Lipid platinum prodrug assisted RNA (Ribonucleic Acid) lipid nanoparticles as well as preparation and application thereof

Abstract

The invention discloses a lipid platinum prodrug assisted RNA (Ribonucleic Acid) lipid nanoparticle as well as preparation and application thereof, and is characterized in that the lipid platinum prodrug assisted RNA lipid nanoparticle comprises SM-102, DSPC (Distearoyl Phthalate), cholesterol, DMG-PEG (Dimethyl Glycol-Polyethylene Glycol), a lipid platinum prodrug and RNA; the lipid platinum prodrug comprises a platinum drug and fatty acid, the RNA comprises siRNA and mRNA, the siRNA is used for targeted regulation of tumor immunosuppression related genes (such as PS exposure related genes), and the mRNA is used for coding antigen protein to activate immune response. According to the invention, not only is the protection on RNA improved, but also the adsorption of the LNPs on the RNA is increased, and a foundation is laid for an effective cis-platinum-RNA collaborative delivery system.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more specifically to the preparation and application of a platinum prodrug lipid nanoparticle and an RNA delivery system. Background Art

[0002] Cisplatin, as a first-line clinical drug for chemotherapy, faces serious challenges, including toxicity, drug resistance, and the promotion of immunosuppressive effects in tumor cells. These limitations have prompted people to seek more refined treatment strategies. In recent years, the research focus has been on developing prodrugs and nano-drug delivery systems to precisely control drug release, thereby reducing toxicity and improving treatment effects. In addition, many current studies have also explored the combination of platinum-based chemotherapy with other treatment methods, such as co-administering with hydroxycamptothecin or combining with photothermal therapy, to overcome tumor drug resistance and improve efficacy. A key issue in the process of platinum-induced tumor cell apoptosis is the massive exposure of phosphatidylserine (PS), and this "danger signal" will inhibit the immune response and reduce the overall efficacy of chemotherapy. Currently, there is a lack of an efficient co-delivery system for platinum drugs and nucleic acids, and it is impossible to achieve the synergistic delivery of platinum drugs and functional RNAs. The existence of these problems has severely limited the clinical application effect of cisplatin chemotherapy. Therefore, there is an urgent need to develop a new drug delivery system that can precisely control the release of cisplatin, reduce toxicity, overcome drug resistance, and achieve synergistic delivery with functional RNAs, thereby improving the overall anti-tumor effect. Summary of the Invention

[0003] The purpose of the present invention is to provide a lipid platinum prodrug-assisted RNA lipid nanoparticle to solve the deficiencies of the prior art. It not only improves the protection of RNA but also increases the adsorption of LNPs to RNA, laying a foundation for an effective cisplatin-RNA synergistic delivery system.

[0004] Another purpose of the present invention is a preparation method of the lipid platinum prodrug-assisted RNA lipid nanoparticle.

[0005] Another purpose of the present invention is the application of the lipid platinum prodrug-assisted RNA lipid nanoparticle.

[0006] The present invention adopts the following technical solution to achieve the above purpose: A lipid platinum prodrug-assisted RNA lipid nanoparticle, characterized in that it comprises SM-102, DSPC, cholesterol, DMG-PEG, a lipid platinum prodrug, and RNA; the lipid platinum prodrug comprises a platinum drug and a fatty acid, and the RNA comprises siRNA and mRNA, wherein the siRNA targets and regulates tumor immunosuppression-related genes (such as PS exposure-related genes), and the mRNA encodes an antigen protein to activate the immune response.

[0007] As a further illustration of the above solution, the fatty acid is a straight-chain or branched-chain fatty acid with a carbon chain length of C6-C24.

[0008] Further, the platinum-based drug is cisplatin oxide or oxaliplatin oxide, and the fatty acid is one or more of lauric acid, palmitic acid, stearic acid, 2-hexylundecanoic acid, or 2-hexadecylstearic acid.

[0009] Further, the mass ratio of SM-102, DSPC, cholesterol, and DMG-PEG is 50:10:38.5:1.5, and the mass proportion of the lipid-platinum prodrug is 5%-30%.

[0010] Preferably, the mass proportion of the lipid-platinum prodrug is 10%-20% to optimize the encapsulation efficiency and stability.

[0011] Further, the particle size of the RNA lipid nanoparticles is 50-150 nm, the surface potential is +10 mV to +30 mV, and the encapsulation efficiency ≥85%; the RNA lipid nanoparticles are modified with targeting ligands (such as folic acid, transferrin) to enhance tumor tissue targeting.

