A nanocomplex loaded with PDGF-BB mRNA and its preparation method and application

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a method for preparing a nanocomposite carrying PDGF-BB mRNA, the preparation method comprising: 1) preparing LNP carrying PDGF-BB mRNA: quickly mixing a lipid phase containing 7C1 cationic liposomes with an aqueous phase containing PDGF-BB mRNA to obtain LNPmRNA; 2) preparing a PLGA gas core core by a double emulsion method; 3) mixing the PLGA gas core core in step 2) with the LNPmRNA in step 1), and standing to obtain a LNPmRNA@PLGA nanocomposite. The LNPmRNA@PLGA nanocomposite of the present invention has the advantages of protection, targeting, visibility and responsiveness. It is combined with in vitro focused ultrasound, which can not only realize the visual delivery of drugs, but also qualitatively and semi-quantitatively evaluate the neovascularization in advanced atherosclerotic plaques, and improve the cell transfection efficiency under the targeted microbubble destruction effect of in vitro focused ultrasound, accurately and efficiently overexpress the target protein, achieve the goal of drug treatment, and reduce systemic toxic side effects.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a nanocomplex carrying PDGF-BB mRNA and a preparation method and application thereof. Background Art

[0002] Atherosclerosis (AS) vulnerable plaques are insidious and often lead to serious consequences such as acute myocardial infarction, transient ischemic attack and stroke. At present, the most attention in the treatment of AS is still the prevention and intervention of a series of events caused by low-density lipoprotein oxidative stress. Although these measures have achieved good therapeutic effects, for advanced vulnerable plaques with complex internal components and various intertwined mechanisms, drug treatment alone cannot achieve completely satisfactory results, and the risks of surgical intervention will still bring a heavy burden to patients and their families. The typical characteristics of vulnerable plaques are large lipid cores, thin fibrous caps, pathological new blood vessels and inflammatory cell infiltration. More and more evidence shows that the integrity of pathological new blood vessels is impaired and the leakage of vascular walls is an important factor contributing to plaque instability and rupture. Therefore, for advanced AS plaques, a plan to restore the integrity of the structure and function of new blood vessels in the plaque and break the vicious cycle of hypoxia-angiogenesis has therapeutic potential. The thickness of the fibrous cap is another key factor affecting the stability of the plaque. Cells from various sources and collagen fibers secreted by the cells together form the plaque fibrous cap. The fibrous cap covers a large amount of lipids in the deep part to play a supporting role. Its thickness is closely related to the stability of the plaque. If the fibrous cap is too thin, it cannot protect the safety and stability of the plaque. If the fibrous cap is too thick, it will aggravate the internal hypoxia of the plaque.

[0003] Therefore, combining the two major influencing factors of advanced vulnerable plaques - neovascularization and fibrous cap thickness - to promote the maturation of neovascularization, alleviate vessel wall hypoxia, control angiogenesis, and appropriately increase the thickness of the fibrous cap to protect the plaque is a new strategy worth developing to stabilize vulnerable plaques.

[0004] mRNA has great potential in a range of applications including viral vaccines, protein replacement therapy, cancer immunotherapy, cell reprogramming and genome editing, becoming a new drug for the prevention and treatment of diseases. To ensure that mRNA works in vivo, a safe, effective and stable delivery system is required to protect the nucleic acid from degradation and allow cells to release mRNA.

[0005] With the continuous development of nanotechnology, the successful application of lipid nanoparticles (LNP)-loaded mRNA technology to treat the new coronavirus has once again become a research hotspot. Although there have been a large number of experimental studies on the use of lipid nanoparticles to encapsulate mRNA, most of their functions are relatively simple, such as lack of targeting, low efficiency in transfecting cells, premature degradation, and inability to be visually delivered in vivo. These shortcomings not only limit the efficacy of the drug, but may even cause systemic side effects to the patient, increasing the physical and economic burden on the patient. Summary of the invention

[0006] The present invention aims to provide a nanocomplex loaded with PDGF-BB mRNA and a preparation method and application thereof, in order to provide a nanocomplex with the advantages of targeting, protection, visualization and responsiveness, and to provide a new drug design idea for the treatment of vulnerable plaques in the late stage of atherosclerosis.

