Multifunctional cascade reactive nanoscale ultrasonic contrast agent as well as preparation method and application thereof

Abstract

The invention relates to a multifunctional cascade reactive nanoscale ultrasonic contrast agent as well as a preparation method and application thereof. According to the contrast agent (GHB-NPs), chitosan loaded with Basigin-siRNA and modified with hyaluronidase serves as a core, phospholipid-polyethylene glycol-2GSH serves as a shell membrane, perfluoropentane is wrapped in the shell membrane, and the particle size ranges from 106 nm to 295 nm. According to the GHB-NPs prepared by the invention, DSPE-PEG2k-2GSH on the surface of the GHB-NPs can react with highly expressed gamma-GGT on the surface of tumor cells, electrostatic repulsion is generated to expose hyaluronidase, so that the GHB-NPs permeates into the deep part of tumor tissues, and the GHB-NPs has targeting and high efficiency in tumor treatment. The compound can be used in combination with UTMD, glycolysis related protein is reduced by inhibiting a beta-catenin / c-Myc pathway, the yield of lactic acid and ATP is reduced, and treatment of tumor diseases is facilitated. Experiments prove that GHB-NPs + UTMD has an obvious treatment effect on melanoma model mice, and can be used for preparing antitumor drugs to realize integration of tumor diagnosis and treatment.

Description

Technical Field

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

[0002] The heterogeneity and complexity of tumor metabolism pose a major challenge to tumor therapy. When tumors proliferate at a rate that exceeds angiogenesis, tumor cells away from blood vessels become hypoxic, which promotes glycolysis and lactate secretion through monocarboxylate transporter 4 (MCT4). Tumor cells close to blood vessels can obtain oxygen and absorb high-concentration lactate in the tumor microenvironment with the help of monocarboxylate transporter 1 (MCT1) to generate ATP, a process known as "lactate shuttling". 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 disruption of the MCT1 / 4-basigin interaction hinders tumor progression. Therefore, basigin may serve as a target for disrupting MCT expression on the cell membrane, however, siRNA treatment has limited effect. Ultrasound (US)-mediated gene delivery shows important clinical potential by transiently disrupting the vascular endothelial barrier and promoting cellular internalization by enhancing cell membrane permeability. Furthermore, US combined with nanoscale ultrasound contrast agents can promote various cellular responses, including immunogenic cell death, epithelial-mesenchymal transition, and antitumor drug resistance. To date, studies on the effects of US stimulation on metabolic symbiosis are lacking.

[0003] γ-Glutamyltranspeptidase (γ-GGT) is overexpressed on the outer surface of endothelial cells and on the cell membrane 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 tissue, resulting in charge conversion. However, the dense extracellular matrix of tumor tissue hinders the penetration of nanomaterials into deeper areas, thereby limiting their efficacy. Hyaluronic acid (HA) is ubiquitous in the extracellular matrix of various solid tumors. And it seriously hinders the penetration of nanomedicines. Hyaluronidase (HAase) has attracted widespread attention due to its ability to degrade HA. Direct injection of HAase is less effective due to the presence of proteases in the blood and insufficient targeting. To meet these challenges, it is crucial to develop a tumor microenvironment-responsive HAase release system.

[0004] Chinese patent document CN109260480A discloses a chitosan nano-scale ultrasound contrast agent carrying doxorubicin, and its preparation method and application. The nano-scale ultrasound contrast agent is chitosan as the shell membrane, doxorubicin and perfluoropropane gas are wrapped inside the chitosan shell membrane, and has a strong enhanced imaging ability. However, the nano-scale ultrasound contrast agent prepared by the invention is in the form of nano-vacuole, and the perfluoropropane nano-vacuole has a low output rate and poor stability. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a chitosan nano-scale ultrasound contrast agent carrying Basigin-siRNA, and modifies hyaluronidase and γ-GGT enzyme responsive lipids on the outside to improve the targeting and permeability to tumor tissue. The cascade reaction nano-scale ultrasound contrast agent is used to block metabolic symbiosis. The contrast agent has good imaging ability, high biosafety, good stability, and strong ability to inhibit tumor growth.