[0012] A method for preparing lipid-platinum prodrug-assisted RNA lipid nanoparticles, which is characterized in that it comprises the following specific steps:

[0013] S1. Dissolve SM-102, DSPC, cholesterol, and DMG-PEG in ethanol in a certain proportion to prepare standard LNPs; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a certain volume ratio, and the flow rate is 8-10 mL / min; the mass ratio of lipid to mRNA in the final LNPs is (9-11):1.

[0014] S2. For lipid-platinum LNPs, dissolve SM-102, DSPC, cholesterol, and DMG-PEG in DMF in the same proportion as in S1, and add the lipid-platinum prodrug to the lipid-platinum LNPs at a mass ratio of 5%-30%; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a certain volume ratio, and the flow rate is 8-10 mL / min; in the obtained lipid-platinum LNPs, the mass ratio of lipid (excluding lipid Pt) to mRNA is maintained at (9-11):1.

[0015] Preferably, in S1, dissolve SM-102, DSPC, cholesterol, and DMG-PEG in ethanol in a ratio of 50:10:38.5:1.5 to prepare standard LNPs; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a volume ratio of 1:3, and the flow rate is 9 mL / min; the mass ratio of lipid to mRNA in the final LNPs is 10:1.

[0016] Preferably, in S2, for lipid platinum LNPs, SM-102, DSPC, cholesterol, and DMG-PEG are dissolved in DMF in the same ratio of 50:10:38.5:1.5, and the lipid cisplatin prodrug is added to the LNPs at a mass ratio of 5%-30%. The mRNA is dissolved in 10 mM citric acid buffer solution, and the two solutions are mixed at a volume ratio of 1:3 with a flow rate of 9 mL / min. In the resulting LNPs, the mass ratio of lipid (excluding lipid Pt) to mRNA is maintained at 10:1.

[0017] Furthermore, the synthesis steps of the lipid cisplatin prodrug include:

[0018] S1. Dissolve 4-6 g of cisplatin in 45-55 mL of 30% hydrogen peroxide solution, heat to 45-55 °C, stir for 23-25 hours, then lyophilize the reaction mixture, wash the product with cold ether, and dry it under vacuum to obtain yellowish powder of tetravalent cisplatin.

[0019] S2. Modify the tetravalent cisplatin with five different fatty acids, lauric acid, palmitic acid, stearic acid, 2-ethylundecanoic acid, and 2-hexadecylstearic acid. Each fatty acid reacts with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) at a molar ratio of fatty acid:EDC of 1:1.1-1.3 for 2-4 hours. Under a nitrogen atmosphere, the product further reacts with the tetravalent cisplatin in DMF at a molar ratio of 2-4:1 for 23-25 hours. The product is washed with ice water and crystallized, and the crude product is then washed with ether and dried under vacuum for 23-25 hours.

[0020] An application of a lipid platinum prodrug-assisted RNA lipid nanoparticle, characterized in that it is applied in drug delivery, preparation of anti-tumor drugs or tumor vaccines, and is particularly suitable for the treatment of solid tumors (such as lung cancer, liver cancer) or hematological tumors (such as leukemia).

[0021] The beneficial effects that can be achieved by the present invention using the above technical solutions are:

[0022] The present invention uses lipid platinum prodrug-assisted RNA lipid nanoparticles mainly composed of SM-102, DSPC, cholesterol, DMG-PEG, lipid platinum prodrug and RNA, which convert cisplatin into a lipid prodrug, can reduce the toxicity of free cisplatin, and at the same time use LNPs for targeted delivery to reduce damage to normal tissues; lipid nanoparticles can be taken up by tumor cells through endocytosis, bypassing the membrane transporters related to drug resistance, which helps to overcome drug resistance; by co-delivering siRNA and cisplatin prodrug, the exposure of PS can be regulated to reduce the immunosuppressive effect; the LNPs system can encapsulate lipid platinum prodrug and functional RNA (such as siRNA and mRNA) at the same time to achieve the synergistic delivery of platinum drugs and nucleic acids; the addition of lipid platinum prodrug not only improves the protection of RNA, but also increases the adsorption of LNPs to RNA, thereby improving the overall delivery efficiency; through the synergistic effect of these mechanisms, this system can effectively solve the multiple challenges faced by traditional cisplatin chemotherapy and improve the anti-tumor treatment effect. Description of the Drawings

[0023] Figure 1 1H NMR and MS mass spectrometry characterization of Lipid-Pt1

[0024] Figure 2 1H NMR and MS mass spectrometry characterization of Lipid-Pt2

[0025] Figure 3 1H NMR and MS mass spectrometry characterization of Lipid-Pt3

[0026] Figure 4 1H NMR and MS mass spectrometry characterization of Lipid-Pt4

[0027] Figure 5 1H NMR and MS mass spectrometry characterization of Lipid-Pt5

[0028] Figure 6 Lipid-Pt1-5 were added to LNPs at different mass ratios.

