Double-layer protective shell type multifunctional targeted ultrasonic contrast agent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical engineering and ultrasonic molecular imaging, and particularly relates to a double-layer protective shell type multifunctional targeted ultrasonic contrast agent as well as a preparation method and application thereof. The double-layer protective shell type multifunctional targeted ultrasonic contrast agent provided by the invention is a novel ultrasonic microbubble formed by loading a chemotherapeutic drug, an immune checkpoint inhibitor and miRNA on a nano carrier and embedding the chemotherapeutic drug, the immune checkpoint inhibitor and the miRNA into nano liquid drops and the ultrasonic microbubble, has high biological safety, can circulate in vivo for a long time, and can be used as a targeted ultrasonic contrast agent. Moreover, the imaging effect is good, the targeting property is strong, the drug utilization rate is high, tumor diagnosis and treatment under ultrasonic guidance can be realized while the targeting property of chemotherapy drugs, immune checkpoint inhibitors and miRNA is improved, and the problems that tumor ultrasonic diagnosis is difficult, immunosuppressor drugs are easy to degrade, the effect is poor, the targeting property is weak and the like at present are comprehensively and effectively solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering and ultrasonic molecular imaging technology, and in particular, relates to a double-layer protective shell type multifunctional targeted ultrasonic contrast agent and a preparation method and application thereof. Background Art

[0002] Breast Cancer (BC) has become the most common malignant tumor in women. Its incidence rate ranks first among all types of cancer in women, and its mortality rate ranks second, second only to lung cancer. At present, the pathogenesis of breast cancer is still unclear. Traditional treatments include chemotherapy, surgical resection, radiotherapy and endocrine therapy, which can reduce the incidence of breast cancer to a certain extent. However, most patients still face the problem of uncontrolled tumor development or treatment failure, especially HER-2 phenotype and triple-negative breast cancer.

[0003] At present, the commonly used medical imaging diagnostic methods for cancer in clinical practice mainly include ultrasound, CT, X-ray and magnetic resonance imaging. Among them, ultrasound technology has become the primary means of cancer diagnosis and examination due to its advantages such as low price, non-invasiveness, no radiation, and real-time imaging. Ultrasound contrast agents (UCAs) can improve the echo signal contrast between the lesion area and normal physiological tissue, improve the precision and accuracy of clinical ultrasound diagnosis, and have been widely used in cancer diagnosis. At present, the ultrasound contrast agents that have been studied more and entered clinical application are mainly microbubble ultrasound contrast agents, which can achieve higher echo intensity. However, microbubble ultrasound contrast agents have poor stability, lack of targeting, lack of specificity for tissues, and cannot penetrate the human reticuloendothelial system to enter the tissue. They can only perform blood pool angiography. Therefore, this type of contrast agent is limited in the application of ultrasound molecular imaging.

[0004] Chemotherapy plays an important role and significance in the treatment of breast cancer, but it also has the problem of systemic toxic side effects. Programmed death receptor 1 (PD1) immune checkpoint inhibitors are one of the hot spots in the research of breast cancer tumor immunotherapy, and related immunotherapy drugs have been launched, which have benefited many breast cancer patients from immunotherapy. However, in some breast cancer patients, the expression of programmed cell death receptor-ligand 1 (PD-L1) is also increased, T cell dysfunction and immune escape occur. Small molecule blockade and antibody blockade may cause drug resistance and non-specific immune-related adverse reactions. In addition, the low expression of "tumor suppressor gene" microRNA (miR) in BC often makes breast cancer patients less sensitive to chemotherapy drugs and even leads to drug resistance, such as tumor suppressor gene miRNA let-7a. It can be seen that single therapy often has various disadvantages and is often accompanied by various side effects, which cannot meet the treatment needs of breast cancer patients. It is urgent to develop combination therapy to treat breast cancer from multiple angles.

[0005] In summary, the development of a new ultrasound contrast agent with good vascular penetration in tumor tissue, strong targeting, high drug utilization, good imaging effect in tumor tissue, and the ability to reduce the drug resistance of chemotherapy drugs and improve the treatment effect is of great significance for improving the efficiency of tumor immunotherapy drug delivery and realizing the integration of tumor diagnosis and immunotherapy under ultrasound guidance. Summary of the invention

[0006] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a double-layer protective shell multifunctional targeted ultrasound contrast agent, which is a new type of ultrasound microbubble formed by using a nanocarrier to load chemotherapeutic drugs, immune checkpoint inhibitors, and miRNA, and embedding them into nanodroplets and ultrasound microbubbles. The ultrasound contrast agent of the present invention has a double-layer protective shell (ultrasound microbubbles and perfluoroalkane nanodroplets containing cRGD cyclopeptide-polycaprolactone-polyethylene glycol), which not only has high biological safety and can circulate in vivo for a long time, but also has good imaging effect, strong targeting, and high drug utilization rate. It can improve the targeting of chemotherapeutic drugs, immune checkpoint inhibitors and miRNA while realizing ultrasound-guided tumor diagnosis and treatment, and comprehensively and effectively solves the current difficulties in ultrasound diagnosis of tumors, easy degradation of immunosuppressive drugs, and weak targeting.