[0007] In order to achieve the above object, the present invention provides a method for preparing a nanocomplex loaded with PDGF-BB mRNA, comprising the following steps:

[0008] S1. Preparation of LNPs loaded with PDGF-BB mRNA: 7C1 cationic liposomes were mixed with polyethylene glycol and dissolved in anhydrous ethanol to obtain a lipid phase; PDGF-BB mRNA was dissolved in a sodium citrate buffer with a pH of 4 and a concentration of 10 mmol / L to obtain an aqueous phase; the lipid phase and the aqueous phase were quickly mixed to obtain LNP mRNA, and then immediately dialyzed in a PBS buffer with a pH of 7.4 overnight to obtain an LNP mRNA solution;

[0009] S2, preparing the PLGA gas core: adding PLGA to dichloromethane, adding perfluoropentane after fully dissolving, ultrasonically shaking until the solution becomes a white emulsion, adding the polyvinyl alcohol solution to the white emulsion, ultrasonically shaking again, and then adding the isopropanol solution, and evaporating the dichloromethane at low temperature; centrifuging and washing at low temperature after the dichloromethane is evaporated, collecting the precipitate, and resuspending and preserving it in a sodium citrate buffer with a pH of 4 and a concentration of 10 mmol / L to obtain a PLGA gas core solution; this step is performed on ice throughout the entire process;

[0010] S3, preparing LNPmRNA@PLGA nanocomplex: mixing the LNPmRNA solution in step S1 with the PLGA core solution in step S2, and letting the mixture stand to obtain the LNPmRNA@PLGA nanocomplex.

[0011] Optionally, the mass ratio of 7C1 cationic liposomes in step S1 to PLGA in step S2 is 0.5 to 32:1.

[0012] Optionally, the mass ratio of 7C1 cationic liposomes in step S1 to PLGA in step S2 is 16:1.

[0013] Optionally, in step S1, during the mixing of the lipid phase and the aqueous phase, the flow rate of the lipid phase is 3 mL / min, and the flow rate of the aqueous phase is 9 mL / min.

[0014] Optionally, in step S1, the mass ratio of 7C1 cationic liposome to polyethylene glycol is 4:1.

[0015] Optionally, in step S1, the volume ratio of the lipid phase to the aqueous phase is 1:3.

[0016] Optionally, in step S2, the conditions for ultrasonic oscillation after the addition of perfluoropentane are: power 90w / s, and ultrasonication at a frequency of running for 5s and stopping for 5s for 1min.

[0017] Optionally, in step S2, the conditions for ultrasonic oscillation after the polyvinyl alcohol solution is added are: power 90w / s, and ultrasonication for 3 minutes at a frequency of running for 5s and stopping for 5s.

[0018] Optionally, in step S2, the mass concentration of the polyvinyl alcohol solution is 4%.

[0019] Optionally, in step S2, the mass concentration of the isopropanol solution is 2%.

[0020] Optionally, in step S2, after the dichloromethane is evaporated, the centrifugal speed is 5000 rpm and the centrifugal time is 10 min.

[0021] The present invention also provides a PDGF-BB mRNA-loaded nanocomplex prepared by the above preparation method. The PDGF-BB mRNA-loaded nanocomplex has the advantages of targeting, protection, visualization and responsiveness, and can be used in the preparation of drugs for treating vulnerable plaques in the late stage of atherosclerosis.

[0022] The working principle and beneficial effects of this program are as follows: This program targets the two major factors that affect plaque stability - pathological neovascularization and fibrous cap thickness. On the one hand, it promotes pericyte recruitment, strengthens the interaction between neovascular endothelial cells and pericytes in atherosclerotic plaques, restores the integrity and function of the neovascular walls, relieves hypoxia to inhibit pathological angiogenesis, and prevents leakage of vascular contents to stabilize plaques; on the other hand, it stimulates vascular smooth muscle cells to transform from a contractile phenotype to a synthetic phenotype, and gradually migrate to the damaged part of the plaque to form a fibrous cap in the endothelium to stabilize the plaque.