[0029] Figure 7 Particle size and zeta potential of LNPs prepared with different ratios of Lipid-Pt

[0030] Figure 8 Efficiency of Lipid-Pt1-5 transfected with siRNA at different ratios

[0031] Figure 9 Stability of LNPs and Pt-LNPs in serum medium

[0032] Figure 10EGFP mRNA expression levels of LNPs in 4T1 cells after the introduction of Lipid-Pt4. Detailed implementation manners

[0033] In the description of the present invention, it should be noted that for orientation terms, such as the terms "center", "horizontal", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., the orientation and position relationships indicated are based on the orientation or position relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and should not be construed as limiting the specific protection scope of the present invention.

[0034] In addition, such terms as "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, the meaning of "at least" is one or more than one, unless otherwise specifically defined.

[0035] In the present invention, unless otherwise clearly specified and limited, such terms as "assembled", "connected" and "joined" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection or an integral connection; it may also be a mechanical connection; it may be directly connected or connected through an intermediate medium, and it may be internally connected and communicated between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0036] In the invention, unless otherwise specified and limited, the first feature being "above" or "below" the second feature may include direct contact between the first and second features, or may include the situation where the first and second features are not in direct contact but in contact through other features therebetween. Moreover, the first feature being "above", "below" and "on the upper surface" of the second feature includes that the first feature is directly above and obliquely above the second feature, or simply means that the horizontal height of the first feature is higher than that of the second feature. The first feature being "above", "below" and "on the lower surface" of the second feature includes that the first feature is directly below or obliquely below the second feature, or simply means that the horizontal height of the first feature is lower than that of the second feature.

[0037] The following further describes the specific implementation manners of the present invention in conjunction with the drawings of the specification, making the technical solutions and their beneficial effects of the present invention clearer and more definite. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention.

[0038] The present invention is a lipid platinum prodrug-assisted RNA lipid nanoparticle, which comprises SM-102, DSPC, cholesterol, DMG-PEG, lipid platinum prodrug and RNA; the lipid platinum prodrug comprises a platinum drug and a fatty acid, the RNA comprises siRNA and mRNA, wherein the siRNA targets and regulates tumor immunosuppression-related genes (such as PS exposure-related genes), and the mRNA encodes an antigen protein to activate an immune response. The fatty acid is a straight-chain or branched-chain fatty acid with a carbon chain length of C6-C24. The platinum drug is cisplatin oxide or oxaliplatin oxide, and the fatty acid is one or more of lauric acid, palmitic acid, stearic acid, 2-hexylundecanoic acid or 2-hexadecylstearic acid. The fatty acid is a straight-chain or branched-chain fatty acid with a carbon chain length of C6-C24. The platinum drug is cisplatin oxide or oxaliplatin oxide, and the fatty acid is one or more of lauric acid, palmitic acid, stearic acid, 2-hexylundecanoic acid or 2-hexadecylstearic acid. The mass ratio of SM-102, DSPC, cholesterol and DMG-PEG is 50:10:38.5:1.5, and the mass proportion of the lipid platinum prodrug is 5%-30%. The particle size of the RNA lipid nanoparticle is 50-150 nm, the surface potential is +10 mV to +30 mV, and the encapsulation efficiency is ≥85%; the RNA lipid nanoparticle is modified with a targeting ligand (such as folic acid, transferrin) to enhance tumor tissue targeting.