[0007] At the same time, the purpose of the present invention is also to provide a method for preparing the above-mentioned double-layer protective shell type multifunctional targeted ultrasound contrast agent.

[0008] At the same time, the purpose of the present invention is also to provide the application of the above-mentioned double-layer protective shell type multifunctional targeted ultrasound contrast agent.

[0009] One of the purposes of the present invention is achieved by the following technical solution:

[0010] A double-layer protective shell multifunctional targeted ultrasound contrast agent, wherein the double-layer protective shell multifunctional targeted ultrasound contrast agent is prepared by loading a chemotherapeutic drug, an immune checkpoint inhibitor and miRNA on a nanocarrier, then embedding them into nanodroplets, and further mixing them with ultrasound microbubbles to obtain the agent;

[0011] The chemotherapy drug is one or more of doxorubicin, doxorubicin hydrochloride, paclitaxel, and gemcitabine; the immune checkpoint inhibitor is PD-L1 siRNA; the miRNA is miRNA let7a; the nanocarrier is cellulose nanocrystals; the nanodroplets are perfluoroalkane nanodroplets containing cRGD cyclopeptide-polycaprolactone-polyethylene glycol; and the ultrasonic microbubbles are SonoVue.

[0012] The ultrasound contrast agent provided by the present invention adopts cellulose nanocrystals as drug carriers, loads chemotherapeutic drugs, immune checkpoint inhibitors, and miRNA drugs, and embeds them into nanodroplets and ultrasonic microbubbles for preparation. Among them, the nanodroplets used in the present invention contain polymer cRGD cyclopeptide-polycaprolactone-polyethylene glycol. Among the polymers, polycaprolactone (PCL) has excellent properties such as good biocompatibility, good biodegradability, and high safety, and is a widely used controlled release carrier. Polyhexylene glycol (PEG) is a hydrophilic polymer with good stability and is not easy to cause immune response. Based on the use of PCL's excellent compatibility, good biodegradability, high safety and other excellent properties and the use of non-immunogenic polymer material PEG, the present invention further combines the targeting of cancer cell integrin α v β3 of cRGD cyclic peptide to form a cRGD cyclic peptide-polycaprolactone-polyethylene glycol high molecular polymer. The prepared nanocarrier has the performance of non-immunogenicity in vivo, and also ensures the efficient performance of the drug efficacy and rapid and safe degradation of the loaded drug. Therefore, the present invention uses nano droplets to enhance the targeting of the loaded drug, improve the accumulation of the drug in the tumor tissue, and reduce the toxicity in the body, thereby achieving the effect of drug release in the tumor.

[0013] Further, the drugs loaded on the drug carrier of the present invention include chemotherapeutic drugs, immune checkpoint inhibitors and miRNA at the same time. By loading the above-mentioned drugs, after tail vein injection, by giving a certain amount of energy of ultrasonic irradiation and cRGD peptide and tumor cell directional targeting in the local tumor through the body surface, the microbubbles are ruptured under ultrasound, releasing nano droplets, and the droplets enter the inside of the tumor tissue and vaporize and expand into micron bubbles, and then rupture to release drugs, and the drugs are inserted into tumor cell DNA to prevent replication and transcription to inhibit tumor proliferation, silence PD-L1, and block PD-1 / PD-L1 immune checkpoints to improve the immune microenvironment inside the tumor tissue, so as to achieve the purpose of killing tumor cells. At the same time, the ultrasonic irradiation formed during ultrasound contrast diagnosis can cause mechanical vibration to destroy the structure of the cancer cell membrane, resulting in cancer cell death. Therefore, the present invention can improve the drug treatment effect from multiple angles.

[0014] As a further preferred embodiment, the chemotherapy drug is doxorubicin hydrochloride.