[0023] Thus, in this scheme, PDGF-BB mRNA is used as the drug core, 7C1 ionizable liposomes are used to encapsulate mRNA, and combined with the PLGA gas core core in a positive and negative charge attraction manner. 7C1 LNP not only has good pH sensitivity, but also has the function of specifically targeting vascular endothelial cells, and finally forms a LNPmRNA@PLGA nano delivery platform with multiple effects. Under the triggering of in vitro focused ultrasound, the PLGA gas core core is "timed and fixed point" blasted to release LNPmRNA, so that it transfects the new blood vessel endothelial cells of the plaque, promotes the overexpression of PDGF-BB in the vascular endothelium, thereby starting the dual effects of "promoting vascular maturation" and "increasing the thickness of the fiber cap", and finally achieving the therapeutic goal of stabilizing the plaque. Moreover, compared with the traditional lipid nanoparticle synthesis scheme, the lipid nano synthesis of this scheme only requires two lipid components, and no additional targeting ligand is required to be connected, which improves the safety of the drug in vivo; and in step S1 of this scheme, under the premise that the previous drugs (lipid phase and water phase) are ready, the synthesis of lipid nanoparticles can be completed within 1 minute, which greatly improves the pharmaceutical efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the preparation process of LNPmRNA@PLGA nanocomplex in the present invention;

[0025] Figure 2 Nanoflow cytometry and quantitative analysis of LNPmRNA@PLGA nanocomplexes in various embodiments of the present invention;

[0026] Figure 3 Confocal microscopy images and quantitative analysis images of LNPmRNA@PLGA nanocomplexes in various embodiments of the present invention;

[0027] Figure 4 It is a transmission electron micrograph of three nanoparticles, LNPmRNA, PLGA gas core, and LNPmRNA@PLGA, in Example 1 of the present invention;

[0028] Figure 5 The particle size statistics and potential statistics of the three nanoparticles LNPmRNA, PLGA gas core, and LNPmRNA@PLGA in Example 1 of the present invention are respectively;

[0029] Figure 6 This is a quantitative analysis diagram of the stability of the LNPmRNA@PLGA nanocomplex in Example 1 of the present invention;

[0030] Figure 7 This is the mRNA gel electrophoresis diagram of LNPmRNA and LNPmRNA@PLGA nanocomplex before and after the lipid membrane is destroyed by Triton 100 in Example 1 of the present invention;

[0031] Figure 8 The in vitro ultrasound imaging and quantitative analysis diagram of the LNPmRNA@PLGA nanocomplex in Example 1 of the present invention;

[0032] Fig. 9 This is a quantitative analysis diagram of the activity of LNPmRNA@PLGA nanocomplexes of different concentrations on vascular endothelial cells in the present invention;

[0033] Fig.10 This is a quantitative analysis diagram of the blood toxicity detection of different concentrations of LNPmRNA@PLGA nanocomplexes in the present invention on mice;

[0034] Fig.11 This is the HE staining image of the main organs of mice after administration of LNPmRNA@PLGA nanocomplex in the present invention. DETAILED DESCRIPTION

[0035] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0036] The present invention provides a method for preparing a nanocomplex loaded with PDGF-BB mRNA, comprising the following steps:

[0037] (1) Preparation of LNPs loaded with PDGF-BB mRNA: 7C1 cationic liposomes and polyethylene glycol were mixed at a mass ratio of 4:1 and dissolved in anhydrous ethanol to obtain a lipid phase; PDGF-BB mRNA was dissolved in a sodium citrate buffer with a pH of 4 and a concentration of 10 mmol / L to obtain an aqueous phase; the lipid phase and the aqueous phase were rapidly mixed by microfluidics at a volume ratio of 1:3, wherein the flow rate of the lipid phase was 3 mL / min and the flow rate of the aqueous phase was 9 mL / min to obtain LNP mRNA, which was then immediately dialyzed overnight in a PBS buffer with a pH of 7.4 to obtain an LNP mRNA solution.