[0006] Terminology Note:

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

[0008] DSPE-PEG2k-NH 2 :Phospholipid-polyethylene glycol-amino

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

[0010] Room temperature: has the meaning commonly known 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 comprises the following steps:

[0015] Phospholipid-polyethylene glycol-amino (DSPE-PEG2k-NH 2) was dissolved in chloroform, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine and Nα,Nε-bis-Boc-L-lysine were added, the reaction was carried out at room temperature for 4 hours, and the solvent was removed by distillation under reduced pressure to obtain product 1; product 1 was re-dissolved in dichloromethane, trifluoroacetic acid was added, the reaction was carried out at room temperature for 0.5 hours, and 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, the reaction was carried out at room temperature for 4 hours, and the solvent was removed by distillation under reduced pressure to obtain product 3; product 3 was re-dissolved in acetyl chloride / methanol mixed solution, the reaction was carried out at room temperature for 0.5 hours, and 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, and after dialysis and freeze-drying, phospholipid-polyethylene glycol-2GSH (DSPE-PEG2k-2GSH) was obtained.

[0016] Preferably according to the present invention, the mass volume ratio of the phospholipid-polyethylene glycol-amino group and chloroform is (80-120):3, in mg / mL; more preferably 100:3, in mg / mL.

[0017] Preferably according to the present invention, the chemical equivalent ratio of the 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); further preferably, it is 1:3:3:1.1.

[0018] Preferably according to the present invention, 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; more preferably 3: 2: 0.5: 3: 3: 3.

[0019] Preferably according to the present invention, the chemical equivalent ratio of the phospholipid-polyethylene glycol-amino group, 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 acetyl chloride / methanol mixed solution is obtained by mixing acetyl chloride and methanol in a volume ratio of 1:6.

[0021] Preferably according to the present invention, the molecular weight cut-off 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 ultrasound contrast agent.

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

[0024] The preparation method of the multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA comprises the following steps:

[0025] (1) adding chitosan to acetic acid and fully dissolving it to obtain a chitosan solution;

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

[0027] (3) adding basigin-siRNA and chitosan solution to the suspension in sequence, homogenizing, and obtaining an emulsion;

[0028] (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;

[0029] (5) dispersing the chitosan loaded with Basigin-siRNA in deionized water, adding hyaluronidase, incubating for 15 to 60 minutes under ice bath conditions, and then centrifuging at high speed to obtain chitosan loaded with Basigin-siRNA and modified with hyaluronidase;

[0030] (6) Chitosan loaded with Basigin-siRNA and modified with hyaluronidase was dispersed in deionized water, phospholipid-polyethylene glycol-2GSH was added, and the mixture was incubated in an ice bath for 6 to 12 hours, and then centrifuged at high speed and washed to obtain a multifunctional cascade reactive nanoscale ultrasound 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), in g / mL; preferably, 0.15 mL: 0.006 ml: 0.004 g: 2.7 mL.

[0033] Preferably, in step (2), the homogenization is specifically performed by using an ultrasonic cell disruptor at a power of 100 W for 5 to 10 min under ice bath conditions, with oscillation and pauses performed alternately during the treatment process, and the oscillation or pause time is 5 to 15 s; preferably, the treatment is performed for 5 to 10 min, with oscillation and pauses performed alternately during the treatment process, and the oscillation or pause time is 10 s.

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

[0035] Preferably, in step (3), the homogenization is specifically performed by using an ultrasonic cell disruptor under ice bath conditions at a power of 65 to 100 W for 1 to 5 min, with oscillation and pauses performed alternately during the treatment process, and the oscillation or pause time is 5 to 10 s; preferably, the power is 65 W, the treatment is performed for 3 min, with oscillation and pauses performed alternately during the treatment process, and the oscillation or pause time is 8 s.

[0036] Preferably according to the present invention, in steps (4) to (6), the speed of the low-speed centrifugation is 100 to 300 rpm for 1 to 5 min, and the speed of the high-speed centrifugation is 12000 to 13000 rpm for 5 to 10 min; preferably 300 rpm for 3 min and 13000 rpm for 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), in mg / mL; preferably, 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), in mg / mL; preferably, 4.5 mg: 3 mL: 0.15 mg.