[0015] As a preferred embodiment, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand a as shown in SEQ ID NO:1 and an antisense strand a as shown in SEQ ID NO:2; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand b as shown in SEQ ID NO:3 and an antisense strand b as shown in SEQ ID NO:4; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand c as shown in SEQ ID NO:5 and an antisense strand c as shown in SEQ ID NO:6; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand d as shown in SEQ ID NO:7 and an antisense strand d as shown in SEQ ID NO:8.

[0016] As a preferred embodiment, the miRNA let7a is a double-stranded RNA consisting of a sense strand e having a nucleotide sequence as shown in SEQ ID NO:9 and an antisense strand e having a nucleotide sequence as shown in SEQ ID NO:10.

[0017] As a preferred embodiment, in the cRGD cyclic peptide-polycaprolactone-polyethylene glycol, the average molecular weight of polycaprolactone is 5000 g / mol, and the average molecular weight of polyethylene glycol is 2000 g / mol.

[0018] The second object of the present invention is achieved by adopting the following technical solution:

[0019] The preparation method of the above-mentioned double-layer protective shell type multifunctional targeted ultrasound contrast agent comprises the following steps:

[0020] (1) preparing the nanocarrier into a nanocarrier suspension; adding a polyethyleneimine solution to the nanocarrier suspension, stirring and centrifuging, resuspending, then adding an immune checkpoint inhibitor and miRNA, mixing and incubating at low temperature, and then adding a chemotherapeutic drug and mixing to obtain a nanodrug;

[0021] (2) stirring and dissolving cRGD cyclic peptide-polycaprolactone-polyethylene glycol in a first solvent, then evaporating the first solvent, adding DEPC water to dissolve, adding the first solvent again and stirring, then evaporating the first solvent, adding the nano drug described in step (1) to the solution obtained after evaporation and stirring, and then adding a perfluorocarbon compound for ultrasonic emulsification to obtain targeted modified nano droplets;

[0022] (3) Mixing the ultrasonic microbubbles with the targeted modified nanodroplets obtained in step (2) to obtain a double-layer protective shell multifunctional targeted ultrasound contrast agent.

[0023] The above-mentioned preparation method provided by the present invention is to mix cellulose nanocrystals and polyethyleneimine to construct a nanocarrier in the form of a cationic polymer, and then load chemotherapeutic drugs, immune checkpoint inhibitors and miRNA through electrostatic layer-by-layer self-assembly technology, and introduce cRGD cyclic peptide-polycaprolactone-polyethylene glycol to form targeted modified nanodroplets after solvent evaporation and ultrasonic emulsification, and further mix ultrasonic microbubbles to prepare an ultrasonic contrast agent. The preparation method of the present invention has a simple process, and the obtained ultrasonic contrast agent has a double-layer protective shell of ultrasonic microbubbles and perfluoroalkane nanodroplets of cRGD cyclic peptide-polycaprolactone-polyethylene glycol. It not only has high biological safety and can circulate in vivo for a long time, but also has a high drug loading capacity of the obtained contrast agent, which can achieve rapid and accurate step-by-step drug delivery in a targeted tumor area under the guidance of external ultrasonic imaging, and realize the integrated diagnosis and treatment of tumors.

[0024] Furthermore, the ultrasound contrast agent prepared by the above method of the present invention, due to the low boiling point of perfluorocarbon compounds, will change from liquid to gas after ultrasonic emulsification, and the change in particle size will enhance the intensity of its ultrasonic echo, achieving the effect of contrast imaging inside the tumor. In addition, the ultrasound contrast agent provided by the present invention can be imaged by ultrasound machine before rupture, and after entering the tumor tissue, it will rupture after ultrasonic irradiation and accumulate in the tumor cells, thereby enhancing the immunotherapy effect. Therefore, the present invention successfully combines ultrasound contrast agents with tumor immunotherapy, chemotherapy and gene therapy, realizing the integrated diagnosis and treatment of tumors.

[0025] As a preferred solution, in step (1), the dosage ratio of nanocarrier, immune checkpoint inhibitor, miRNA, and chemotherapeutic drug is (4-6) mg: (80-120) μL: (80-120) μL: (1-3) mg; the temperature of the low-temperature incubation is -5-5°C, and the time is 10-30 min. More preferably, the dosage ratio of nanocarrier, immune checkpoint inhibitor, miRNA, and chemotherapeutic drug is 5 mg: 100 μL: 100 μL: 1 mg.

[0026] As a preferred embodiment, in step (1), the mass ratio of the nanocarriers in the nanocarrier suspension to the polyethyleneimine in the polyethyleneimine solution is (4-6):(10-20), more preferably 5:15.