[0038] (2) Preparation of PLGA gas core: PLGA was added to dichloromethane, and perfluoropentane was added after it was fully dissolved. The solution was ultrasonically oscillated for 1 min at a power of 90 w / s with a frequency of running for 5 s and stopping for 5 s to turn the solution into a white emulsion. A 4 wt % polyvinyl alcohol solution was added to the white emulsion. The solution was ultrasonically oscillated for 3 min at a power of 90 w / s with a frequency of running for 5 s and stopping for 5 s. A 2 wt % isopropanol solution was then added. The dichloromethane was evaporated at low temperature for 24 hours. The solution was then centrifuged at 5000 rpm for 10 min. After two washes, the precipitate was collected and resuspended in a sodium citrate buffer solution with a pH of 4 and a concentration of 10 mmol / L to obtain a PLGA gas core solution. This step was performed on ice throughout the entire process.

[0039] (3) Preparation of LNPmRNA@PLGA nanocomplex: The LNPmRNA solution in step (1) and the PLGA core solution in step (2) were mixed and allowed to stand at room temperature for 30 minutes to obtain the LNPmRNA@PLGA nanocomplex.

[0040] Wherein, the mass ratio of 7C1 cationic liposome in step (1) to PLGA in step (2) is 0.5-32:1, preferably 16:1.

[0041] The LNPmRNA@PLGA nanocomplex of the present invention has advantages of protection, targeting, visibility and responsiveness, wherein the protection is embodied in that the ionizable LNP is protonated and positively charged at low pH values, but they remain neutral at physiological pH values. Neutral LNP interacts less with the anionic membrane of blood cells, which can improve the biocompatibility of LNP and facilitate the delivery of mRNA in vivo. In addition, by adding a biodegradable lipid component to the ionizable LNP, the overall tolerance and rapid metabolism of the LNP can be improved, and mRNA can be released.

[0042] Targeting is reflected in that the mRNA molecule must reach specific target cells and produce enough effective protein to exert a therapeutic effect. The targeting of LNP can be obtained by modifying or assembling lipid components, for example, by targeting specific cells through antibody encapsulation. In addition, targeting can also be achieved by adjusting the ratio of lipid components. The 7C1 to be used in the present invention is to achieve targeting of vascular endothelial cells by adjusting the lipid components. 7C1 is a polymer made of ionizable low molecular weight polyamines and lipids, which can target endothelial cells in multiple organs and participate in processes such as angiogenesis signals, vascular permeability, and inflammation.

[0043] Visibility is reflected in the following: Perfluoropentane (PFP) is a substance that can change from liquid to gas at 29°C. Therefore, when the PLGA gas core wrapped with PFP enters the body of mice (body temperature 37°C), the PLGA core will gradually increase in size and form a gas core. In this way, not only can good ultrasound enhanced imaging function be achieved to visualize drug delivery, but also the density of new blood vessels in advanced AS plaques can be further evaluated.

[0044] The responsiveness is reflected in the following aspects: the shock waves generated by ultrasound-targeted microbubble / nanobubble destruction (UTMD / UTND) technology will induce transient micropore formation on adjacent cell membranes, which can increase the transfection rate of mRNA to neovascular endothelial cells. In addition, the local thermal and mechanical effects produced by ultrasonic cavitation can further enhance the permeability of cell membranes and increase the transfection rate of LNP to cells. Compared with traditional chemical methods, electroporation and other methods, one of the significant advantages of UTMD / UTND as a gene transfection technology is the use of biocompatible microbubbles or nanobubbles as carriers, which not only reduces immune response and toxicity, but also achieves precise delivery of genes or drugs through ultrasound.