[0039] The application of the above-mentioned multifunctional cascade reactive nano-scale 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 described in detail in the present invention are all carried out according to conventional operations in the art.

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

[0043] 1. The multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA prepared by the present invention has DSPE-PEG2k-2GSH on its surface that can react with the highly expressed γ-GGT on the surface of tumor cells, resulting in electrostatic repulsion and exposing hyaluronidase, so that GHB-NPs can penetrate deep into tumor tissue, which is targeted and highly effective in the treatment of tumors.

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

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

[0046] 4. The multifunctional cascade reaction nano-scale ultrasound contrast agent (GHB-NPs) carrying basigin-siRNA provided by the present invention can be used in combination with ultrasound targeted blasting technology (UTMD) to downregulate glycolysis-related proteins by inhibiting the β-catenin / c-Myc pathway, reduce lactate and ATP production, and facilitate the treatment of tumor diseases. In addition, the present invention has experimentally proved that GHB-NPs+UTMD has a significant therapeutic effect on melanoma model mice, and can be used to prepare anti-tumor drugs to achieve integrated tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum identification of the targeting material phospholipid-polyethylene glycol-2GSH (DSPE-PEG2k-2GSH).

[0048] Figure 2 It is a synthesis model diagram of a multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA and the materials used.

[0049] Figure 3 It is the characterization and detection of multifunctional cascade reactive nano-scale ultrasound contrast agents carrying Basigin-siRNA;

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

[0051] Figure 4 is the encapsulation efficiency of the multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA;

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

[0053] Figure 5 It is the charge conversion ability of the multifunctional cascade reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA;

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

[0055] Figure 6 It is the cumulative release capacity of multifunctional cascade-reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0056] Figure 7 It is the ability of multifunctional cascade reactive nanoscale ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA to degrade hyaluronic acid.

[0057] Figure 8 It is the targeting ability of multifunctional cascade reactive nanoscale ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA.

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

[0059] Fig. 9 This is an in vivo ultrasound imaging image of a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0060] Fig.10 It is a study on the inhibition of glycolysis by ultrasound-targeted microbubble destruction technology of multifunctional cascade-reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA;

[0061] In the figure, A is the immunoblotting results under different conditions, and B is the bar chart analysis corresponding to Figure A;

[0062] Fig.11 The multifunctional cascade-reactive nanoscale ultrasound contrast agent carrying Basigin-siRNA treats tumors in vivo under different conditions;

[0063] In the figure, A is the tumor growth of each group during the treatment, and B is the weight of the in vitro tumor of each group after the treatment. DETAILED DESCRIPTION

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

[0065] Materials and reagents involved in the examples are common commercially available products unless otherwise specified; experimental operations involved in the examples are performed according to routine operations in the art unless otherwise specified.

[0066] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

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

[0068] Unless otherwise specified, the drugs and reagents involved in this embodiment are all common commercially available products.

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

[0070] 1. Synthesis of targeting material phospholipid-polyethylene glycol-2GSH (DSPE-PEG2k-2GSH), the specific method is as follows:

[0071] 100 mg of phospholipid-polyethylene glycol-amino (DSPE-PEG2k-NH 2) was dissolved in 3 ml of chloroform, and then 3.0 eq. of EDC, 3.0 eq. of 4-dimethylaminopyridine and 1.1 eq. of Nα, Nε-bis-Boc-L-lysine were added, and the reaction was carried out at room temperature for 4 h, and the solvent was removed by distillation under reduced pressure to obtain product 1; product 1 was re-dissolved in 2 ml of dichloromethane, 0.5 ml of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 0.5 h, and the solvent was removed by distillation under reduced pressure to obtain product 2; product 2 was re-dissolved in 3 ml of dimethylformamide, 2.2 eq. of PyBOP, 3.0 eq. of triethylamine were added and 2.2 eq. of GSH group, reacted at room temperature for 4 h, and distilled off the solvent under reduced pressure to obtain product 3; product 3 was re-dissolved in 3 ml of acetyl chloride / methanol mixed solution, reacted at room temperature for 0.5 h, and distilled off the solvent under reduced pressure to obtain product 4; product 4 was re-dissolved in 3 ml of methanol, and the pH was adjusted to neutral with sodium hydroxide aqueous solution, transferred to a dialysis bag (molecular weight cutoff = 2000 Da) and dialyzed in pure water for 24 h, the dialysate was collected and freeze-dried to obtain phospholipid-polyethylene glycol-2GSH (DSPE-PEG2k-2GSH).