[0027] As a preferred solution, in step (2), the ratio of cRGD cyclic peptide-polycaprolactone-polyethylene glycol, the first solvent, and the perfluorocarbon compound is (4-6) mg: (0.4-0.6) mL: (15-25) μL. More preferably, the ratio of cRGD cyclic peptide-polycaprolactone-polyethylene glycol, the first solvent, and the perfluorocarbon compound is 5 mg: 0.5 mL: 20 μL.

[0028] As a preferred embodiment, in step (2), the first solvent is one or more of acetone, methanol, ethanol, and dimethyl sulfoxide, more preferably acetone; the perfluorocarbon compound is one or more of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane, more preferably perfluorohexane; the power of the ultrasonic emulsification is 50 to 200 W; and the time of the ultrasonic emulsification is 1 to 5 s.

[0029] As a preferred solution, in step (3), the mixing volume ratio of ultrasonic microbubbles to targeted modified nanodroplets is (0.8-1.2):(0.8-1.2), more preferably 1:1.

[0030] As a preferred solution, the ultrasonic microbubbles are prepared by dissolving a tube of SonoVue powder in 5 mL of normal saline;

[0031] The third object of the present invention is achieved by adopting the following technical solution:

[0032] The double-layer protective shell multifunctional targeted ultrasound contrast agent is used in the preparation of diagnostic reagents for ultrasound diagnosis of breast cancer and / or pharmaceutical preparations for the treatment of breast cancer.

[0033] The beneficial effects of the technical solution of the present invention are:

[0034] The double-layer protective shell multifunctional targeted ultrasound contrast agent provided by the present invention is a composite ultrasound contrast agent and has a double-layer protective shell. Among them, on the one hand, the ultrasound microbubbles can pass through the vascular endothelial cells to reach the extravascular target under the action of ultrasound, target the tumor tissue through high permeability and retention effect and enter the tumor tissue gap. On the other hand, the perfluorocarbon compound forms nanodroplets after ultrasonic emulsification, which has good phase change properties. It changes from liquid to gas under ultrasonic irradiation to form gas-containing vesicles. Under the action of ultrasonic irradiation and cyclic peptide cRGD, the nanocontrast agent is enriched in the tumor tissue, and the imaging effect is effectively exerted through the aggregation of the contrast agent, providing strong evidence for tumor diagnosis.

[0035] Therefore, the ultrasound contrast agent prepared by the present invention can perform fixed-point imaging and step-by-step targeted drug delivery to the lesion site under the guidance of ultrasound imaging, thereby maximizing drug utilization. By allowing the contrast agent provided by the present invention to penetrate into the tumor tissue through the vascular system under ultrasound conditions, accurate step-by-step drug delivery to the targeted tumor area can be achieved under the guidance of external ultrasound contrast imaging conditions, significantly improving the anti-tumor effect of immunotherapy drugs, and realizing the integration of tumor diagnosis and treatment. Therefore, the ultrasound contrast agent provided by the present invention has broad application prospects in the field of ultrasonic molecular imaging and / or tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is an image of the double-layer protective shell multifunctional targeted ultrasound contrast agent in Example 1 of the present invention under a fluorescence microscope;

[0037] Figure 2 This is an agarose gel image of RNA in the supernatant of the double-layer protective shell type multifunctional targeted ultrasound contrast agent in Experimental Example 1 of the present invention;

[0038] Figure 3 This is an in vitro ultrasound contrast imaging diagram of the double-layer protective shell type multifunctional targeted ultrasound contrast agent in Experimental Example 2 of the present invention at different MI values ​​in B mode and contrast mode;

[0039] Figure 4 The effects of the double-layer protective shell multifunctional targeted ultrasound contrast agent in Experimental Example 3 of the present invention and other experimental groups on the weight of mice;

[0040] Figure 5 This is the effect of the double-layer protective shell multifunctional targeted ultrasound contrast agent in Experimental Example 3 of the present invention and other experimental groups on the tumor volume of mice. DETAILED DESCRIPTION

[0041] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and drawings. However, it should be understood by those skilled in the art that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following embodiments are conventional methods unless otherwise specified; the raw materials used are materials commonly used in the art and available to the public through commercial channels unless otherwise specified.

[0042] In the following embodiments, cellulose nanocrystals are from Shansi New Material Technology Co., Ltd. In other embodiments, cellulose nanocrystals may also be made of cellulose nanocrystal raw materials from other sources, as long as they can achieve the loading of nano drugs, and the invention is not particularly limited thereto.