[0045] In summary, when the nanocomplex (LNPmRNA@PLGA) composed of LNPmRNA and PLGA gas core flows through the blood throughout the body, the nanocarrier has good biocompatibility and effectively protects mRNA; by adjusting the lipid composition, it can accurately target and transfect vascular endothelial cells; using liquid fluorocarbon (PFP) to form the gas core can visualize the delivery process and evaluate the density of new blood vessels; combined with focused ultrasound to trigger the cavitation effect of the gas core, further improve the transfection rate, which is more efficient and accurate than ordinary in vivo transfection. Therefore, LNPmRNA@PLGA can not only promote the maturation of new blood vessels in vulnerable plaques in vivo, but also increase the thickness of the fibrous cap, and play a role in stabilizing plaques.

[0046] The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range according to the description herein, and are not limited to the specific values ​​​​exemplified below.

[0047] In the following examples, PDGF-BB mRNA was purchased from Suzhou Haixing Biotechnology Co., Ltd., and 7C1 cationic liposomes were purchased from Shanghai Shuna Biotechnology Co., Ltd.

[0048] Example 1

[0049] This embodiment provides a nanocomposite loaded with PDGF-BB mRNA, namely, LNPmRNA@PLGA nanocomposite. The preparation process of the nanocomposite is as follows: Figure 1 As shown ( Figure 1 In the above, "LNP" refers to LNP mRNA, and "PLGA" refers to PLGA gas core), the preparation method of the nanocomposite specifically comprises the following steps:

[0050] (1) Preparation of LNPs loaded with PDGF-BB mRNA: 375 μg of 7C1 cationic liposomes were mixed with 125 μg of polyethylene glycol (C14PEG2000) and dissolved in 250 μL of anhydrous ethanol to obtain a lipid phase; 100 μg of PDGF-BB mRNA was dissolved in 750 μL of sodium citrate buffer at pH 4 and a concentration of 10 mmol / L to obtain an aqueous phase; the lipid phase and the aqueous phase were quickly mixed by microfluidics (the volume ratio of the lipid phase to the aqueous phase was 1:3) to obtain 1 mL of LNPmRNA mixed solution, which was then immediately dialyzed overnight in PBS buffer at pH 7.4 to obtain a LNPmRNA solution, and the LNPmRNA solution was sterile filtered through a 0.22 μm filter for later use. In the process of mixing the lipid phase and the aqueous phase, the flow rate of the lipid phase was 3 mL / min and the flow rate of the aqueous phase was 9 mL / min.

[0051] (2) Preparation of PLGA core: 350 μg PLGA (polylactic acid-co-glycolic acid) was added to 10 μL dichloromethane. After fully dissolved, 10 μL perfluoropentane (PFP) was added. The solution was ultrasonically shaken at a power of 90 w / s for 5 s on and off for 1 min to turn it into a white emulsion. 50 μL 4 wt % polyvinyl alcohol solution was added to the white emulsion. The solution was ultrasonically shaken at a power of 90 w / s for 3 min at a power of 5 s on and off for 5 s. Then, 100 μL 2 wt % isopropanol solution was added. The dichloromethane was evaporated at low temperature for 24 hours. Then, the solution was centrifuged at 5000 rpm for 10 min. After washing twice, the precipitate was collected and resuspended in 1 mL of sodium citrate buffer with a pH of 4 and a concentration of 10 mmol / L to obtain a PLGA core solution. This step was performed on ice throughout the entire process.

[0052] (3) Preparation of LNPmRNA@PLGA nanocomplex: 149 μL of the LNPmRNA solution filtered in step (1) (149 μL of LNPmRNA solution corresponds to 55.9 μg of 7C1 cationic liposomes) and 10 μL of the PLGA gas core solution in step (2) (10 μL of PLGA gas core solution corresponds to 3.5 μg of PLGA) were mixed and allowed to stand at room temperature for 30 min to obtain the LNPmRNA@PLGA nanocomplex. Thus, in this embodiment, the mass ratio of 7C1 cationic liposomes to PLGA is actually 16:1.