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

[0073] DSPE-PEG2k-2GSH was identified by HNMR. Figure 1 As shown. Figure 1 It can be seen that the peak corresponding to the GSH polypeptide was observed in the final product, indicating that the material was synthesized correctly and DSPE-PEG2k-2GSH was successfully synthesized.

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

[0075] like Figure 2 As shown, a method for preparing a multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA comprises the following steps:

[0076] (1) adding 20 mg of chitosan into 1 mL of acetic acid (concentration of 1%) and fully dissolving the chitosan to obtain a chitosan solution;

[0077] (2) Dispersing 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 treating with an ultrasonic cell disruptor at a power of 100 W for 5 min under ice bath conditions, with shaking and pausing alternately during the treatment process, and the shaking or pausing time is 10 s to obtain a suspension;

[0078] (3) 100 μL of basigin-siRNA and 225 μL of the chitosan solution obtained in step (1) were sequentially added to 2.7 mL of the suspension obtained in step (2) to make the final concentration of basigin-siRNA in the suspension 250 μM, and the suspension was treated with an ultrasonic cell disruptor at a power of 65 W for 3 min under ice bath conditions, with alternating shaking and pausing during the treatment process, and the shaking or pausing time was 8 s to obtain an emulsion;

[0079] (4) After the emulsion was allowed to stand at room temperature, it was centrifuged at 300 rpm for 3 min, the middle layer was collected and then centrifuged at 13000 rpm for 5 min, and the precipitate was obtained to obtain chitosan loaded with Basigin-siRNA;

[0080] (5) 4.5 mg of chitosan loaded with Basigin-siRNA was dispersed in 3 mL of deionized water, 4 mg of hyaluronidase was added, and the mixture was incubated for 30 min using a magnetic stirrer under ice bath conditions, and then centrifuged at 13,000 rpm for 5 min to obtain the precipitate to obtain chitosan loaded with Basigin-siRNA and modified with hyaluronidase;

[0081] (6) 4.5 mg of chitosan loaded with Basigin-siRNA and modified with hyaluronidase was dispersed in 3 mL of deionized water, and 0.15 mg of phospholipid-polyethylene glycol-2GSH was added. After incubation for 12 h using a magnetic stirrer under ice bath conditions, the mixture was centrifuged at 13,000 rpm for 5 min and washed three times with phosphate buffer to remove impurities, thereby obtaining a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

[0082] According to the existing method, FAM-siRNA is used to replace basigin-siRNA to label GHB-NPs to obtain FAM-labeled GHB-NPs for later use.

[0083] Example 3

[0084] A method for preparing a multifunctional cascade reactive nanoscale 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 nanoscale 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 nanoscale 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 nanoscale 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 nanoscale 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 nanoscale 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 only in step (6), 0.15 mg of DSPE-PEG2k-2GSH is replaced with 0.15 mg of DSPE-PEG2k-NH 2 , and obtained an ultrasound contrast agent (DHB-NPs) coated with DSPE-PEG2k-NH2.

[0097] According to the existing method, FAM-siRNA is used to replace basigin-siRNA to label DHB-NPs to obtain FAM-labeled DHB-NPs for later use.

[0098] Comparative Example 2

[0099] A method for preparing an ultrasound contrast agent, the steps are as described in Example 2, except that in step (3), basigin-siRNA is not added, and the ultrasound contrast agent (GH-NPs) not carrying basigin-siRNA is obtained.

[0100] Comparative Example 3

[0101] A method for preparing an ultrasound contrast agent, the steps are as described in Example 2, except that in step (5), hyaluronidase is not added, and an ultrasound contrast agent (GB-NPs) without modified hyaluronidase is obtained.

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

[0103] 1. Structural observation of GHB-NPs

[0104] The GHB-NPs prepared in Example 2 were diluted with PBS buffer and then observed. The transmission electron microscopy of the GHB-NPs was as follows: Figure 3 As shown in A.