[0043] In the cRGD cyclic peptide-polycaprolactone-polyethylene glycol used in the following examples, the average molecular weight of polycaprolactone is 5000 g / mol, and the average molecular weight of polyethylene glycol is 2000 g / mol. The polymer is denoted as cRGD-PCL5K-PEG2K, which is provided by Xi'an Ruixi Biotechnology Co., Ltd., with the product number R-CW-PP07K.

[0044] The SonoVue used in the following examples is a commercial product. The commercial SonoVue is a white powder, and each commercial SonoVue contains 59 mg SF6 gas and 25 mg lyophilized powder (lyophilized powder composition: polyethylene glycol 4000, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol sodium, palmitic acid). When used, according to the preparation method specified in the instructions, a SonoVue is mixed with 5 mL of normal saline to prepare ultrasonic microbubbles.

[0045] In the present invention, the PD-L1 siRNA that can be used is a double-stranded RNA consisting of a sense strand a as shown in SEQ ID NO:1 and an antisense strand a as shown in SEQ ID NO:2; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand b as shown in SEQ ID NO:3 and an antisense strand b as shown in SEQ ID NO:4; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand c as shown in SEQ ID NO:5 and an antisense strand c as shown in SEQ ID NO:6; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand d as shown in SEQ ID NO:7 and an antisense strand d as shown in SEQ ID NO:8.

[0046] In the present invention, miRNA let7a is a double-stranded RNA consisting of a sense strand e having a nucleotide sequence as shown in SEQ ID NO:9 and an antisense strand e having a nucleotide sequence as shown in SEQ ID NO:10.

[0047] The nucleotide sequences of SEQ ID NO:1 to SEQ ID NO:10 of the present invention are obtained by replacing "U" in RNA with "T".

[0048] The nucleotide sequence of the sense strand a shown in SEQ ID NO: 1 is: 5'-CTGACATTCATCTTCCGTTTATT-3', and the sequence before replacement is 5'-CUGACAUUCAUCUUCCGUUUATT-3';

[0049] The nucleotide sequence of the antisense strand a shown in SEQ ID NO: 2 is: 5'-TAAACGGAAGATGAATGTCAGTT-3', and the sequence before replacement is 5'-UAAACGGAAGAUGAAUGUCAGTT-3';

[0050] The nucleotide sequence of the sense strand b shown in SEQ ID NO: 3 is: 5'-CGAATTACTGTGAAAGTCAATTT-3', and the sequence before replacement is 5'-CGAAUUACUGUGAAAGUCAAUTT-3';

[0051] The nucleotide sequence of the antisense strand b shown in SEQ ID NO: 4 is: 5'-ATTGACTTTCACAGTAATTCGTT-3', and the sequence before replacement is 5'-AUUGACUUUCACAGUAAUUCGTT-3';

[0052] The nucleotide sequence of the sense strand c shown in SEQ ID NO:5 is: 5'-GAGGTAATCTGGACAAACA-3',

[0053] The sequence before replacement was 5′-GAGGUAAUCUGGACAAACA-3′;

[0054] The nucleotide sequence of the antisense strand c shown in SEQ ID NO:6 is: 5'-TGTTTGTCCAGATTACCTC-3',

[0055] The sequence before replacement was 5′-UGUUUGUCCAGAUUACCUC-3′;

[0056] The nucleotide sequence of the sense strand d shown in SEQ ID NO:7 is: 5'-GACCTATATGTGGTAGAGTATTT-3', and the sequence before replacement is 5'-GACCUAUAUGUGGUAGAGUAUTT-3';

[0057] The nucleotide sequence of the antisense strand d shown in SEQ ID NO:8 is: 5'-ATACTCTACCACATATAGGTCTT-3', and the sequence before replacement is 5'-AUACUCUACCACAUAUAGGUCTT-3';

[0058] The nucleotide sequence of the sense strand e shown in SEQ ID NO:9 is: 5'-TGAGGTAGTAGGTTGTATAGTT-3', and the sequence before replacement is 5'-UGAGGUAGUAGGUUGUAUAGUU-3';

[0059] The nucleotide sequence of the antisense strand e shown in SEQ ID NO: 10 is: 5'-AACTATACAACCTACTACCTCA-3', and the sequence before replacement is 5'-AACUAUACAACCUACUACCUCA-3'.

[0060] Example 1

[0061] This embodiment provides a double-layer protective shell multifunctional targeted ultrasound contrast agent, which is prepared by loading chemotherapy drugs, immune checkpoint inhibitors and miRNA on nanocarriers, then embedding them into nanodroplets, and further mixing them with ultrasound microbubbles to prepare;

[0062] Among them, the chemotherapy drug is doxorubicin hydrochloride; the immune checkpoint inhibitor is PD-L1 siRNA; the miRNA is miRNAlet7a; the nanocarrier is cellulose nanocrystals; the nanodroplets are perfluoroalkane nanodroplets containing cRGD cyclic peptide-polycaprolactone-polyethylene glycol (cRGD-PCL5K-PEG2K); and the ultrasonic microbubbles are SonoVue.

