the treatment ended on the 14th day. The results are as Figure 11 shown

[0155] As Figure 11 shown in A, compared with the control group, the GH-NPs group had almost no inhibitory effect on tumor growth, and the GHB-NPs+US group had the most significant inhibitory ability on tumor growth.

[0156] As Figure 11As can be seen from Table B, the tumor weight of the GHB-NPs+US group (0.13±0.05 g) was significantly lower than that of the GHB-NPs group (0.58±0.15 g) and the GH-NPs+US group (0.53±0.11 g).

[0157] The above data fully demonstrate that the multifunctional cascade reaction nano-ultrasound contrast agent (GHB-NPs) carrying basigin-siRNA provided by the present invention can be used in combination with ultrasound-targeted destruction technology (UTMD). By inhibiting the β-catenin / c-Myc pathway, it downregulates glycolysis-related proteins, reduces lactic acid and ATP production, which is beneficial to the treatment of tumor diseases. And the present invention has experimentally proven that GHB-NPs+UTMD has an obvious therapeutic effect on melanoma model mice, can be used to prepare anti-tumor drugs, and realizes the integration of tumor diagnosis and treatment.

[0158] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA, characterized in that: The core is chitosan loaded with Basigin-siRNA and modified with hyaluronidase, the shell membrane is phospholipid-polyethylene glycol-2GSH, and the inside of the shell membrane is wrapped with perfluoropentane; the particle size of the multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA is 106-295nm; The phospholipid-polyethylene glycol-2GSH is characterized by having the following structure: ； The preparation method of the multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA comprises the following steps: (1) adding chitosan into acetic acid and fully dissolving the chitosan to obtain a chitosan solution; (2) dispersing perfluorohexane, Tween 20 and lecithin in deionized water and homogenizing to obtain a suspension; (3) adding basigin-siRNA and chitosan solution to the suspension in sequence and homogenizing to obtain an emulsion; (4) After the emulsion is allowed to stand at room temperature, it is centrifuged at low speed, the middle layer is collected, and then centrifuged at high speed to obtain the precipitate to obtain chitosan loaded with Basigin-siRNA; (5) Dispersing the chitosan loaded with Basigin-siRNA in deionized water, adding hyaluronidase, incubating in an ice bath for 15 to 60 minutes, and then centrifuging at high speed to obtain chitosan loaded with Basigin-siRNA and modified with hyaluronidase; (6) Chitosan loaded with Basigin-siRNA and modified with hyaluronidase was dispersed in deionized water, and phospholipid-polyethylene glycol-2GSH was added. After incubation in an ice bath for 6-12 h, high-speed centrifugation and washing were performed to obtain multifunctional cascade reactive nanoscale ultrasound contrast agent GHB-NPs carrying Basigin-siRNA.

2. The multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA according to claim 1, characterized in that: The phospholipid-polyethylene glycol-2GSH is prepared according to the following method: The phospholipid-polyethylene glycol-amino was dissolved in chloroform, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine and Nα,Nε-bis-Boc-L-lysine were added, and the reaction was carried out at room temperature for 4 hours. The solvent was removed by distillation under reduced pressure to obtain product 1; product 1 was re-dissolved in dichloromethane, trifluoroacetic acid was added, and the reaction was carried out at room temperature for 0.5 hours. The solvent was removed by distillation under reduced pressure to obtain product 2; product 2 was re-dissolved in dimethylformamide, PyBOP, triethylamine and GSH group were added, and the reaction was carried out at room temperature for 4 hours. The solvent was removed by distillation under reduced pressure to obtain product 3; product 3 was re-dissolved in acetyl chloride / methanol mixed solution, and the reaction was carried out at room temperature for 0.5 hours. The solvent was removed by distillation under reduced pressure to obtain product 4; product 4 was re-dissolved in methanol and the pH was adjusted to neutral. After dialysis and freeze-drying, phospholipid-polyethylene glycol-2GSH was obtained.

3. The multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA as claimed in claim 2, characterized in that: Meet one or more of the following conditions: a. The mass volume ratio of phospholipid-polyethylene glycol-amino and chloroform is (80-120):3, in mg / mL; b. The chemical equivalent ratio of phospholipid-polyethylene glycol-amino, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine and Nα,Nε-bis-Boc-L-lysine is 1:(2.5~3.5):(2.5~3.5):(1~1.1); c. The volume ratio of chloroform, dichloromethane, trifluoroacetic acid, dimethylformamide, acetyl chloride / methanol mixed solution and methanol is 3: (1.5-2.5): (0.4-0.6): (2.5-3.5): 3; d. The chemical equivalent ratio of phospholipid-polyethylene glycol-amino, PyBOP, triethylamine and GSH groups is 1:(2~2.5):(2.5~3.5):(2~2.5); e. The molecular weight cut-off for dialysis is 1500~2500Da.

