Novel nucleic acid delivery system based on phosphatidylmitoxantrone as well as preparation method and application of novel nucleic acid delivery system

Abstract

The invention discloses a novel nucleic acid delivery system based on phosphatidylmitoxantrone as well as a preparation method and application of the novel nucleic acid delivery system based on phosphatidylmitoxantrone, and phosphatidylmitoxantrone (PMA) is used as a novel cationic lipid to construct a novel lipidosome nano nucleic acid delivery system by utilizing the enzymatic response release property and ionizable cationic phospholipid characteristics of phosphatidylmitoxantrone (PMA). PMA not only can be used as a novel cationic lipid entrapped nucleic acid, but also can be used as an auxiliary lipid component to promote nucleic acid delivery, so that the synergistic anti-tumor effect of nucleic acid and mitoxantrone is realized. The system can respond to high expression of phospholipase D (PLD) in tumor tissues, promote hydrolysis of PMA and release chemotherapeutic drug mitoxantrone, dual functions of nucleic acid delivery and drug release are achieved, the purpose of double-target inhibition of tumor growth is achieved, and a new strategy is provided for cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel nucleic acid delivery system based on phosphatidylmitoxantrone, and a preparation method and application thereof. Background Art

[0002] In recent years, with the continuous development of gene therapy technology, nucleic acids have shown great potential in the field of gene therapy, especially in anti-tumor therapy. However, the application of nucleic acids in vivo faces many challenges, including easy degradation, difficulty in effectively entering cells, and difficulty in achieving efficient release. Traditional nucleic acid delivery systems are difficult to meet the needs of efficient delivery and combined therapy at the same time, limiting the breadth and effectiveness of their clinical applications. In order to improve the delivery efficiency of nucleic acids, researchers have tried to use nanotechnology to construct a variety of delivery systems, including delivery systems based on liposomes, polymers, and inorganic nanoparticles. However, these systems still have some shortcomings in practical applications, such as low drug loading capacity, poor targeting, and large toxic side effects. Therefore, the development of a new and efficient nucleic acid delivery system that can achieve targeted delivery and synergistic therapy in tumor tissues has become a hot spot and difficulty in current research.

[0003] Research progress in nucleic acid delivery systems shows that lipid-based delivery systems are a hot topic in current research. These systems can improve the efficiency of nucleic acid encapsulation, protect nucleic acids from degradation, enhance cellular uptake and endosomal escape by optimizing the structure and composition of lipids, thereby improving the efficacy of gene therapy. Research on nucleic acid delivery systems is constantly progressing, and the development of new delivery vectors and strategies is expected to solve the problems existing in existing systems and provide safer and more effective solutions for gene therapy.

[0004] Tumors are composed of many different cells, which may maintain their growth and survival through different signaling pathways and mechanisms. Therefore, nucleic acid therapy with a single target may only affect a small number of cells in the tumor, while other unaffected cells may bypass the inhibited signaling pathways through other pathways, thereby developing drug resistance. Nucleic acid therapy alone may not be sufficient to overcome the multiple defense mechanisms of tumors. Combination therapy can enhance the therapeutic effect through drug interactions of different mechanisms. Summary of the invention

[0005] The purpose of the present invention is to provide a novel nucleic acid delivery system based on phosphatidylmitoxantrone, and a preparation method and application thereof. Phosphatidylmitoxantrone can be used as a cationic lipid or auxiliary lipid of the nucleic acid delivery system, thereby solving the deficiencies of the nucleic acid delivery system in the prior art in terms of drug stability, delivery efficiency, low anti-tumor effect, easy development of drug resistance, etc.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a novel nucleic acid delivery system based on phosphatidylmitoxantrone is provided, comprising a nanocarrier and a negatively charged component; the nanocarrier comprises: phosphatidylmitoxantrone, cholesterol, an auxiliary lipid or a cationic lipid, and an amphiphilic compound, wherein the negatively charged component is a cargo molecule; the cationic lipid comprises: a lipid that continues to have cationic properties without being affected by pH changes or an ionic lipid that is converted to cationic properties by pH changes; the auxiliary lipid is a neutral auxiliary lipid, comprising: a lipid that continues to have neutral properties without being affected by pH changes or an ionic lipid that is converted to neutral properties by pH changes; the cargo molecule is selected from: at least one of nucleic acids or nucleic acid analogs, peptides, proteins, lipids, chemical compounds, pharmaceutically active agents, their biosimilars, their biooptimizations, their biological derivatives, and their bioequivalents; the nucleic acid or nucleic acid analog is selected from The invention relates to a novel nanoparticle delivery system comprising: at least one of siRNA, mRNA, shRNA, lncRNA, pDNA, polyIC, CpG or cyclic dinucleotide; the molar ratio of phosphatidyl mitoxantrone to cholesterol is 5:1 to 1:1; when the phosphatidyl mitoxantrone is used as a cationic lipid component, the N / P ratio of the constructed nanocarrier to the cargo molecule is 1:1 to 70:1, wherein N / P is the molar ratio of nitrogen atoms in the cationic lipid in the nanocarrier to phosphoric acid in the cargo molecule; when the phosphatidyl mitoxantrone is used as an auxiliary lipid component, the N / P ratio of the constructed nanocarrier to the cargo molecule is 1:1 to 70:1, wherein N / P is the molar ratio of nitrogen atoms in the auxiliary lipid in the nanocarrier to phosphoric acid in the cargo molecule; the novel nucleic acid delivery system simultaneously releases nucleic acid drugs and the chemotherapy drug mitoxantrone under the action of phospholipase D highly expressed in tumor tissue, thereby achieving a synergistic anti-tumor effect.