[0063] Furthermore, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand d having a nucleotide sequence as shown in SEQ ID NO:7 and an antisense strand d having a nucleotide sequence as shown in SEQ ID NO:8.

[0064] The nucleotide sequence of the sense strand d shown in SEQ ID NO:7 is:

[0065] 5'-GACCTATATGTGGTAGAGTATTT-3';

[0066] The nucleotide sequence of the antisense strand d shown in SEQ ID NO:8 is:

[0067] 5'-ATACTCTACCACATATAGGTCTT-3'.

[0068] miRNA let7a is a double-stranded RNA consisting of a sense strand e as shown in the nucleotide sequence of SEQ ID NO:9 and an antisense strand e as shown in the nucleotide sequence of SEQ ID NO:10.

[0069] The nucleotide sequence of the sense strand e shown in SEQ ID NO:9 is:

[0070] 5'-TGAGGTAGTAGGTTGTATAGTT-3';

[0071] The nucleotide sequence of the sense strand e shown in SEQ ID NO: 10 is:

[0072] 5'-AACTATACAACCTACTACCTCA-3'.

[0073] The preparation method of the above-mentioned double-layer protective shell type multifunctional targeted ultrasound contrast agent specifically comprises the following steps:

[0074] (1) Prepare cellulose nanocrystals (CNC) into a CNC suspension with a concentration of 5 mg / mL using DEPC water; add 1 mL of CNC suspension to a container, add 1 mL of 15 mg / mL PEI solution at a stirring speed of 200 rpm, stir for 30 min, then centrifuge at 10000 rpm and 4°C for 10 min, discard the supernatant, resuspend the precipitate with 1 mL of DEPC water, and then centrifuge, resuspend, centrifuge, and resuspend again according to the above steps to obtain a resuspension. Add 100 μL of PD-L1 siRNA (100 pmol) and 100 μL of miRNA let7a (100 pmol) to the above resuspension, pipet and vortex to mix, incubate on ice for 20 min, centrifuge, discard the supernatant, and obtain a precipitate. 1 mL of 1 mg / mL doxorubicin hydrochloride solution (DOX·HCL) was added dropwise to the above precipitate, and the mixture was evenly mixed by blowing and vortexing. The mixture was centrifuged, the supernatant was discarded, and the mixture was resuspended in 1 mL of DEPC water to obtain the nanomedicine.

[0075] (2) Take 5 mg of cRGD-PCL5K-PEG2K polymer in a sterile enzyme-free round-bottom flask, add 0.5 mL of acetone, stir for 15 minutes, and then perform rotary evaporation until the acetone in the flask is completely evaporated, then add 1 mL of DEPC water to dissolve and stir for another 15 minutes, and add 0.5 mL of acetone while stirring during this stirring process. After the stirring is completed, perform rotary evaporation again until the acetone is completely evaporated, add the nanodrug obtained in step (1) to the solution after rotary evaporation, stir for 15 minutes, and then add 20 uL of perfluorohexane, emulsify for 1 second under the condition of an ultrasonic power of 100 W, and obtain targeted modified nanodroplets.

[0076] (3) Take 5 mL of normal saline to fully dissolve the commercially available SonoVue powder to obtain ultrasonic microbubbles, and mix the ultrasonic microbubbles with the targeted modified nanodroplets obtained in step (2) at a volume ratio of 1:1 to obtain a double-layer protective shell multifunctional targeted ultrasound contrast agent.

[0077] The fluorescence microscope image of the double-layer protective shell multifunctional targeted ultrasound contrast agent of Example 1 is as follows: Figure 1 As shown. Figure 1 It can be seen that the double-layer protective shell type multifunctional targeted ultrasound contrast agent prepared by the present invention achieves successful drug loading.

[0078] Experimental Example 1: RNA loading amount determination experiment

[0079] The RNA loading amount in the double-layer protective shell multifunctional targeted ultrasound contrast agent of Example 1 was determined by agarose gel. Figure 2 shown. Figure 2 The test results show that when 100 μL of PD-L1 siRNA (100 pmol) and 100 μL of miRNA let7a (100 pmol) are loaded using the ratio in Example 1 of the present invention, that is, when the mass ratio of ultrasound contrast agent to RNA is 2:1, the free RNA content in the supernatant is the lowest, and the optimal loading amount can be obtained at this time.