[0053] Example 2

[0054] The difference between this embodiment and embodiment 1 is that the amount of LNPmRNA solution taken in step (3) of this embodiment is different from that in step (3) of embodiment 1, and the rest are the same. Specifically, in step (3) of this embodiment, 4.6 μL of LNPmRNA solution is taken, so that in this embodiment, the mass ratio of 7C1 cationic liposome to PLGA is actually 0.5:1.

[0055] Example 3

[0056] The difference between this embodiment and embodiment 1 is that the amount of LNPmRNA solution taken in step (3) of this embodiment is different from that in step (3) of embodiment 1, and the rest are the same. Specifically, in step (3) of this embodiment, 9.3 μL of LNPmRNA solution is taken, so that in this embodiment, the mass ratio of 7C1 cationic liposome to PLGA is actually 1:1.

[0057] Example 4

[0058] The difference between this embodiment and embodiment 1 is that the amount of LNPmRNA solution taken in step (3) of this embodiment is different from that in step (3) of embodiment 1, and the rest are the same. Specifically, in step (3) of this embodiment, 18.6 μL of LNPmRNA solution is taken, so that in this embodiment, the mass ratio of 7C1 cationic liposome to PLGA is actually 2:1.

[0059] Example 5

[0060] The difference between this embodiment and embodiment 1 is that the amount of LNPmRNA solution taken in step (3) of this embodiment is different from that in step (3) of embodiment 1, and the rest are the same. Specifically, in step (3) of this embodiment, 37.2 μL of LNPmRNA solution is taken, so that in this embodiment, the mass ratio of 7C1 cationic liposome to PLGA is actually 4:1.

[0061] Example 6

[0062] The difference between this embodiment and embodiment 1 is that the amount of LNPmRNA solution taken in step (3) of this embodiment is different from that in step (3) of embodiment 1, and the rest are the same. Specifically, in step (3) of this embodiment, 74.5 μL of LNPmRNA solution is taken, so that in this embodiment, the mass ratio of 7C1 cationic liposome to PLGA is actually 8:1.

[0063] Example 7

[0064] The difference between this embodiment and embodiment 1 is that the amount of LNPmRNA solution taken in step (3) of this embodiment is different from that in step (3) of embodiment 1, and the rest are the same. Specifically, in step (3) of this embodiment, 298 μL of LNPmRNA solution is taken, so that in this embodiment, the mass ratio of 7C1 cationic liposome to PLGA is actually 32:1.

[0065] Experimental example

[0066] The LNPmRNA@PLGA nanocomplexes obtained in Examples 1-7 were subjected to nanoflow cytometry. Figure 2 As shown ( Figure 2 The "LNP:PLGA" in the figure refers to 7C1 cationic liposome:PLGA). Figure 2 It can be found that when the mass ratio of 7C1 cationic liposomes to PLGA is 16:1 and 32:1, the binding rate of LNPmRNA to the PLGA core is the highest. Since there is no significant difference in the statistical analysis of the two dosage ratios, the mass ratio of 7C1 cationic liposomes to PLGA is finally selected as 16:1.

[0067] The fluorescence binding of LNPmRNA and PLGA core core of the LNPmRNA@PLGA nanocomplex obtained in Example 1-7 under confocal microscopy is shown as follows: Figure 3 As shown ( Figure 3 In the figure, "PLGA" in the upper figure refers to PLGA gas core; "LNP:PLGA" in the lower figure refers to 7C1 cationic liposome:PLGA), which is consistent with Figure 2 The results shown are consistent.