[0008] In the present invention, the phosphatidyl mitoxantrone (PMA) refers to a prodrug formed by bonding a phospholipid compound and mitoxantrone through a phosphorus-oxygen bond under the catalytic action of phospholipase D (PLD). The specific preparation process of the phosphatidyl mitoxantrone can be found in patent application CN105963708A. The phosphatidyl mitoxantrone can be hydrolyzed and release anti-tumor drugs under the catalytic action of phospholipase D highly expressed in tumor tissues.

[0009] In some embodiments of the present invention, the molar ratio of phosphatidylmitoxantrone to cholesterol is 5:1 to 1:1.

[0010] In some embodiments of the present invention, when the phosphatidyl mitoxantrone is used as the cationic lipid component, the molar ratio of the phosphatidyl mitoxantrone to the auxiliary lipid is 5:1 to 1:1.

[0011] In some embodiments of the present invention, when the phosphatidyl mitoxantrone is used as an auxiliary lipid component, the molar ratio of the phosphatidyl mitoxantrone to the cationic lipid is 1:1 to 1:5.

[0012] In some embodiments of the present invention, the cationic lipid is selected from 1,2-diindoleethoxy-3-dimethylaminopropane, 1,2-dioleoyl-3-trimethylammonium salt propane, trimethyl-2,3-dioleyloxypropylammonium chloride, trimethyl-2,3-dioleyloxypropylammonium bromide, trimethyl-2,3-dioleyloxypropylammonium trifluoroacetate, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium bromide, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide, dimethyl-4-hydroxyethyl ... 2,3-dioleyloxypropylammonium bromide, dimethyl-5-hydroxypentyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dixadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide, N-(2-spermine formyl)-NN-dioctadecylglycinamide, 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3B-[N-(N,N-dimethylaminoethylcarbamoyl)]cholesterol, lipid poly-L-lysine and stearylamine, dimethyl dioctadecylammonium bromide, 1,2-dioleoyl-3-trimethylpropane ammonium , 3β-[N-(N',N'-dimethylaminoethane)carbamoylcholine, 1,2-dioleoyloxy-3-dimethylpropane ammonium, 1,2-di-O-octadecenyl-3-trimethylpropane ammonium, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine Acid choline, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyl]-benzamide, 1,2-dimyristoyl-3-dimethylammonium propane, 1,2-dipalmitoyl-3-dimethylammonium propane, 1,2-distearoyl-3-dimethylammonium propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium, 1,2-stearoyl-3-trimethylammonium propane, 1,2-dipalmitoyl-3-trimethylammonium propane, 1,2-dimyristoyl-3-trimethylammonium propane, N4-cholesterol spermine, but is not limited thereto.

[0013] In some preferred embodiments of the present invention, the cationic lipid is 1,2-diindoleethoxy-3-dimethylaminopropane.

[0014] In an embodiment of the present invention, the helper lipid is a neutral helper lipid.

[0015] In some embodiments of the present invention, the neutral helper lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA) and / or phosphatidylcholine (PC), but is not limited thereto.

[0016] In some preferred embodiments of the present invention, the neutral helper lipid is dilinoleoylphosphatidylethanolamine.

[0017] In some preferred embodiments of the present invention, the neutral helper lipid is distearoylphosphatidylcholine.

[0018] In some embodiments of the present invention, the amphiphilic compound is selected from DSPE-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, myristoyl phosphatidylethanolamine PEG, distearoyl phosphatidylethanolamine-polyethylene glycol and dilauroyl phosphatidyl Z alcoholamine-polyethylene glycol, but is not limited thereto.

[0019] In some preferred embodiments of the present invention, the amphiphilic compound is DSPE-PEG.

[0020] In some embodiments of the present invention, the cargo molecule is selected from at least one of nucleic acids, peptides, proteins, lipids, chemical compounds, pharmaceutically active agents, biosimilars thereof, biooptimized products thereof, biological derivatives thereof, and bioequivalents thereof.

[0021] In some preferred embodiments of the present invention, the cargo molecule is a nucleic acid or a nucleic acid analog.

