A cascade response type diagnosis and treatment integrated ultrasonic microbubble, a preparation method and application thereof

Abstract

This invention discloses a cascade-response integrated ultrasound microbubble for diagnosis and treatment, its preparation method, and its applications. From the inside out, it comprises a perfluoropentane phase-change core, a drug-loaded PLGA mesoporous nanoparticle intermediate layer, and a lipid shell modified with folic acid and a transmembrane peptide dual ligand. An MMP-2-responsive peptide chain crosslinking agent is embedded in the lipid layer. This invention utilizes the MMP-2 enzyme in the tumor microenvironment to trigger the outer layer's peeling, exposing the transmembrane peptide for enhanced targeting. Then, ultrasound triggers the core's phase-change explosion to release the drug, achieving a closed-loop "identification-confirmation-treatment" cascade. This microbubble exhibits good stability and high drug delivery efficiency, with a cumulative release rate exceeding 85% after 48 hours under dual stimulation, demonstrating significant in vivo tumor-suppressing effects. This invention provides an intelligent ultrasound microbubble platform for precision tumor diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and ultrasound molecular imaging, specifically to a cascade-response integrated diagnostic and therapeutic ultrasound microbubble, its preparation method, and its application. Background Technology

[0002] Ultrasound microbubble contrast agents, as a core carrier of therapeutic platforms, have shown great potential in tumor imaging and drug delivery in recent years. However, current technologies still face the following key bottlenecks:

[0003] The contradiction between stability and responsiveness: Although traditional lipid microbubbles have good acoustic responsiveness, they are not stable enough in blood circulation and are prone to rupture before reaching the target tissue; while microbubbles using harder shell materials (such as polymers) have improved stability, they are difficult to release drugs effectively under ultrasound.

[0004] Bottleneck of low drug delivery efficiency: Conventional microbubbles have limited drug loading capacity, and drug release mainly relies on the inertial cavitation effect of high sound pressure (>1 MPa). This non-specific blasting not only poses a risk of damage to surrounding normal tissues, but also leads to a narrow therapeutic window.

[0005] Insufficient targeting: Existing active targeting strategies are mostly single-ligand modifications, which are difficult to overcome the heterogeneity of the tumor microenvironment; and there is a lack of real-time feedback mechanism for the drug release process, making it difficult to form a closed loop of "diagnosis-treatment-reassessment".

[0006] Limitations of existing therapeutic microbubbles: Existing patents, such as CN108721648B, disclose multifunctional microbubbles integrating ultrasound / fluorescence dual-modal imaging and gene therapy / photodynamic therapy. However, the simple superposition of functional modules leads to complex structures and difficulties in quality control. Other studies have adopted a strategy guided by MRI / ultrasound dual-modal imaging, but MRI equipment is expensive and complex to operate, limiting its clinical application.

[0007] Therefore, developing an ultrasonic microbubble that combines good cyclic stability, controllable and precise drug release capability, and efficient integrated diagnosis and treatment function is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] The purpose of this invention is to provide a cascaded response-type diagnostic and therapeutic integrated ultrasound microbubble, its preparation method, and its application. The technical problems to be solved by this invention include: (1) improving the stability of microbubbles in blood circulation while ensuring their controllable rupture at the target site; (2) improving the delivery efficiency and penetration depth of drugs in tumor tissues; (3) realizing real-time imaging feedback during the treatment process; and (4) simplifying the preparation process and ensuring batch-to-batch stability.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] The first aspect of this invention provides a cascade-response therapeutic ultrasound microbubble, wherein the microbubble comprises, from the inside out: a core layer, an intermediate drug-loaded layer, and an outer shell layer; the core layer is a perfluoropentane or perfluorohexane phase-change droplet; the intermediate drug-loaded layer is a PLGA mesoporous nanoparticle encapsulating a drug active ingredient; and the outer shell layer is a dual-ligand modified lipid monolayer containing a matrix metalloproteinase (MMP)-responsive peptide chain crosslinking agent.