[0080] Experimental Example 2: In vitro imaging experiment

[0081] Referring to the preparation method of Example 1, the double-layer protective shell multifunctional targeted ultrasound contrast agent (denoted as SR-CPRDA) prepared in Example 1 was injected into the small capsule at the head of the rubber-tipped dropper, and ultrapure water was used as the background. In vitro ultrasound detection was performed using a Doppler color ultrasound diagnostic instrument (HITACHI, Japan) in B mode and contrast mode, and the ultrasound contrast enhancement effect under different mechanical indexes (MI was 0.08, 0.20, 0.40, 0.60, 0.80 and 1.00, respectively) was studied. The in vitro ultrasound contrast imaging of the double-layer protective shell multifunctional targeted ultrasound contrast agent of this example is shown in FIG. Figure 3 shown.

[0082] Depend on Figure 3 It can be seen that in B mode and contrast mode, the ultrasound image enhancement effect becomes brighter in color and clearer in image layer details as MI increases, indicating that the double-layer protective shell multifunctional targeted ultrasound contrast agent has good imaging effect under ultrasound, and the ultrasound echo intensity is MI-dependent.

[0083] Experimental Example 3: Study on Biosafety and Tumor Treatment Effect

[0084] Female Balb / c mice inoculated with 4T1 cells (mouse breast cancer cells) and successfully bearing tumors were randomly divided into 6 groups, with 6 mice in each group. The experimental groups were: (1) saline group; (2) free doxorubicin group (DOX); (3) nanodrug group (CPDA) obtained in step (1) of Example 1 of the present invention; (4) targeted modified nanodroplet group (R-CPRDA) obtained in step (2) of Example 1 of the present invention; (5) double-layer protective shell multifunctional targeted ultrasound contrast agent group (SR-CPRDA) of Example 1; (6) ultrasound + double-layer protective shell multifunctional targeted ultrasound contrast agent group (SR-CPRDA+US) of Example 1. Except for the saline group, each group was given a different preparation containing an effective dose of 5 mg / kg doxorubicin by tail vein injection, once every 3 days, and monitored continuously for 20 days. At the same time, for the group requiring ultrasound stimulation, 1 hour after the administration of the preparation, a power of 1 W / cm 2 The tumor site was stimulated by ultrasound for 2 minutes. The weight and tumor volume of mice were measured every two days. The results were as follows: Figure 4 , Figure 5 shown.

[0085] Depend on Figure 4 The test results of the mouse weight showed that the weight trend of the mice in the ultrasound + double-layer protective shell multifunctional targeted ultrasound contrast agent group of Example 1 was the same as that in the normal saline group, indicating that the ultrasound plus the ultrasound contrast agent of the present invention had good in vivo safety. This also confirmed that the ultrasound contrast agent of the present invention has good biosafety and can be further applied to in vivo treatment.

[0086] Depend on Figure 5The tumor volume test results show that the free DOX group has a certain tumor treatment effect, but the experimental group of ultrasound plus double-layer protective shell multifunctional targeted ultrasound contrast agent in Example 1 of the present invention has the slowest tumor volume growth compared with other groups, and it is in a decreasing trend, indicating that the double-layer protective shell multifunctional targeted ultrasound contrast agent in Example 1 has the best anti-tumor effect. This phenomenon shows that after ultrasonic irradiation, the perfluoroalkane compounds vaporize to produce ultrasonic cavitation effect, releasing the carried chemotherapy drugs and RNA around the tumor cells. The released chemotherapy drugs, siRNA, and miRNA can enter the blood vessel wall and even the tissue gap, prevent tumor cell DNA replication, specifically target PD-L1 on the surface of tumor cells and silence it, block the PD-1 / PD-L1 immune checkpoint, so as to improve the immune microenvironment inside the tumor tissue, and finally significantly enhance the inhibitory effect on the tumor.