[0022] In some preferred embodiments of the present invention, the nucleic acid or nucleic acid analog is selected from siRNA, mRNA, shRNA, lncRNA, pDNA, polyIC, CpG, and cyclic dinucleotide, but is not limited thereto.

[0023] In some embodiments of the present invention, the basic principle of nucleic acid delivery is as follows: the present invention mainly concentrates the negatively charged components to be delivered on nucleic acid molecules, such as pDNA, siRNA, etc. According to the above, the phosphatidyl mitoxantrone in the nucleic acid delivery vector can form a complex with the negatively charged nucleic acid molecules, thereby improving its stability and loading capacity and reducing the risk of nucleic acid molecules being degraded. After the complex is engulfed by the phagocytic vesicle, it is finally transported to the lysosome. Under the acidic conditions of the lysosome, the phosphatidyl mitoxantrone containing multiple secondary amine structures will trigger the "proton sponge effect", which improves the in vivo release and transfection efficiency of nucleic acid molecules. When the nucleic acid molecule is siRNA, the released siRNA is directly delivered to the cytoplasm, forms a RISC complex with the mRNA of the target gene, and mediates the degradation of the target gene mRNA, thereby playing a biological role in knocking down the expression of specific genes. At the same time, the PLD enzyme highly expressed in the tumor site hydrolyzes the phosphatidyl mitoxantrone to release the anti-tumor drug mitoxantrone, which exerts its anti-tumor effect by inducing DNA damage, interfering with RNA and inhibiting topoisomerase II, so that the two effective substances of mitoxantrone and siRNA achieve a synergistic anti-tumor effect and enhance the efficacy. The delivery principle of other nucleic acid molecules is the same and will not be repeated.

[0024] In some embodiments of the present invention, the particle size of the nano-delivery system is 10 to 350 nm.

[0025] In some embodiments of the present invention, the N / P ratio of the liposome constructed by using the nanocarrier phosphatidylmitoxantrone as the cationic lipid component to the cargo molecule is 1:1 to 70:1, and the content percentage of the amphiphilic compound in the cationic liposome is 0.5% to 15%.

[0026] In some embodiments of the present invention, the N / P ratio of the liposome constructed by using the nanocarrier phosphatidylmitoxantrone as an auxiliary lipid component to the cargo molecule is 1:1 to 70:1, and the content percentage of the amphiphilic compound in the cationic liposome is 0.5% to 15%.

[0027] Furthermore, in some embodiments of the present invention, the lipid composition includes not only the necessary additional components of the basic structure, but also functionally modified materials such as TAT, RGD, ANG, folic acid, transferrin and targeted monoclonal antibodies, as well as traditional liposome materials such as phosphatidylethanolamine, deoxycholic acid, cholesterol sulfate, etc. to adjust the performance.

[0028] According to a second aspect of the present invention, there is provided a method for preparing a novel nucleic acid delivery system based on phosphatidylmitoxantrone, comprising the following steps:

[0029] S1, preparing a lipid composition: when phosphatidyl mitoxantrone is used as an auxiliary lipid component: weighing a cationic lipid, phosphatidyl mitoxantrone, an amphiphilic complex and cholesterol, and mixing them to obtain a lipid composition; when phosphatidyl mitoxantrone is used as a cationic lipid component: weighing phosphatidyl mitoxantrone, a neutral auxiliary lipid, an amphiphilic complex and cholesterol, respectively, and mixing them to obtain a lipid composition;

[0030] S2, preparing a mixed lipid membrane: dissolving the lipid composition in an organic solvent, and removing the organic solvent by reducing pressure or blowing with nitrogen to obtain a mixed lipid membrane;

[0031] S3, re-dissolution and ultrasonic treatment: re-dissolve the mixed lipid film with a buffer solution under low-power ultrasonic conditions, and then perform high-power ultrasonic treatment to obtain a crude nanoformulation solution;

[0032] S4, preparing a nanoformulation by an extruder: preparing the crude nanoformulation solution into a nanoformulation with uniform particle size by an extruder, namely, a phosphatidylmitoxantrone-based nucleic acid delivery liposome;

[0033] S5, mixing the nucleic acid delivery liposome with the cargo molecule, and standing at room temperature for not less than 20 minutes to obtain a phosphatidylmitoxantrone-based nucleic acid delivery system.

[0034] In step S2, the organic solvent is an organic solvent that can dissolve lipids, and can be selected from: tetrahydrofuran, chloroform, dichloromethane, methanol, ethanol, but it should be understood that this is only for example and not for limitation.

[0035] In the process of preparing the nanoparticles in step S4, the complex is mainly formed by charge interaction, and the mixture is left standing at room temperature for not less than 20 minutes mainly to allow the charge interaction to form the complex more fully so as not to cause precipitation.