[0011] Preferably, the dual ligands are folic acid and the cell-penetrating peptide TAT.

[0012] Preferably, the amino acid sequence of the MMP-responsive peptide crosslinking agent is GPLGVRGC.

[0013] Preferably, the active pharmaceutical ingredient is selected from docetaxel, paclitaxel, doxorubicin, or siRNA.

[0014] Specifically, the cascaded response-type integrated ultrasound microbubble for diagnosis and treatment comprises, from the inside out:

[0015] (1) Core layer: Perfluoropentane (PFP) or perfluorohexane (PFH) phase change droplets with a particle size of 50-200 nm;

[0016] (2) Intermediate drug-carrying layer: PLGA mesoporous nanoparticles encapsulating the active pharmaceutical ingredient. The nanoparticles have a positive charge on their surface and a particle size of 100-300 nm. They are self-assembled on the periphery of the core layer through electrostatic adsorption and hydrophobic interaction.

[0017] (3) Outer shell: a lipid monolayer modified with two ligands, wherein the lipid layer contains distearate phosphatidylcholine (DSPC), dipalmitoylphosphatidylethanolamine (DPPE) and cholesterol, wherein the two ligands are folic acid (FA) and cell-penetrating peptide (TAT), and the lipid layer also contains a matrix metalloproteinase (MMP-2) responsive peptide chain crosslinker with the amino acid sequence GPLGVRGC, as shown in SEQ ID NO.1.

[0018] Specifically, the PLGA has a molecular weight of 10,000-50,000 Da, and the molar ratio of lactic acid to glycolic acid in the PLGA is 50:50 or 75:25.

[0019] A second aspect of the present invention also provides a method for preparing the above-mentioned cascaded response-type integrated diagnostic and therapeutic ultrasound microbubbles, comprising the following steps:

[0020] (1) PLGA mesoporous nanoparticles encapsulating drug active ingredients and having a positively charged surface were prepared by a double emulsification method;

[0021] (2) Preparation of perfluoropentane lipid microbubbles using microfluidic technology;

[0022] (3) PLGA nanoparticles are self-assembled on the surface of lipid microbubbles by electrostatic adsorption;

[0023] (4) Folic acid, TAT and MMP-responsive peptide crosslinking agents are coupled to the surface of microbubbles via amide bonds.

[0024] Preferably, in step (3), the mass ratio of PLGA nanoparticles to lipid microbubbles is 1:2 to 1:5.

[0025] Preferably, in step (4), the amount of the MMP-responsive peptide chain crosslinking agent is 0.5-5 mol of the total molar amount of phospholipids.

[0026] Specifically, the preparation method of the above-mentioned cascaded response-type integrated diagnostic and therapeutic ultrasound microbubble includes the following steps:

[0027] Step 1: PLGA mesoporous nanoparticles encapsulating active pharmaceutical ingredients are prepared using a modified double emulsification method (W / O / W). The emulsification parameters are adjusted to make the nanoparticle surface positively charged.

[0028] Step 2: Mix perfluoropentane with phospholipid materials (DSPC, DPPE) and prepare PFP lipid microbubbles using microfluidic technology. Then, use ultrasonic treatment to homogenize the particle size.

[0029] Step 3: Mix the PLGA nanoparticles obtained in Step 1 with the PFP lipid microbubbles obtained in Step 2 at a mass ratio of 1:2 to 1:5, and use ultrasonic oscillation to allow the nanoparticles to be assembled on the surface of the microbubbles through electrostatic adsorption.

[0030] Step 4: Mix folic acid-PEG2000-succinimide ester, TAT-PEG2000-maleimide and MMP-2 responsive peptide chain crosslinking agent in proportion, and react at 4°C with stirring for 12-24 hours. The mixture is then coupled to the surface of the microbubble through amide bonds to obtain the target microbubble.

[0031] The third aspect of this application also provides the application of the aforementioned cascaded response-type therapeutic ultrasound microbubbles in the preparation of tumor diagnostic and therapeutic reagents.