[0105] Depend on Figure 3 As shown in A, GHB-NPs are round in shape, with a smooth and transparent surface and a clear core-shell structure, and a narrow band can be observed outside (red arrow).

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

[0107] Depend on Figure 3 B and C show that the particle size of GHB-NPs is 285.4±10.93nm, the polymer dispersion index is 0.212±0.02, and the Zeta potential is -(22.20±1.28)mV.

[0108] 2. Dilute the GHB-NPs prepared in Example 2 with PBS buffer and drop them onto a glass slide. Observe them under a fluorescence microscope. The results are as follows: Figure 3 As shown in D.

[0109] Depend on Figure 3 As can be seen from D, FAM-siRNA is green, Dil-labeled DSPE-PEG2k-2GSH is red, and the FAM / Dil graph 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 reactive nanoscale ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA

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

[0112] Depend on Figure 4 A shows that when the final concentration of basigin-siRNA is 250 μM, the basigin-siRNA encapsulation efficiency is the highest.

[0113] 2. The hyaluronidase encapsulation efficiency of GHB-NPs prepared in Examples 2, 6 to 8 was determined using the BCA kit and method. The results are as follows: Figure 4 As shown in B.

[0114] Depend on Figure 4 B shows that when the mass of hyaluronidase is 5 mg, the encapsulation efficiency of hyaluronidase is the highest.

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

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

[0117] Depend on Figure 5 A shows that the surface charge of GHB-NPs in a 10U / mL γ-GGT solution changes from negative to positive, and its surface charge in a 0.05U / mL γ-GGT solution is a stable negative charge. At the same time, the nanoscale ultrasound contrast agent without DSPE-PEG2k-2GSH is a stable positive charge, indicating that the charge transformation is the result of the reaction between DSPE-PEG2k-2GSH and γ-GGT.

[0118] Depend on Figure 5 B shows that the peripheral narrow band of GHB-NPs disappeared in 10U / mL γ-GGT solution, while the narrow band was still visible in 0.05U / mL γ-GGT solution, proving that DSPE-PEG2k-2GSH can be shed layer by layer in the tumor microenvironment.

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

[0120] The GHB-NPs prepared in Example 2 were placed in a dialysis bag, and the dialysis bag was placed in 0.05U / mL and 10U / mL γ-GGT solutions, respectively, and then placed in a constant temperature shaking incubator, shaken at 37°C and 200rpm, and the drug release capacity of the GHB-NPs was measured. The specific method is as follows:

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

[0122]

[0123] Where R is the cumulative drug release rate; V e is the displacement volume of deionized water; V 0 is the total volume of the release medium; Ci is the hyaluronidase concentration of 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 PBS is replaced, and the results are shown in Figure 6 shown.

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

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

[0126] 2×10 4B16F10 cells / well were seeded in a 24-well plate, and PBS buffer (Control), hyaluronidase (HAase), 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, respectively. The unbound contrast agent was washed away with PBS buffer, and the cells were stained with DAPI dye. The degradation ability was analyzed using a confocal microscope. The results are shown in Figure 7 shown.

[0127] Depend on Figure 7 It can be seen that the hyaluronidase group was used as the positive control group, and the hyaluronidase degradation level after GHB-NPs treatment was roughly similar to that of the positive group. This confirms that GHB-NPs can 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 can achieve responsive exposure of hyaluronidase at the tumor site.

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

[0129] 1. Targeting ability of γ-GGT response

[0130] 2×10 5 MDA-MB-231 cells / 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 any reagents were used as a 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, respectively. The results are shown in FIG. Figure 8 As shown in A and B.

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

[0132] 2. Targeting capability of hyaluronidase response

[0133] 2×10 5MDA-MB-231 cells / 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 used to co-culture the cells for 6 h. At the same time, cells without any reagents were used as a 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, respectively. The results are shown in FIG. Figure 8 As shown in C and D.

[0134] Depend on Figure 8 As shown in C and D, the internalization uptake of GHB-NPs by B16F10 cells is significantly higher than that of 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 capability of multifunctional cascade reactive nanoscale ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA

[0136] The GHB-NPs prepared in Example 2 were placed in a circular hole made of agar gel, and the contrast mode of the GE logiq E9 ultrasonic diagnostic instrument was used, with a frequency of 9.0 MHz and a mechanical index (MI) of 0.5. The two-dimensional and ultrasound contrast modes were observed simultaneously, and the parameter settings were adjusted. The image data was stored in the internal workstation of the ultrasound instrument. PBS buffer was used as the control group. The results are shown in FIG. Fig. 9 shown.