[0036] According to the third aspect of the present invention, there is provided a novel nucleic acid delivery system for use in the preparation of anti-tumor drugs, wherein the novel nucleic acid delivery system simultaneously releases nucleic acid drugs and the chemotherapy drug mitoxantrone under the action of phospholipase D highly expressed in tumor tissues, thereby achieving a synergistic anti-tumor effect.

[0037] In some embodiments of the present invention, depending on the different targets of nucleic acids and nucleic acid analogs, they have inhibitory effects on a variety of tumor cells, including pancreatic cancer cells, breast cancer cells, colon cancer cells, cervical cancer cells, brain glioma cells, lymphoma cells, ovarian cancer cells, gastric cancer cells, prostate cancer cells, and skin cancer cells, but are not limited to these.

[0038] In this application, phosphatidylmitoxantrone can replace other cationic lipids to become a new type of cationic lipid to achieve effective encapsulation of nucleic acids.

[0039] In this application, phosphatidylmitoxantrone can replace auxiliary lipids and cooperate with other cationic lipids to assist in the construction of nucleic acid delivery vectors.

[0040] In this application, the above-mentioned phosphatidyl anti-tumor prodrug-based nucleic acid delivery system is prepared into an anti-tumor drug, the system self-assembles into nano-liposomes in an aqueous phase, and the liposomes encapsulate nucleic acids to form nano-complexes. When the nano-complexes are targeted and enter tumor tissues, under the catalysis of phospholipase D highly expressed in tumor tissues, synergistic nucleic acid drugs and chemotherapy drugs mitoxantrone are released simultaneously, thereby avoiding the treatment complexity caused by different pharmacokinetics of biodistribution in combined administration and solving the problem of multi-drug delivery and safe and effective release in anti-tumor treatment.

[0041] According to a fourth aspect of the present invention, a drug is provided, comprising the nucleic acid delivery system as described above.

[0042] In some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0043] As a new type of drug delivery phospholipid, phosphatidyl mitoxantrone (PMA) can be used as a new type of cationic lipid for nucleic acid delivery system because of its good enzymatic response release and ionization properties. It can adsorb negatively charged nucleic acids and achieve specific drug release under the action of phospholipase D (PLD) highly expressed in tumor tissues, significantly improving drug accumulation and therapeutic effects in tumor sites. In addition, phosphatidyl mitoxantrone can also be combined with other lipid materials to construct a stable nano-delivery system, further improving the delivery efficiency and anti-tumor activity of nucleic acids.

[0044] The key invention point of the present invention is that phosphatidyl mitoxantrone is selected as a nucleic acid delivery carrier for the first time to prepare a nucleic acid delivery system. First, the phosphatidyl mitoxantrone molecule contains multiple secondary amine groups, which can be protonated in an acidic environment, so that the compound presents positive charge, replaces the existing cationic lipids to become a new type of cationic lipids, effectively encapsulates nucleic acids, and enhances the delivery efficiency of nucleic acids. Secondly, phosphatidyl mitoxantrone is obtained by phosphatidylcholine modification design, it contains phospholipid structure, shows good amphipathicity and biocompatibility, and can be used as auxiliary phospholipids to enhance the construction and stability of nanocarriers. Phosphatidyl mitoxantrone is formed by phosphatidylcholine and mitoxantrone coupled by phosphodiester bond, can be hydrolyzed under the action of PLD enzymes highly expressed in tumor tissue specificity, and promotes the release of cargo molecules and mitoxantrone. And the "proton sponge effect" of phosphatidyl mitoxantrone helps to solve the problem of cargo molecules escaping lysosomal hydrolysis, and then interferes with gene expression, and realizes dual mechanism to inhibit tumor growth in this process. The interaction between phosphatidylmitoxantrone and the PLD enzyme on the cell membrane is expected to improve the uptake of nucleic acid drugs by tumor cells. This study solves the problem of low efficacy of single-target treatment of nucleic acid drugs in anti-tumor treatment and safe and effective drug release, which not only helps to reduce costs and improve treatment safety, but also provides new strategies and directions for the field of nucleic acid drug delivery.

[0045] Since phosphatidyl mitoxantrone will not be hydrolyzed and released in normal tissues, the nonspecific distribution of chemotherapy drugs and nucleic acid drugs in the body is reduced, ensuring that the active ingredients of the drugs act on the target cells at the same location at the same time, avoiding the complexity of combined treatment due to different pharmacokinetic biodistribution;

[0046] Since phosphatidylmitoxantrone in the carrier component is also a drug with anticancer activity, the total drug loading in the nucleic acid delivery carrier provided by the present invention can exceed 50% (w / w%), reducing excipient-related side effects and improving drug safety.

[0047] The present invention realizes the replacement of existing cationic lipids by phosphatidylmitoxantrone. As a new type of ionizable cationic lipid, phosphatidylmitoxantrone realizes the loading and delivery of nucleic acid drugs while synergistically enhancing the synergy between phosphatidylmitoxantrone and nucleic acid drugs, thereby reducing costs and enhancing the delivery efficiency of nucleic acid drugs, breaking through the domestic patent barriers in the field of ionizable cationic lipids, and contributing new solutions to the development of the domestic nucleic acid drug delivery field.