[0087] In summary, the double-layer protective shell-type multifunctional targeted ultrasound contrast agent with adjustable particle size provided by the present invention is a composite ultrasound contrast agent with a simple process and a high drug loading capacity. It can achieve rapid and accurate step-by-step drug delivery in targeted tumor areas under the guidance of external ultrasound imaging, effectively solving the current problems of complex preparation of ultrasound contrast agents, difficulty in ultrasound diagnosis of tumors, easy degradation and lack of specificity of immunotherapy drugs, and realizing the integration of tumor diagnosis and immunotherapy under ultrasound guidance.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A double-layer protective shell multifunctional targeted ultrasound contrast agent, characterized in that: The double-layer protective shell multifunctional targeted ultrasound contrast agent is prepared by loading the chemotherapy drug, immune checkpoint inhibitor and miRNA on a nanocarrier, then embedding them into nanodroplets, and further mixing them with ultrasound microbubbles to obtain the contrast agent. The chemotherapy drug is one or more of doxorubicin, doxorubicin hydrochloride, paclitaxel, and gemcitabine; the immune checkpoint inhibitor is PD-L1 siRNA; the miRNA is miRNA let7a; the nanocarrier is cellulose nanocrystals; the nanodroplets are perfluoroalkane nanodroplets containing cRGD cyclopeptide-polycaprolactone-polyethylene glycol; and the ultrasonic microbubbles are SonoVue.

2. The double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 1, characterized in that: The PD-L1 siRNA is a double-stranded RNA consisting of a sense strand a as shown in SEQ ID NO:1 and an antisense strand a as shown in SEQ ID NO:2; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand b as shown in SEQ ID NO:3 and an antisense strand b as shown in SEQ ID NO:4; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand c as shown in SEQ ID NO:5 and an antisense strand c as shown in SEQ ID NO:6; or, the PD-L1 siRNA is a double-stranded RNA consisting of a sense strand d as shown in SEQ ID NO:7 and an antisense strand d as shown in SEQ ID NO:

8.

3. The double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 1, characterized in that: The miRNA let7a is a double-stranded RNA consisting of a sense strand e as shown in the nucleotide sequence of SEQ ID NO:9 and an antisense strand e as shown in the nucleotide sequence of SEQ ID NO:

10.

4. The double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 1, characterized in that: In the cRGD cyclic peptide-polycaprolactone-polyethylene glycol, the average molecular weight of polycaprolactone is 5000 g / mol, and the average molecular weight of polyethylene glycol is 2000 g / mol.

5. A method for preparing a double-layer protective shell multifunctional targeted ultrasound contrast agent as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: (1) preparing the nanocarrier into a nanocarrier suspension; adding a polyethyleneimine solution to the nanocarrier suspension, stirring and centrifuging, resuspending, then adding an immune checkpoint inhibitor and miRNA, mixing and incubating at low temperature, and then adding a chemotherapeutic drug and mixing to obtain a nanodrug; (2) stirring and dissolving cRGD cyclic peptide-polycaprolactone-polyethylene glycol in a first solvent, then evaporating the first solvent, adding DEPC water to dissolve, adding the first solvent again and stirring, then evaporating the first solvent, adding the nano drug described in step (1) to the solution obtained after evaporation and stirring, and then adding a perfluorocarbon compound for ultrasonic emulsification to obtain targeted modified nano droplets; (3) Mixing the ultrasonic microbubbles with the targeted modified nanodroplets obtained in step (2) to obtain a double-layer protective shell multifunctional targeted ultrasound contrast agent.

6. The method for preparing the double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 5, characterized in that: In step (1), the dosage ratio of the nanocarrier, immune checkpoint inhibitor, miRNA, and chemotherapy drug is (4-6) mg: (80-120) μL: (80-120) μL: (1-3) mg; the temperature of the low-temperature incubation is -5-5°C, and the time is 10-30 min.

7. The method for preparing the double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 5, characterized in that: In step (2), the usage ratio of cRGD cyclic peptide-polycaprolactone-polyethylene glycol, the first solvent, and the perfluorocarbon compound is (4-6) mg: (0.4-0.6) mL: (15-25) μL.

8. The method for preparing the double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 5, characterized in that: In step (2), the first solvent is one or more of acetone, methanol, ethanol, and dimethyl sulfoxide; the perfluorocarbon compound is one or more of perfluorohexane, perfluoropentane, decafluoropentane, and perfluoropropane; the power of the ultrasonic emulsification is 50 to 200 W, and the time of the ultrasonic emulsification is 1 to 5 s.

9. The method for preparing the double-layer protective shell multifunctional targeted ultrasound contrast agent according to claim 5, characterized in that: In step (3), the mixing volume ratio of ultrasonic microbubbles and targeted modified nanodroplets is (0.8-1.2): (0.8-1.2).

10. An application of the double-layer protective shell multifunctional targeted ultrasound contrast agent as claimed in any one of claims 1 to 4 or the double-layer protective shell multifunctional targeted ultrasound contrast agent prepared by the preparation method as claimed in any one of claims 5 to 9, characterized in that: Application in the preparation of diagnostic reagents for ultrasonic diagnosis of breast cancer and / or pharmaceutical preparations for the treatment of breast cancer.

Images