Application of amino-containing PEGylated 1, 2-glyceryl dimyristate in preparation of carrier for targeted delivery of antitumor drugs

Abstract

The invention provides an application of pegylated 1, 2-glyceryl dimyristate (DMG-PEG2K-NH2) with an amino group in preparation of a carrier serving as a targeted delivery anti-tumor drug. The invention also provides a tumor targeting liposome nanoparticle, the tumor targeting liposome nanoparticle is composed of pegylated 1, 2-glyceryl dimyristate with amino and lipid, and the lipid is any one or a combination of more than two of ionizable cationic lipid, cationic lipid, anionic lipid, cholesterol, phospholipid and pegylated lipid. In the drug delivery system provided by the invention, the introduction of DMG-PEG2K-NH2 not only endows the liposome nanoparticles with excellent tumor targeting performance, but also provides reaction active sites for further functional modification of the liposome. The tumor targeted liposome nanoparticles provided by the invention can be used for targeted delivery of various antitumor drugs, and have great clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a drug delivery carrier, specifically to the use of amino-containing polyethylene glycol 1,2-dimyristyl glyceride in the preparation of a carrier for targeted delivery of anti-tumor drugs. Background Art

[0002] Nucleic acids, as important biological macromolecules in organisms, not only play a core role in the storage and transmission of genetic information, but also are deeply involved in important physiological activities such as gene expression regulation, protein synthesis, cell signal transduction, energy metabolism, and immune response. They are crucial to maintaining the normal functions of organisms.

[0003] In this context, in recent years, an emerging gene therapy technology has gradually emerged, aiming to use foreign nucleic acid drugs to intervene in the treatment of genetic defect diseases or complex diseases that are difficult to cure by traditional means (such as cancer). However, nucleic acid molecules are unstable in the body and are easily degraded by nucleases. Moreover, nucleic acid molecules are negatively charged and it is difficult for them to penetrate the cell membrane and enter the cell to exert their biological functions. Therefore, nucleic acid drugs require suitable delivery vectors to deliver them intact and efficiently to the target cells. However, how to accurately and targetedly deliver nucleic acid drugs to specific organs or tissues outside the liver is still a major challenge currently faced.

[0004] Liposome nanoparticles are the most popular and promising nucleic acid drug delivery system in recent years. At present, studies have reported that by adjusting the lipid composition, liposome nanoparticles can be effectively delivered to normal tissues of the body such as the liver, spleen, and lungs (Nature Communications, 2024, 15: 5659; Nature Nanotechnology, 2020, 15: 313–320). However, there are few reports on drug delivery systems targeting extrahepatic tumors and other diseased tissues. Summary of the invention

[0005] In view of the above technical problems in the prior art, the present invention provides a polyethylene glycol-containing 1,2-dimyristyl glyceride (DMG-PEG 2K -NH2) in the preparation of a carrier for targeted delivery of anti-tumor drugs. The use of this amino-containing polyethylene glycol 1,2-dimyristoyl glyceride in the preparation of a carrier for targeted delivery of anti-tumor drugs is to solve the technical problem that the drug delivery carrier in the prior art is not effective in delivering anti-tumor drugs.

[0006] The present invention provides a polyethylene glycol-containing 1,2-dimyristyl glyceride (DMG-PEG 2K -NH2) in the preparation of a carrier for targeted delivery of anti-tumor drugs.

[0007] Furthermore, the carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 5-50:50-95.

[0008] Furthermore, the carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 10-20:80-90.

[0009] Furthermore, the lipid is any one of ionizable cationic lipids, cationic lipids, anionic lipids, cholesterol, phospholipids, and pegylated lipids, or a combination of two or more thereof.

[0010] Furthermore, DLin-MC3-DMA, cholesterol, distearoylphosphatidylcholine, amino-PEGylated 1,2-dimyristylglycerol and DMG-mPEG 2K The composition has a molar ratio of 50:28.5:10:10:1.5 or 50:18.5:10:20:1.5.