[0048] The present invention simultaneously realizes the phospholipase D-triggered response release of the chemotherapy drug mitoxantrone in tumor tissue and the effective delivery of nucleic acid drugs, achieving a synergistic dual-target treatment effect, avoiding the limitation of low efficacy of nucleic acid drugs alone, opening up new ideas for the development of tumor therapeutic drugs, and having good application prospects in clinical tumor treatment.

[0049] In summary, a novel nucleic acid delivery system based on phosphatidylmitoxantrone and a preparation method thereof and application thereof in the preparation of anti-tumor drugs provided by the present invention have the advantages of high safety, low side effects and high efficacy, can achieve the specific release of chemotherapy drugs and nucleic acid drugs at the tumor site, exert a synergistic effect, and solve the problems of nucleic acid delivery, effective release and low efficacy in anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Transmission electron microscopy images of nucleic acid delivery liposome formulations with phosphatidylmitoxantrone as the cationic lipid are shown;

[0051] Figure 2 shows the particle size diagram of nucleic acid delivery liposome formulations with phosphatidylmitoxantrone as the cationic lipid;

[0052] Figure 3 The particle size diagram of liposome preparations containing siRNA phosphatidylmitoxantrone as cationic lipid is shown;

[0053] Figure 4 The gel electrophoresis diagram showing the detection of liposome siRNA complexing ability of phosphatidylmitoxantrone as a cationic lipid;

[0054] Figure 5 The graph shows the results of CCK-8 experiments of liposome formulations containing siRNA and phosphatidylmitoxantrone as cationic lipids. DETAILED DESCRIPTION

[0055] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0056] Example 1 Preparation of Nucleic Acid Delivery Liposomes Using Phosphatidylmitoxantrone as an Auxiliary Lipid

[0057] Weigh 31.8 mg of Dlin-MC3-DMA, 2.44 mg of DSPE-mPEG2000, 25.34 mg of neutral auxiliary lipid phosphatidyl mitoxantrone and 9.36 mg of cholesterol, add 12 mL of chloroform, and perform ultrasound at room temperature until completely dissolved as the oil phase. Prepare 24 mL of 10 mM HEPES buffer solution (pH 6.5) as the aqueous phase. After removing tetrahydrofuran from the oil phase by reduced pressure evaporation, a dry lipid film is obtained. Subsequently, the film is immersed in the aqueous phase and continuously ultrasound is performed at 100 W for 5 minutes to redissolve the lipid film. When the solution becomes a white turbid solution, increase the ultrasound power to 250 W, perform 90 cycles (10 seconds per cycle, including 5 seconds of ultrasound and 5 seconds of rest) to obtain a crude nanoformulation solution. Finally, the crude nanoformulation is processed into a nanoformulation with uniform particle size by an extruder, and phosphatidyl mitoxantrone is used as an auxiliary lipid to construct siRNA delivery liposomes, which are stored in a refrigerator at 4 ° C away from light.

[0058] Example 2 Preparation of Nucleic Acid Delivery Liposomes Constructed by Using Phosphatidylmitoxantrone as Cationic Lipid

[0059] Weigh 31.2 mg of phosphatidyl mitoxantrone, 1.56 mg of DSPE-mPEG2000, 3.56 mg of neutral auxiliary lipid DOPE and 7.5 mg of cholesterol, add 12 mL of tetrahydrofuran, and perform ultrasound at room temperature until completely dissolved as the oil phase. Prepare 24 mL of 10 mM HEPES buffer solution (pH 6.5) as the aqueous phase. After removing the tetrahydrofuran in the oil phase by reduced pressure evaporation, a dry lipid film is obtained. Subsequently, the film is immersed in the aqueous phase and subjected to continuous ultrasound at 100 W for 5 minutes to redissolve the lipid film. When the solution becomes a blue turbid solution, increase the ultrasound power to 250 W, perform 90 cycles (10 seconds per cycle, including 5 seconds of ultrasound and 5 seconds of rest) to obtain a crude nanoformulation solution. Finally, the crude nanoformulation is processed into a nanoformulation with uniform particle size by an extruder, and phosphatidyl mitoxantrone is used as a cationic lipid to construct siRNA delivery liposomes, which are stored in a refrigerator at 4 ° C away from light.