[0137] Depend on Fig. 9 It can be seen that GHB-NPs showed enhanced ultrasound imaging, while the control group did not show enhanced imaging.

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

[0139] 5×10 5 B16F10 cells / well were seeded in 6-well plates. When the cells grew to a density of 50-70%, three groups of different treatments were given as follows:

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

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

[0142] GH-NPs+US group: added ultrasound contrast agent without basigin-siRNA (Comparative Example 2) and subjected to ultrasound irradiation;

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

[0144] After processing, the results are as follows Fig.10 shown.

[0145] Depend on Fig.10 It 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, ultrasound-targeted microbubble destruction technology downregulated the expression of glycolysis-related proteins by inhibiting the β-catenin / c-Myc pathway.

[0146] Example 14: Evaluation of the ability of multifunctional cascade-reactive nanoscale ultrasound contrast agents (GHB-NPs) carrying Basigin-siRNA to inhibit tumor growth in vivo

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

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

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

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

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

[0152] GHB-NPs group: GHB-NPs were added but no ultrasound irradiation was performed;

[0153] GHB-NPs+US group: GHB-NPs were added and ultrasound irradiated;

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

[0155] Depend on Fig.11 A shows that 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 effect on tumor growth.

[0156] Depend on Fig.11 B shows that the tumor weight of the GHB-NPs+US group (0.13±0.05g) was significantly lower than that of the GHB-NPs group (0.58±0.15g) and the GH-NPs+US group (0.53±0.11g).

[0157] The above data fully demonstrate that the multifunctional cascade reaction nano-scale ultrasound contrast agent (GHB-NPs) carrying basigin-siRNA provided by the present invention can be used in combination with ultrasound targeted blasting technology (UTMD), which can inhibit the β-catenin / c-Myc pathway to downregulate glycolysis-related proteins, reduce lactate and ATP production, and is beneficial to the treatment of tumor diseases. In addition, the present invention has proved through experiments that GHB-NPs+UTMD has a significant therapeutic effect on melanoma model mice, and can be used to prepare anti-tumor drugs to achieve integrated tumor diagnosis and treatment.

[0158] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A targeting material phospholipid-polyethylene glycol-2GSH, characterized in that: Has the following structure:

2. The method for preparing the targeting material phospholipid-polyethylene glycol-2GSH according to claim 1, characterized in that: The steps include: 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 preparation method according to 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 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:(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 preparation method according to 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 (80-120):3, the 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. Use of the targeting material phospholipid-polyethylene glycol-2GSH according to claim 1 in the preparation of ultrasound contrast agents.

6. 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.

7. The method for preparing the multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA according to claim 6 comprises the following steps: (1) adding chitosan to acetic acid and fully dissolving it 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, homogenizing, and obtaining 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 for 15 to 60 minutes under ice bath conditions, 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, phospholipid-polyethylene glycol-2GSH was added, and the mixture was incubated in an ice bath for 6 to 12 hours, and then centrifuged at high speed and washed to obtain a multifunctional cascade reactive nanoscale ultrasound contrast agent (GHB-NPs) carrying Basigin-siRNA.

8. The preparation method according to claim 7, 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 performed by using an ultrasonic cell disruptor at a power of 100 W for 5 to 10 min in an ice bath, with shaking and pausing alternately during the treatment process, and the shaking or pausing time is 5 to 15 s; 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 to 300 rpm for 1 to 5 min, and the high-speed centrifugation speed is 12000 to 13000 rpm for 5 to 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), and the unit is 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), and the unit is mg / mL.

9. The preparation method according to claim 8, 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 min in an ice bath, with shaking and pausing alternately during the treatment process, and the shaking or pausing time is 10 s; 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.

10. Use of the multifunctional cascade reactive nano-scale ultrasound contrast agent carrying Basigin-siRNA according to claim 1 in the preparation of anti-tumor drugs.

Images