[0032] Preferably, the method of using the application is as follows: first, low mechanical index ultrasound is used to image and locate the tumor site enriched with microbubbles, and after confirmation, the method is switched to high mechanical index ultrasound irradiation to trigger drug release, wherein the low mechanical index ultrasound has an MI of 0.1-0.3 and the high mechanical index ultrasound has an MI of 0.8-1.2.

[0033] The application of the cascade-response integrated ultrasound microbubbles for diagnosis and treatment is particularly evident in their use in the preparation of integrated tumor diagnostic and therapeutic reagents. The specific method of use is as follows: after intravenous injection of the microbubbles, low mechanical index ultrasound (MI 0.1-0.3) is first used for Doppler imaging to locate the tumor sites where microbubbles are enriched; after confirmation, the process is switched to high mechanical index ultrasound (MI 0.8-1.2) irradiation for 5-15 minutes to trigger drug release; after treatment, low mechanical index ultrasound imaging can be performed again to assess the drug release effect.

[0034] The beneficial effects of this invention are:

[0035] Compared with the prior art, the present invention has the following significant advantages:

[0036] (1) Cascaded response design, intelligent release: The first step of "de-causing" is triggered by the high expression of MMP-2 in the tumor microenvironment, exposing TAT to enhance active targeting; then, ultrasound is used to trigger the second step of liquid-gas phase change drug release. This stepwise response mechanism avoids premature drug leakage and improves delivery efficiency. In vitro release experiments show that under dual stimulation of MMP-2 and ultrasound, the cumulative drug release rate reaches more than 85% after 48 hours, while the single stimulation group is only about 30%.

[0037] (2) Integrated imaging and treatment with precise control: Conventional microbubbles require sound pressure levels three times higher than the imaging threshold to break down, which can easily cause tissue damage. This invention utilizes a phase transition mechanism to clearly monitor the location of microbubbles at low MI (0.3). Once the location of the microbubble is confirmed to have reached the tumor site, the power is increased to trigger the phase transition and treatment, achieving a precise closed loop of "what is seen is what is treated". This feature can significantly reduce off-target toxic side effects.

[0038] (3) Balance between stability and responsiveness: Traditional lipid microbubbles have poor stability, while polymeric microbubbles are difficult to rupture. This invention adopts a nested structure of "hard shell drug delivery + soft shell stealth": the PLGA mesoporous nanoparticle layer provides a stable drug carrier and an outer lipid anchoring site; the perfluoropentane core is stable at room temperature and undergoes a phase transition only under ultrasonic irradiation; this design makes the microbubbles significantly more stable in blood circulation than ordinary lipid microbubbles, while maintaining good ultrasonic responsiveness. Experiments show that the particle size change of the microbubbles of this invention in serum at 37°C is less than 10% within 6 hours, while traditional lipid microbubbles rupture by more than 50% within 2 hours.

[0039] (4) Assembly process innovation: Compared with the complex process that requires direct connection of lipids and drugs through covalent bonds, the present invention utilizes charge adsorption and ligand-receptor interaction for modular assembly. Each functional layer can be optimized independently, the preparation process is simple, the batch-to-batch stability is good, and it is suitable for large-scale production. Attached Figure Description

[0040] Figure 1This is a schematic diagram of the structure of the microbubbles of the present invention (three-layer structure: PFP core, PLGA drug-carrying layer, and dual-ligand lipid shell).

[0041] Explanation of reference numerals in the attached figures:

[0042] 1. Cell-penetrating transcellular adenocarcinoma (TAT); 2. Inner core layer; 3. Intermediate drug-carrying layer; 4. Folic acid; 5. Drug; 6. MMP2-responsive peptide chain. Detailed Implementation

[0043] The following detailed description, in conjunction with embodiments, illustrates a cascade-response integrated diagnostic and therapeutic ultrasound microbubble and its preparation method provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0044] This invention addresses the aforementioned problems through a dual strategy of "defensive shell design" and "cascading release mechanism." The technical concept is as follows:

[0045] The three-layer structure is designed as follows: the inner core layer 2 is a perfluoropentane (PFP) phase change nanodroplet, the middle drug-loaded layer 3 is a PLGA (polylactic acid-glycolic acid copolymer) mesoporous nanoparticle aggregate layer loaded with chemotherapy drugs 5 (such as docetaxel), and the outer shell is a lipid monolayer modified with folic acid 4 (FA) and cell-penetrating peptide TAT 1 dual ligands, and a matrix metalloproteinase (MMP-2) responsive peptide chain crosslinker (MMP2 responsive peptide chain 6) is embedded in the lipid monolayer.

[0046] Its mechanism of action is as follows: After entering the bloodstream, the outer lipid layer of microbubbles provides concealment and stability ("cloak of invisibility"). Upon reaching the tumor site, the MMP-2 enzyme overexpressed in the tumor microenvironment cleaves the peptide chain crosslinking agent, triggering the outer lipid layer to peel off (first step response), exposing the TAT membrane-penetrating peptide in the middle layer, which enhances the adhesion and internalization of microbubbles with tumor cells. At this point, the microbubbles still maintain their intact structure and can be imaged at high resolution under low mechanical index ultrasound (MI 0.1-0.3). After confirming that the microbubbles are enriched in the target area, diagnostic high-intensity ultrasound (MI 0.8-1.2) is applied, triggering a liquid-gas phase transition of perfluoropentane, generating a cavitation effect, bursting the PLGA mesoporous nanoparticle layer, releasing the drug, and generating an acoustic pore effect to promote drug penetration (second step response). The core innovation of this design lies in cascading "environmental response" and "external field triggering," realizing a closed-loop diagnostic and therapeutic logic of "arrival-identification-confirmation-release."

[0047] Example 1: Preparation of docetaxel-loaded cascade-response ultrasonic microbubbles

[0048] The structure of microbubbles is shown in the attached figure. Figure 1 As shown, the specific preparation steps are as follows:

[0049] (1) Preparation of PLGA mesoporous nanoparticles: 50 mg PLGA (50:50, Mw=30 kDa) and 5 mg docetaxel were dissolved in 4 mL of dichloromethane and slowly added dropwise to 10 mL of aqueous phase containing 1% polyvinyl alcohol (PVA). The mixture was ultrasonically treated in an ice bath (200 W, 5 min) to form a pre-emulsion. The pre-emulsion was slowly added to a 0.3% PVA aqueous solution and ultrasonically treated again (150 W, 3 min). The mixture was stirred overnight at room temperature to evaporate the organic solvent. After centrifugation and washing (12000 rpm, 15 min), the PLGA nanoparticles were obtained by freeze-drying. The particle size was measured to be 185±25 nm by dynamic light scattering, and the surface Zeta potential was +18.5 mV.

[0050] (2) Preparation of PFP lipid microbubbles: DSPC, DPPE and cholesterol were mixed in chloroform at a mass ratio of 60:25:15, evaporated to form a film, and then vacuum dried overnight. PBS buffer was added for hydration, perfluoropentane gas was introduced, and microbubbles were prepared at 4°C using a microfluidic device. The microbubbles were then sonicated to homogenize the particle size to 1-3 μm.

[0051] (3) Self-assembly: PLGA nanoparticles (5 mg) were resuspended in 2 mL PBS and mixed with PFP microbubbles (2 × 10^8). The mixture was then sonicated in an ice bath for 30 minutes. The nanoparticles adhered to the surface of the microbubbles through electrostatic adsorption, forming a "microbubble-nanoparticle" complex.

[0052] (4) Dual-ligand modification: Folic acid-PEG2000-succinimide ester (FA-PEG2000-NHS, 0.5 μmol, which can react with the free amino groups on DPPE in the microbubble component), TAT-PEG2000-maleimide (TAT-PEG2000-Mal, 0.5 μmol) and MMP-2 responsive peptide chain crosslinking agent (GPLGVRGC-modified DSPE, 1.0 μmol, containing thiol groups, which can react with the Mal groups on TAT-PEG2000-Mal) were dissolved in 1 mL of PBS and added to the microbubble suspension from step (3). The mixture was stirred at 4°C for 24 hours. The unbound ligands were removed by washing with PBS three times to obtain the target microbubbles (denoted as FA / TAT / MMP-MBs).