[0060] Example 3 Preparation of TAT-modified phosphatidylmitoxantrone as auxiliary lipid to construct nucleic acid delivery liposomes

[0061] Weigh Dlin-MC3-DMA 31.8mg, DSPE-mPEG2000-TAT 2.44mg, neutral auxiliary lipid phosphatidyl mitoxantrone 25.34mg and cholesterol 9.36mg, add 12mL chloroform, and ultrasonicate to completely dissolve at room temperature as oil phase. Prepare 24mL 10mM HEPES buffer solution (pH6.5) as aqueous phase. After removing tetrahydrofuran in the oil phase by reduced pressure evaporation, a dry lipid film is obtained. Subsequently, the film is immersed in the aqueous phase and 100W continuous ultrasound is performed for 5 minutes to redissolve the lipid film. When the solution becomes a white turbid solution, the ultrasonic power is increased to 250W, and 90 cycles (10 seconds per cycle, wherein ultrasound is performed for 5 seconds, and intervals are performed for 5 seconds) are performed to obtain a coarse nano preparation solution. Finally, the crude nanoformulation was processed into a nanoformulation with uniform particle size by an extruder, and TAT-modified phosphatidylmitoxantrone was used as an auxiliary lipid to construct siRNA delivery liposomes, which were stored in a refrigerator at 4°C away from light.

[0062] Example 4 Preparation of TAT-modified phosphatidylmitoxantrone as cationic lipid to construct nucleic acid delivery liposomes

[0063] Weigh 31.2 mg of phosphatidyl mitoxantrone, 1.56 mg of DSPE-mPEG2000-TAT, 3.56 mg of neutral auxiliary lipid DOPE and 7.5 mg of cholesterol, add 12 mL of tetrahydrofuran, and perform ultrasound at room temperature until completely dissolved as the oil phase. Prepare 24 mL of 10 mM HEPES buffer solution (pH 6.5) as the aqueous phase. After removing tetrahydrofuran from the oil phase by reduced pressure evaporation, a dry lipid film is obtained. Subsequently, the film is immersed in the aqueous phase and subjected to continuous ultrasound at 100 W for 5 minutes to redissolve the lipid film. When the solution becomes a blue turbid solution, increase the ultrasound power to 250 W, perform 90 cycles (10 seconds per cycle, including 5 seconds of ultrasound and 5 seconds of rest) to obtain a crude nanoformulation solution. Finally, the crude nanoformulation is processed into a nanoformulation with uniform particle size by an extruder, and TAT-modified phosphatidyl mitoxantrone is used as a cationic lipid to construct siRNA delivery liposomes, which are stored in a refrigerator at 4 ° C away from light.

[0064] Example 5 Morphological characterization of nucleic acid delivery liposomes constructed using phosphatidylmitoxantrone as cationic lipid

[0065] The morphology of phosphatidylmitoxantrone siRNA delivery liposomes (from Example 1) was studied using a transmission electron microscope (JEM-1400). 20 μL of the sample was dripped onto a copper grid, stained, and observed. Figure 1 As shown, the phosphatidylmitoxantrone liposomes were smooth and round with good overall uniformity.

[0066] Example 6 Study on the average particle size, PDI and zeta potential of siRNA delivery liposomes constructed using phosphatidylmitoxantrone as a cationic lipid

[0067] The dynamic scattering method was used to measure the particle size using a Malvern laser particle size analyzer.

[0068] 1) Determination of average particle size and PDI

[0069] The target sample (from Example 2) was diluted with deionized water to a concentration of 200 μg / mL. Before measuring, the sample pool was thoroughly cleaned 5 times with deionized water to ensure that there was no interference from impurities. Take 20 μL of the prepared liposome preparation, dilute it to 1 mL with deionized water, and add it to the cleaned sample pool. Place the sample pool in a Malvern particle size potential analyzer, and the measurement conditions are: 173° scattering angle, 25°C. Each sample was measured three times, and each measurement contained 10 cycles to ensure the accuracy and repeatability of the data. The results are as follows. Figure 2 As shown, the relative particle size of the nanoparticles after loading siRNA ( Figure 3 ), the particle size increased from 113.3 nm to 148.8 nm, further indicating that siRNA was encapsulated, resulting in an increase in particle size. At the same time, the particle size also met the standards for drug delivery liposomes.

[0070] 2) Determination of zeta potential

[0071] 1 mL of the liposome preparation diluted 50 times with deionized water was added to the measurement container to avoid bubbles in the container. Each sample was measured three times.

[0072] Example 7 Preparation of siRNA-carrying liposome formulation containing phosphatidylmitoxantrone as cationic lipid

[0073] 1OD siRNA (dry powder) was centrifuged at 4000rpm for 1min, 125μL DEPC water was added to 1OD siRNA to prepare a siRNA solution with a concentration of 20μM, and empty phosphatidylmitoxantrone delivery liposome nanoformulation was added at N / P ratios of 5, 10, 20, 30, and 50, mixed evenly, and incubated at room temperature for 20min to obtain a liposome preparation containing siRNA and phosphatidylmitoxantrone as a cationic lipid.