[0039] The following are the implementation examples:

[0040] Synthesis of lipid cisplatin prodrug:

[0041] Synthesize tetravalent cisplatin prodrug. Dissolve 5 g of cisplatin in 50 ml of 30% hydrogen peroxide solution, heat to 50 °C, stir for 24 hours, then freeze-dry the reaction mixture, wash the product with cold ether, and dry it under vacuum to obtain a pale yellow powdery tetravalent cisplatin. Then, use five different fatty acids, lauric acid, palmitic acid, stearic acid, 2-hexylundecanoic acid and 2-hexadecylstearic acid, to modify tetravalent cisplatin. Each fatty acid reacts with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) at a molar ratio of fatty acid:EDC of 1:1.2 for 3 hours. Under a nitrogen atmosphere, the product further reacts with tetravalent cisplatin in DMF at a molar ratio of 3:1 for 24 hours. The product is crystallized after washing with ice water, and the crude product is washed with ether, and then dried under vacuum for 24 hours. The structure of the final product was confirmed by 1H NMR and MS spectra, and the 1H NMR and MS spectra are as Figure 1-5 shown.

[0042] Preparation of lipid nanoparticles (LNPs):

[0043] First, dissolve SM-102, DSPC, cholesterol, and DMG-PEG in ethanol at a ratio of 50:10:38.5:1.5 to prepare standard LNPs. The mRNA is dissolved in 10 mM citrate buffer solution, and the two solutions are mixed at a volume ratio of 1:3 with a flow rate of 9 mL / min. The final lipid-to-mRNA mass ratio of the LNPs is 10:1. For lipid-platinum LNPs, dissolve SM-102, DSPC, cholesterol, and DMG-PEG in DMF at the same ratio of 50:10:38.5:1.5. As Figure 6 shown, add different amounts of lipid-platinum, and add Lipid-Pt1-5 to the LNPs at a mass ratio of 5%-30%. The mRNA is dissolved in 10 mM citrate buffer solution, and the two solutions are mixed at a volume ratio of 1:3 with a flow rate of 9 mL / min. In the resulting LNPs, the mass ratio of lipid (excluding lipid Pt) to mRNA remains at 10:1. At the highest ratio of 30%, all five lipids can still be almost completely encapsulated in the empty LNPs (all encapsulation rates are greater than 98%, Table 1).

[0044] Table 1 Encapsulation efficiency of lipid cisplatin prodrugs in blank LNPs Cisplatin prodrug Encapsulation efficiency Lipid-Pt1 98.91±1.44 Lipid-Pt2 98.25±0.96 Lipid-Pt3 98.15±0.67 Lipid-Pt4 99.07±0.77 Lipid-Pt5 99.72±0.26 。

[0045] Preparation and characterization of LNPs containing siRNA (siEGFP):

[0046] LNPs containing siRNA (siEGFP) (platinum LNPs) were prepared by adding different additional ratios of lipid cisplatin in the formulation at a lipid / mRNA mass ratio of 10:1. As Figure 7 shown, compared with LNPs without Pt lipid, the particle size and zeta potential of Pt-LNPs changed slightly, but generally still remained in the range of particle size 80-150 nm and zeta potential -10-10 mV. Then, using cells stably expressing EGFP as a model, the efficiency of lipid-platinum LNPs was studied. As Figure 8 shown, at different ratios, Lipid-Pt1-Lipid-Pt5 all showed a trend of increasing siRNA efficiency, among which, lipid cisplatin 4 (5%-15%) had the most significant effect.

[0047] Preparation and characterization of LNPs containing EGFP mRNA (mEGFP):

[0048] We further studied the effect of Lipid-Pt4 on mRNA delivery, prepared LNPs containing cisplatin prodrugs under optimized conditions, and detected the stability of Pt-LNPs in serum medium;Figure 9 It is shown that free mRNA degrades rapidly in serum, while Pt-LNPs can protect mRNA and keep it stable within 12 hours. LNPs containing platinum can further enhance the protective effect of LNPs, and no obvious degradation is observed even after 24 hours. Finally, taking EGFP mRNA (mEGFP) in 4T1 cells as a model, as Figure 8 shown, the expression level of LNPs increased by 20%-30% after introducing Lipid-Pt4 ( Figure 10 ).

[0049] These examples detail the synthesis of lipid platinum prodrugs, the preparation method of LNPs, and the performance of the system in siRNA and mRNA delivery, fully demonstrating the feasibility and effectiveness of this technical solution.

[0050] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention.

Claims

1. A lipid-platinum prodrug-assisted RNA lipid nanoparticle, characterized in that, It includes SM-102, DSPC, cholesterol, DMG-PEG, lipid platinum prodrug and RNA; the lipid platinum prodrug includes a platinum drug and a fatty acid, and the RNA includes siRNA and mRNA, wherein the siRNA targets and regulates tumor immunosuppression-related genes, and the mRNA encodes an antigen protein to activate the immune response.