[0068] The LNPmRNA, PLGA gas core and LNPmRNA@PLGA in Example 1 were detected by transmission electron microscopy (scale: 200nm, 600nm, 100nm). The results are as follows Figure 4 As shown in the figure, the upper left is a single LNPmRNA, which is circular; the lower left is a single PLGA gas core, which is circular; the right picture is LNPmRNA@PLGA, the green arrow indicates the LNPmRNA attached to the PLGA gas core, and the surrounding is scattered LNPmRNA, which shows that the present invention successfully carries LNPmRNA on the PLGA gas core. In addition, the average particle size and charge of LNPmRNA, PLGA gas core and LNPmRNA@PLGA are shown in Table 1 and Figure 5 As shown ( Figure 5 In the figure, "LNP" refers to LNPmRNA; "PLGA" refers to PLGA gas core), which shows that the particle size of the LNPmRNA@PLGA nanocomposite of the present invention is uniform, relatively stable, and has no agglomeration phenomenon.

[0069] Table 1 Average particle size and charge data of LNPmRNA, PLGA gas core and LNPmRNA@PLGA

[0070] Size(nm) Zata LNPmRNA 109±8.9 -13.3±0.85 PLGA air core 452±8.7 +5.6±0.46 (LNPmRNA@PLGA) 511±49.6 -0.8±0.21

[0071] The stability of the LNPmRNA@PLGA nanocomplex obtained in Example 1 is shown in Figure 6 As shown by Figure 6 It can be seen that the LNPmRNA@PLGA nanocomplex can be maintained for 36 h in both PBS buffer and serum. Figure 7 The mRNA gel electrophoresis diagram of LNPmRNA and LNPmRNA@PLGA nanocomplex before and after the lipid membrane was destroyed by Triton 100 ( Figure 7 In the figure, "LNP" refers to LNPmRNA, "LNP+T" refers to LNPmRNA+T), channels 1 and 3 are without the addition of Triton 100, while channels 2 and 4 are with the addition of Triton 100, which is a substance that can destroy the lipid layer. Figure 7 It can be seen that in channels 1 and 3 without the addition of Triton 100, the lipid layer can completely wrap the mRNA and the mRNA will not leak, so no bands can be displayed on the gel electrophoresis. However, in channels 2 and 4 with the addition of Triton 100, bands appeared on the gel electrophoresis due to the leakage of mRNA, which shows that the LNPmRNA@PLGA nanocomplex has good protection for mRNA. Figure 8As shown, the ultrasound imaging effect of the nanocomposite began to weaken at 8 minutes and completely disappeared at 30 minutes, indicating the long-term effect of the nanocomposite in vitro ultrasound imaging effect.

[0072] At the same time, the effects of different concentrations of the LNPmRNA@PLGA nanocomplex obtained in Example 1 on the activity of vascular endothelial cells are shown in the following table. Fig. 9 As shown ( Fig. 9 In the table, "control" represents normal cultured cells without drug addition). No matter it is a low concentration, a medium concentration or a high concentration, it has no significant effect on the activity of vascular endothelial cells, indicating that the nanocomplex of the present invention has cell safety (low concentration: 1 μg / mL; medium concentration: 2 μg / mL; high concentration 4 μg / mL; this drug concentration refers to the concentration of the mRNA contained). In addition, the LNPmRNA@PLGA nanocomplex obtained in Example 1 is also biosafe for mice at different concentrations. When the LNPmRNA@PLGA nanocomplex of different concentrations (low concentration: 100 μg / mL; medium concentration: 200 μg / mL; high concentration: 400 μg / mL; this drug concentration refers to the concentration of the mRNA contained) enters the body of mice through the tail vein, there is no significant change in the blood routine, liver and kidney function or coagulation function of the mice compared with the control group, as shown in FIG. Fig.10 As shown ( Fig.10 In the figure, "sham" means that the mice in this group were injected with an equal volume of normal saline through the tail vein. Meanwhile, the HE staining images of the main organs of the mice also showed that there was no obvious damage to the organs, such as Fig.11 Therefore, Figure 9-11 All these indicate that the nanocomplex has good biosafety.