[0074] Example 8 - Study on the siRNA complexing ability of liposomes with phosphatidylmitoxantrone as a cationic lipid

[0075] Having a strong nucleic acid complexing ability is a key factor for the success of nucleic acid-carrying cationic liposome preparations. The nucleic acid complexing ability of the cationic liposomes of the present invention was examined by agarose gel electrophoresis using the N / P ratio as an indicator. Weigh 1.2 g of agarose and dissolve it in 100 mL of 1×TAE solution. Heat it in a microwave oven to completely dissolve the agarose particles. Add 10 μL of nucleic acid dye (gelred) to the agarose gel while it is hot, add the gel to the gel tank, insert a comb, and pull out the comb after it is naturally dried. A mixture of the nucleic acid-carrying cationic liposome preparation (40 μL) prepared by the cationic liposomes and siRNA of Example 2 with different N / P ratios as described in Example 7 and 2 μL of 6× loading buffer was added to the agarose gel wells, and the electrophoresis voltage was set to 120 V for the electrophoresis experiment. Electrophoresis was performed at room temperature for 15 minutes. Pure siRNA solution and completely demulsified solution of nucleic acid-carrying cationic liposome preparation with the same concentration were used as controls. The results are as follows Figure 4 As shown in the figure, with the increase of N / P, the brightness of the band is significantly weakened. When N / P is 20:1, the band is extremely weak, indicating that only a small part of free siRNA exists in the system. No obvious band is observed when N / P is 30:1 and 50:1, indicating that most siRNA is encapsulated. This shows that the nucleic acid-loaded cationic liposome preparation of the present invention can complex most siRNA when N / P is 20-50, and has a strong siRNA complexing ability.

[0076] Example 9 - Study on the cytotoxicity of liposomes containing siRNA and phosphatidylmitoxantrone as cationic lipid on tumor cells

[0077] The CCK-8 method was used to detect the killing effect of liposome preparations containing siRNA phosphatidylmitoxantrone as cationic lipids with an N / P ratio of 20:1 (from Example 7) on Hela cells and MDA-MB-231 cells with high expression of PLD enzyme. The siRNA drug concentration range was set to 32.5nM to 130nM. After the cells were cultured for 48 hours after administration, CCK-8 reagent was added, the absorbance was measured, and the cell inhibition effect was calculated. The results are shown in Figure 5 As shown, with the increase of administration concentration, the inhibition rate of samples on both tumor cells gradually increased, among which the highest concentration sample had a cell inhibition rate on MDA-MB-231 cells that was about 40% higher than that on Hela cells, indicating that there was a significant correlation between the expression level of PLD (phospholipase D) and the toxic sensitivity of cells to the samples, thereby indirectly confirming that PMA, as a cationic lipid, can effectively enter cells and can synergize with siRNA to jointly inhibit tumor cell growth.

[0078] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiment of the present invention can also be modified in various ways. That is, all simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A novel nucleic acid delivery system based on phosphatidylmitoxantrone, characterized in that: It comprises a nanocarrier and a negatively charged component; the nanocarrier comprises: phosphatidyl mitoxantrone, cholesterol, auxiliary lipid or cationic lipid, and amphiphilic compound, and the negatively charged component is a cargo molecule; The cationic lipids include: lipids that continue to be cationic regardless of pH changes or ionic lipids that are converted to cationic properties by pH changes; The auxiliary lipid is a neutral auxiliary lipid, including: a lipid that is not affected by pH changes and continues to be neutral or an ionic lipid that is converted to neutral by pH changes; The cargo molecule is at least one selected from: nucleic acid or nucleic acid analogs, peptides, proteins, lipids, chemical compounds, pharmaceutically active agents, biosimilars thereof, biooptimized products thereof, biological derivatives thereof and biological equivalents thereof; The nucleic acid or nucleic acid analog is at least one selected from: siRNA, mRNA, shRNA, lncRNA, pDNA, polyIC, CpG or cyclic dinucleotide; The molar ratio of phosphatidylmitoxantrone to cholesterol is 5:1 to 1:1; When the phosphatidylmitoxantrone is used as the cationic lipid component, the N / P ratio of the constructed nanocarrier to the cargo molecule is 1:1 to 70:1, wherein N / P is the molar ratio of nitrogen atoms in the cationic lipid in the nanocarrier to phosphoric acid in the cargo molecule; When the phosphatidyl mitoxantrone is used as the auxiliary lipid component, the N / P ratio of the constructed nanocarrier to the cargo molecule is 1:1 to 70:1, wherein N / P is the molar ratio of the nitrogen atoms in the auxiliary lipid in the nanocarrier to the phosphoric acid in the cargo molecule; The novel nucleic acid delivery system simultaneously releases nucleic acid drugs and the chemotherapy drug mitoxantrone under the action of phospholipase D highly expressed in tumor tissues, achieving a synergistic anti-tumor effect.

2. The novel nucleic acid delivery system according to claim 1, characterized in that: The phosphatidylmitoxantrone is formed by coupling a phospholipid compound with mitoxantrone through a phosphodiester bond under the catalysis of phospholipase D, and can be hydrolyzed to release antitumor drugs under the catalysis of phospholipase D that is highly expressed in tumor tissues.