2. The lipid-platinum prodrug-assisted RNA lipid nanoparticle according to claim 1, wherein, The fatty acid is a straight-chain or branched-chain fatty acid with a carbon chain length of C6-C24.

3. The lipid platinum prodrug-assisted RNA lipid nanoparticle according to claim 1, characterized in that, The platinum drug is cisplatin oxide or oxaliplatin oxide, and the fatty acid is one or more of lauric acid, palmitic acid, stearic acid, 2-hexylundecanoic acid or 2-hexadecylstearic acid.

4. A lipid-platinum prodrug-assisted RNA lipid nanoparticle according to claim 1, characterized in that, The mass ratio of SM-102, DSPC, cholesterol and DMG-PEG is 50:10:38.5:1.5, and the mass proportion of the lipid platinum prodrug is 5%-30%.

5. A lipid-platinum prodrug-assisted RNA lipid nanoparticle according to claim 1, characterized in that, The particle size of the RNA lipid nanoparticles is 50-150 nm, the surface potential is +10 mV to +30 mV, and the encapsulation efficiency is ≥85%; the RNA lipid nanoparticles are modified with a targeting ligand.

6. A preparation method corresponding to the RNA lipid nanoparticle assisted by the lipid platinum prodrug according to any one of claims 1-5, characterized in that, It includes the following specific steps: S1. Dissolve SM-102, DSPC, cholesterol and DMG-PEG in ethanol in a certain proportion to prepare standard LNPs; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a certain volume ratio, and the flow rate is 8-10 mL / min; the mass ratio of the lipid to mRNA in the final LNPs is (9-11):

1. S2. For lipid platinum LNPs, dissolve SM-102, DSPC, cholesterol and DMG-PEG in DMF in the same proportion as in S1, and add the lipid platinum prodrug to the lipid platinum LNPs at a mass ratio of 5%-30%; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a certain volume ratio, and the flow rate is 8-10 mL / min; in the obtained lipid platinum LNPs, the mass ratio of the lipid to mRNA is maintained at (9-11):

1.

7. The preparation method of the RNA lipid nanoparticle assisted by the lipid platinum prodrug according to claim 6, characterized in that, In S1, dissolve SM-102, DSPC, cholesterol and DMG-PEG in ethanol at a ratio of 50:10:38.5:1.5 to prepare standard LNPs; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a volume ratio of 1:3, and the flow rate is 9 mL / min; the mass ratio of the lipid to mRNA in the final LNPs is 10:

1.

8. The preparation method of the RNA lipid nanoparticle assisted by the lipid platinum prodrug according to claim 6, characterized in that, In S2, for lipid platinum LNPs, dissolve SM-102, DSPC, cholesterol and DMG-PEG in DMF in the same proportion of 50:10:38.5:1.5, and add the lipid cisplatin prodrug to the LNPs at a mass ratio of 5%-30%; dissolve mRNA in 10 mM citrate buffer solution, mix the two solutions in a volume ratio of 1:3, and the flow rate is 9 mL / min; in the obtained LNPs, the mass ratio of the lipid (excluding lipid Pt) to mRNA is maintained at 10:

1.

9. The method for preparing the RNA lipid nanoparticle assisted by the lipid platinum prodrug according to claim 6, wherein The synthesis steps of the lipid cisplatin prodrug include: S1. Dissolve 4 - 6 g of cisplatin in 45 - 55 mL of 30% hydrogen peroxide solution, heat to 45 - 55 °C, stir for 23 - 25 hours, then lyophilize the reaction mixture, wash the product with cold ether, and dry it under vacuum to obtain yellowish - brown powdery tetravalent cisplatin; S2. Modify tetravalent cisplatin using five different fatty acids, namely lauric acid, palmitic acid, stearic acid, 2 - ethylundecanoic acid, and 2 - hexadecylstearic acid. Each fatty acid reacts with 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide (EDC) at a molar ratio of fatty acid:EDC of 1:1.1 - 1.3 for 2 - 4 hours; under a nitrogen environment, the product further reacts with tetravalent cisplatin in DMF at a molar ratio of 2 - 4:1 for 23 - 25 hours; the product is crystallized after being washed with ice - water, the crude product is then washed with ether, and then dried under vacuum for 23 - 25 hours.

10. Use of the lipid platinum prodrug-assisted RNA lipid nanoparticle according to any one of claims 1-8, characterized in that, It is applied in drug delivery, the preparation of anti - tumor drugs or tumor vaccines, and is especially suitable for the treatment of solid tumors or hematological tumors.

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