[0073] In summary, the present invention combines LNPmRNA with the PLGA gas core and combines it with in vitro focused ultrasound, which can not only realize the visualized delivery of drugs, but also qualitatively and semi-quantitatively evaluate the neovascularization in advanced atherosclerotic plaques, and improve the cell transfection efficiency under the targeted microbubble destruction effect of in vitro focused ultrasound, accurately and efficiently overexpress the target protein, achieve the goal of drug treatment, and reduce systemic toxic side effects.

[0074] The above are only embodiments of the present invention. The invention is not limited to the fields involved in this implementation case. The common sense such as the known specific structures and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field to which the invention belongs before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the present invention. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to explain the content of the claims.

Claims

1. A method for preparing a nanocomplex loaded with PDGF-BB mRNA, characterized in that: The following steps are involved: S1. Preparation of LNPs loaded with PDGF-BB mRNA: 7C1 cationic liposomes were mixed with polyethylene glycol and dissolved in anhydrous ethanol to obtain a lipid phase; PDGF-BB mRNA was dissolved in a sodium citrate buffer with a pH of 4 and a concentration of 10 mmol / L to obtain an aqueous phase; the lipid phase and the aqueous phase were quickly mixed to obtain LNP mRNA, and then immediately dialyzed in a PBS buffer with a pH of 7.4 overnight to obtain an LNP mRNA solution; wherein the polyethylene glycol was C14PEG2000, and PDGF-BB mRNA was purchased from Suzhou Haixing Biotechnology Co., Ltd.; S2, preparing the PLGA gas core: adding PLGA to dichloromethane, adding perfluoropentane after fully dissolving, ultrasonically shaking until the solution becomes a white emulsion, adding the polyvinyl alcohol solution to the white emulsion, ultrasonically shaking again, and then adding the isopropanol solution, and evaporating the dichloromethane at low temperature; centrifuging and washing at low temperature after the dichloromethane is evaporated, collecting the precipitate, and resuspending and preserving it in a sodium citrate buffer with a pH of 4 and a concentration of 10 mmol / L to obtain a PLGA gas core solution; this step is performed on ice throughout the entire process; S3, preparing LNPmRNA@PLGA nanocomplex: mixing the LNPmRNA solution in step S1 with the PLGA core solution in step S2, and letting the mixture stand to obtain the LNPmRNA@PLGA nanocomplex.

2. The preparation method according to claim 1, characterized in that: The mass ratio of 7C1 cationic liposomes in step S1 to PLGA in step S2 is 8 to 32:

1.

3. The preparation method according to claim 2, characterized in that: The mass ratio of 7C1 cationic liposomes in step S1 to PLGA in step S2 is 16:

1.

4. The preparation method according to claim 1, characterized in that: In step S1, during the mixing of the lipid phase and the aqueous phase, the flow rate of the lipid phase was 3 mL / min, and the flow rate of the aqueous phase was 9 mL / min.

5. The preparation method according to claim 1, characterized in that: In step S1, the mass ratio of 7C1 cationic liposome to polyethylene glycol is 4:1; and / or, In step S1, the volume ratio of the lipid phase to the aqueous phase is 1:

3.

6. The preparation method according to claim 1, characterized in that: In step S2, the conditions for ultrasonic oscillation after the addition of perfluoropentane are: power 90w / s, ultrasonic oscillation at a frequency of 5s on and 5s off for 1 minute; and / or, In step S2, after the polyvinyl alcohol solution is added, the ultrasonic oscillation conditions are: power 90w / s, and ultrasonic oscillation for 3 minutes at a frequency of running for 5s and stopping for 5s.

7. The preparation method according to claim 1, characterized in that: In step S2, the mass concentration of the polyvinyl alcohol solution is 4%; and / or, In step S2, the mass concentration of the isopropanol solution is 2%.

8. The preparation method according to claim 1, characterized in that: In step S2, after the dichloromethane is evaporated, the centrifugal speed is 5000 rpm and the centrifugal time is 10 min.

9. A nanocomplex containing PDGF-BB mRNA obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the nanocomposite according to claim 9 in preparing a drug for treating vulnerable plaques in late stage of atherosclerosis.

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