3. The novel nucleic acid delivery system according to claim 1, characterized in that: The cationic lipids include: 1,2-diindoleethoxy-3-dimethylaminopropane, 1,2-dioleoyl-3-trimethylammonium salt propane, trimethyl-2,3-dioleyloxypropylammonium chloride, trimethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, dimethyldioctadecylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioleyloxypropylammonium bromide, dimethyl-3-hydroxypropyl-2,3-dioleyloxypropylammonium bromide, dimethyl-4-hydroxy-2.3-dioleyloxypropylammonium bromide, Oleyloxypropylammonium bromide, dimethyl-5-hydroxypentyl-2.3-dioleyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dihexadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-dioctadecyloxypropylammonium bromide, dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide, N-(2-spermine formyl)-NN-dioctadecylglycinamide, 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester, 3B-[N-(N,N-dimethylaminoethylcarbamoyl)]cholesterol, lipid poly-L-lysine and stearylamine, dimethyldioctadecylammonium bromide, 1,2-dioleoyl-3-trimethylpropane ammonium, 3β -[N-(N',N'-dimethylaminoethane)carbamoylcholesterol, 1,2-dioleoyloxy-3-dimethylpropane ammonium, 1,2-di-O-octadecenyl-3-trimethylpropane ammonium, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethyl 1,2-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium, 1,2-dimethyl-3-propane-1-yl, 1,2-dipalmitoyl-3-propane-1-yl, 1,2-distearoyl-3-dimethyl-3-propane-1-yl, 1,2-dipalmitoyl-3-propane-1-yl, 1,2-dimethyl-2,3-bis(oleoyloxy)propane-1-ammonium, 1,2-dimethyl-3-propane-1-yl, 1,2-distearoyl-3-trimethyl-3-propane-1-yl, 1,2-dipalmitoyl-3-trimethyl-3-propane-1-yl, 1,2-dimyristoyl-3-trimethyl-3-propane-1-yl, and N4-cholesterol spermine.

4. The novel nucleic acid delivery system according to claim 1, characterized in that: The neutral auxiliary lipids include: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA) or phosphatidylcholine (PC).

5. The novel nucleic acid delivery system according to claim 1, characterized in that: The amphiphilic compound includes: DSPE-PEG, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, myristoyl phosphatidylethanolamine PEG, distearoyl phosphatidylethanolamine-polyethylene glycol or dilauroyl phosphatidyl Z alcoholamine-polyethylene glycol.

6. The novel nucleic acid delivery system according to claim 1, characterized in that: The particle size of the nucleic acid delivery system is 10 to 350 nm.

7. A method for preparing a novel nucleic acid delivery system according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, preparing a lipid composition: when phosphatidyl mitoxantrone is used as an auxiliary lipid component: weighing a cationic lipid, phosphatidyl mitoxantrone, an amphiphilic complex and cholesterol, and mixing them to obtain a lipid composition; when phosphatidyl mitoxantrone is used as a cationic lipid component: weighing phosphatidyl mitoxantrone, a neutral auxiliary lipid, an amphiphilic complex and cholesterol, respectively, and mixing them to obtain a lipid composition; S2, preparing a mixed lipid membrane: dissolving the lipid composition in an organic solvent, and removing the organic solvent by reducing pressure or blowing with nitrogen to obtain a mixed lipid membrane; S3, re-dissolution and ultrasonic treatment: re-dissolve the mixed lipid film with a buffer solution under low-power ultrasonic conditions, and then perform high-power ultrasonic treatment to obtain a crude nanoformulation solution; S4, preparing a nanoformulation by an extruder: preparing the crude nanoformulation solution into a nanoformulation with uniform particle size by an extruder, namely, a phosphatidylmitoxantrone-based nucleic acid delivery liposome; S5, mixing the nucleic acid delivery liposomes with the cargo molecules and standing at room temperature for not less than 20 minutes to obtain a novel nucleic acid delivery system based on phosphatidylmitoxantrone.

8. Use of the novel nucleic acid delivery system according to any one of claims 1 to 6 in the preparation of anti-tumor drugs, characterized in that: The novel nucleic acid delivery system simultaneously releases nucleic acid drugs and the chemotherapy drug mitoxantrone under the action of phospholipase D highly expressed in tumor tissues, achieving a synergistic anti-tumor effect.

9. The use according to claim 8, characterized in that: According to the different targets of nucleic acids and nucleic acid analogs, the anti-tumor drugs target the following types of cancer: pancreatic cancer, breast cancer, colon cancer, cervical cancer, glioma, lymphoma, ovarian cancer, gastric cancer, prostate cancer, and skin cancer.

10. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the novel nucleic acid delivery system according to any one of claims 1 to 6.

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