[0053] Laser confocal microscopy revealed that the microbubble surface simultaneously exhibited fluorescence from FITC-labeled folic acid and Rhodamine-labeled TAT, confirming successful dual-ligand modification. Particle size analysis showed an average particle size of 2.8 ± 0.5 μm with uniform particle size distribution.

[0054] Example 2: In vitro stability and responsiveness test

[0055] (1) Serum stability: The FA / TAT / MMP-MBs prepared in Example 1 and the control ordinary lipid microbubbles (SonoVue) were added to PBS containing 10% fetal bovine serum and incubated at 37°C. Samples were taken at 0, 1, 2, 4, and 6 hours, and intact microbubbles were counted under a microscope. The results showed that the integrity rate of the microbubbles of the present invention was still 82.3±5.6% after 6 hours, while that of the control group was only 23.5±4.2%, indicating that the microbubbles of the present invention have significantly enhanced serum stability.

[0056] (2) MMP-2 responsiveness test: FA / TAT / MMP-MBs were co-incubated with recombinant human MMP-2 enzyme (100 ng / mL), and samples were taken at 0, 1, 2, and 4 hours. The fluorescence intensity of TAT on the microbubble surface was detected by flow cytometry. The results showed that the fluorescence intensity of TAT gradually increased with the extension of incubation time, reaching 4.2 times the initial value at 4 hours, indicating that after the cross-linked peptide chain was cleaved by MMP-2, the TAT that was originally covered by the lipid layer was exposed. In the control experiment, there was no significant change in fluorescence intensity in the group without MMP-2 enzyme.

[0057] (3) Ultrasound-responsive drug release: FA / TAT / MMP-MBs were pre-incubated for 2 hours in a release medium containing MMP-2 (100 ng / mL) to simulate the "unmasking" process in the tumor microenvironment. Diagnostic ultrasound (frequency 1 MHz, MI 1.0, duty cycle 50%, irradiation for 2 minutes) was then applied. The docetaxel release rate was detected by high-performance liquid chromatography. The results showed that the drug release rate in the MMP-2 pre-incubation + ultrasound irradiation group was 89.3±4.7%; in the MMP-2-only incubation group, it was only 28.6±3.2%; in the ultrasound-only irradiation group (without MMP-2 pre-incubation, i.e., without removal of the outer lipid layer), the release rate was only 15.4±2.1%; and in the control group (without MMP-2, without ultrasound), the release rate was 4.2±1.1%. These results fully demonstrate the effectiveness of the "cascade response" mechanism of the microbubbles in this invention.

[0058] Example 3: In vivo ultrasound imaging and evaluation of antitumor drug efficacy

[0059] Animal model: 4-6 week old female BALB / c nude mice were subcutaneously inoculated with 4T1 breast cancer cells (2×10^6 cells / mouse) in the right anterior axilla. The experiment was carried out when the tumor volume reached 100-150 mm^3.

[0060] Ultrasound imaging experiment: Tumor-bearing mice were injected via tail vein with FA / TAT / MMP-MBs (200 μL, 1×10^8 microbubbles), and real-time imaging was performed using low mechanical index ultrasound (MI 0.2). Before injection, the tumor area showed uniform hypoechoicity with no obvious contrast enhancement signal. 30 seconds after injection of the microbubbles of this invention, punctate contrast enhancement began to appear at the tumor site; 2 minutes after injection, the enhancement reached its peak, and the tumor area showed uniform and significant hyperechoic enhancement signal with clear boundaries, indicating that the microbubbles had efficiently accumulated in the tumor tissue through a cascade response mechanism. 30 minutes after injection, the tumor area still maintained significant hyperechoic enhancement, with a signal intensity of approximately 70% of the peak value, indicating that the microbubbles of this invention have a long retention time at the tumor site. In the control group, after injection of untargeted microbubbles (unmodified FA / TAT / MMP, but with the same structure), only weak enhancement was observed at the tumor site, and the signal basically disappeared within 5 minutes after injection. The above results fully demonstrate that the microbubbles of the present invention achieve efficient active tumor targeting and long-term retention by exposing TAT in response to MMP-2.