4. The multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA as claimed in claim 3, characterized in that: Meet one or more of the following conditions: a. The mass volume ratio of phospholipid-polyethylene glycol-amino and chloroform is 100:3, unit is mg / mL; b. The chemical equivalent ratio of phospholipid-polyethylene glycol-amino, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine and Nα,Nε-bis-Boc-L-lysine is 1:3:3:1.1; c. The volume ratio of chloroform, dichloromethane, trifluoroacetic acid, dimethylformamide, acetyl chloride / methanol mixed solution and methanol is 3:2:0.5:3:3:3; d. The chemical equivalent ratio of phospholipid-polyethylene glycol-amino, PyBOP, triethylamine, and GSH groups is 1:2.2:3.0:2.2; e. The molecular weight cut-off for dialysis is 2500Da.

5. The multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA according to claim 1, characterized in that: Meet one or more of the following conditions: i. In step (1), the concentration of the chitosan solution is 15-20 mg / mL, and the concentration of acetic acid is 0.5-1%; ii. In step (2), the volume mass ratio of perfluorohexane, Tween 20, lecithin and deionized water is (0.1-2): (0.001-0.01): (0.001-0.01): (2-3), in units of g / mL; iii. In step (2), the homogenization is specifically carried out by using an ultrasonic cell disruptor at a power of 100 W for 5 to 10 minutes under ice bath conditions, with shaking and pausing alternately during the treatment process, and the shaking or pausing time is 5 to 15 seconds; iv. In step (3), the volume ratio of basigin-siRNA, chitosan and suspension is 1: (2-2.5): (25-30); v. In step (3), the homogenization is specifically carried out by using an ultrasonic cell disruptor at a power of 65 to 100 W for 1 to 5 min in an ice bath, with shaking and pausing alternately during the treatment process, and the shaking or pausing time is 5 to 10 s; vi. In steps (4) to (6), the low-speed centrifugation speed is 100-300 rpm for 1-5 min, and the high-speed centrifugation speed is 12000-13000 rpm for 5-10 min; vii. In step (5), the mass volume ratio of chitosan loaded with Basigin-siRNA, deionized water, and hyaluronidase is (2-5): (1-3): (2-5), in units of mg / mL; viii. In step (6), the mass volume ratio of chitosan loaded with Basigin-siRNA and modified with hyaluronidase, deionized water, and phospholipid-polyethylene glycol-2GSH is (2-5): (1-3): (0.1-0.3), the unit is mg / mL.

6. The multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA as claimed in claim 5, characterized in that: Meet one or more of the following conditions: i. In step (1), the concentration of the chitosan solution is 20 mg / mL, and the concentration of acetic acid is 1%; ii. In step (2), the volume ratio of perfluorohexane, Tween 20, lecithin and deionized water is 0.15 mL: 0.006 ml: 0.004 g: 2.7 mL; iii. In step (2), the homogenization is specifically carried out by using an ultrasonic cell disruptor at a power of 100 W for 5 to 10 minutes under ice bath conditions, with shaking and pausing alternately during the treatment process, and the shaking or pausing time is 10 seconds; iv. In step (3), the volume ratio of basigin-siRNA, chitosan and suspension is 1:2.25:27; v. In step (3), the homogenization is specifically carried out by using an ultrasonic cell disruptor at a power of 65 W for 3 min in an ice bath, with alternating shaking and pausing during the treatment process, and the shaking or pausing time is 8 s; vi. In steps (4) to (6), the low-speed centrifugation speed is 300 rpm for 3 min, and the high-speed centrifugation speed is 13000 rpm for 5 min; vii. In step (5), the mass volume ratio of chitosan loaded with Basigin-siRNA, deionized water, and hyaluronidase is 4.5 mg: 3 mL: 4 mg; viii. In step (6), the mass volume ratio of chitosan loaded with Basigin-siRNA and modified with hyaluronidase, deionized water, and phospholipid-polyethylene glycol-2GSH is 4.5 mg:3 mL:0.15 mg.

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