[0061] Evaluation of antitumor efficacy: Tumor-bearing mice were randomly divided into 5 groups (n=6): (A) saline control group; (B) free docetaxel group (5 mg / kg); (C) conventional drug-loaded microbubbles (without cascade response function, containing an equal amount of drug) combined with ultrasound irradiation; (D) FA / TAT / MMP-MBs group (without ultrasound irradiation); (E) FA / TAT / MMP-MBs combined with ultrasound irradiation group. Each group was treated once every 3 days for a total of 4 times. Tumor volume and body weight were measured regularly during treatment. Results showed that group E (the group of this invention) had the best tumor-suppressing effect. On day 21 after treatment, the tumor volume in group E was only 0.32 times the initial volume, while groups A, B, C, and D were 5.6 times, 2.8 times, 1.6 times, and 2.1 times the initial volume, respectively. The median survival time of mice in group E was 65% longer than that in group C, and no significant weight loss or other toxic side effects were observed.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cascaded response-type integrated diagnostic and therapeutic ultrasound microbubble, characterized in that, The microbubbles, from the inside out, consist of: a core layer, an intermediate drug-loaded layer, and an outer shell layer; the core layer is a perfluoropentane or perfluorohexane phase change droplet; the intermediate drug-loaded layer is a PLGA mesoporous nanoparticle encapsulating the active pharmaceutical ingredient; the outer shell layer is a lipid monolayer modified with dual ligands, and the lipid monolayer contains a matrix metalloproteinase (MMP)-responsive peptide chain crosslinking agent.

2. The cascaded response integrated diagnostic and therapeutic ultrasound microbubble according to claim 1, characterized in that, The dual ligands are folic acid and the cell-penetrating peptide TAT.

3. The cascaded response integrated diagnostic and therapeutic ultrasound microbubble according to claim 1, characterized in that, The amino acid sequence of the MMP-responsive peptide crosslinker is GPLGVRGC.

4. The cascaded response integrated diagnostic and therapeutic ultrasound microbubble according to claim 1, characterized in that, The active pharmaceutical ingredient is selected from docetaxel, paclitaxel, doxorubicin, or siRNA.

5. A method for preparing a cascaded response-type therapeutic ultrasound microbubble as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) PLGA mesoporous nanoparticles encapsulating drug active ingredients and having a positively charged surface were prepared by a double emulsification method; (2) Preparation of perfluoropentane lipid microbubbles using microfluidic technology; (3) PLGA nanoparticles are self-assembled on the surface of lipid microbubbles by electrostatic adsorption; (4) Folic acid, TAT and MMP-responsive peptide crosslinking agents are coupled to the surface of microbubbles via amide bonds.

6. The preparation method according to claim 5, characterized in that, In step (3), the mass ratio of PLGA nanoparticles to lipid microbubbles is 1:2 to 1:

5.

7. The preparation method according to claim 5, characterized in that, In step (4), the amount of the MMP-responsive peptide chain crosslinking agent used is 0.5-5 mol of the total molar amount of phospholipids.

8. The application of the cascaded response-type therapeutic ultrasound microbubbles as described in any one of claims 1-4 in the preparation of tumor diagnostic and therapeutic reagents.

9. The application according to claim 8, characterized in that, The application is used as follows: first, low mechanical index ultrasound is used to image and locate the tumor site enriched with microbubbles. After confirmation, the application is switched to high mechanical index ultrasound to trigger drug release. The low mechanical index ultrasound has an MI of 0.1-0.3, and the high mechanical index ultrasound has an MI of 0.8-1.2.

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