[0011] The present invention also provides a tumor-targeted liposome nanoparticle, which is composed of amino-containing polyethylene glycol-1,2-dimyristyl glyceride and a lipid group, wherein the lipid is any one of ionizable cationic lipids, cationic lipids, anionic lipids, cholesterol, phospholipids, and polyethylene glycol-containing lipids (PEGylated lipids) or a combination of two or more thereof.

[0012] Furthermore, the carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 5-50:50-95.

[0013] Furthermore, the carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 10-20:80-90.

[0014] Furthermore, DLin-MC3-DMA, cholesterol, distearoylphosphatidylcholine, amino-PEGylated 1,2-dimyristylglycerol and DMG-mPEG 2K The composition has a molar ratio of 50:28.5:10:10:1.5 or 50:18.5:10:20:1.5.

[0015] The present invention provides a polyethylene glycol-containing 1,2-dimyristyl glyceride (DMG-PEG 2K -NH2) is used in the preparation of a carrier for targeted delivery of anti-tumor drugs. The introduction of DMG-PEG-NH2 not only gives liposome nanoparticles excellent tumor targeting properties, but also provides reactive sites for further functional modification of liposomes.

[0016] Compared with the prior art, the technical effect of the present invention is positive and obvious.

[0017] (1) The tumor-targeted liposome nanoparticles provided by the present invention have enhanced tumor targeting properties and can effectively deliver drugs to extrahepatic tumor tissues;

[0018] (2) In the tumor-targeting liposome nanoparticles provided by the present invention, DMG-PEG 2K The introduction of -NH2 also provides a reaction site for the functional modification of liposomes. Through this site, the liposomes can be further modified with hyaluronic acid, and the affinity between hyaluronic acid and tumor CD44-positive cells can be used to promote the effective endocytosis of liposomes in tumor cells, thereby improving the bioavailability and therapeutic effect of drugs.

[0019] (3) The tumor-targeted liposome nanoparticles provided by the present invention can be used not only for the delivery of biological agents such as nucleic acids, but also for the delivery of other anti-tumor drugs such as chemotherapy, and have great clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The commercial LNP loaded with DiR fluorescence (without DMG-PEG) was injected in equal amounts through the tail vein. 2K -NH2 liposome nanoparticles), LNP-10% NH2 (containing 10% molar content of DMG-PEG 2K -NH2) and LNP-20% NH2 (containing 20% ​​molar content of DMG-PEG 2K -NH2), and 5 days later, the fluorescence intensity distribution diagram of the main organs and tumor sites in tumor-bearing (breast cancer) mice.

[0021] Figure 2 This is the fluorescence intensity percentage of major organs and tumor tissues in tumor-bearing (breast cancer) mice 5 days after equal amounts of DiR fluorescent commercial LNP, LNP-10% NH2 and LNP-20% NH2 were injected through the tail vein.

[0022] Figure 3This is a graph showing the fluorescence intensity per unit mass of tumor tissue in tumor-bearing (breast cancer) mice 5 days after the same amount of commercial LNP, LNP-10% NH2 and LNP-20% NH2 loaded with DiR fluorescence were injected through the tail vein.

[0023] Figure 4 These are the particle size analysis results of the prepared LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158 liposome nanoparticles.

[0024] Figure 5 This is the fluorescence intensity distribution diagram of tumor tissue in tumor-bearing (breast cancer) mice 3 days after equal amounts of DiR fluorescent LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158 particles were injected through the tail vein.

[0025] Figure 6 This is a graph of the fluorescence intensity per unit mass of tumor tissue in tumor-bearing (breast cancer) mice 3 days after equal amounts of DiR fluorescent LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158 particles were injected through the tail vein.

[0026] Figure 7 The expression of piR-2158 in mouse tumor tissue 3 days after injection of LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0028] Example 1 Preparation of tumor-targeted liposome nanoparticles

[0029] The MC3 liposome carrier, which is already on the market of Alnylam Pharmaceuticals, was used as a reference control. Its components include DLin-MC3-DMA (purchased from Shanghai McLean Biochemical Technology Co., Ltd.), cholesterol (purchased from Shanghai McLean Biochemical Technology Co., Ltd.), distearoylphosphatidylcholine (DSPC) (purchased from Shanghai McLean Biochemical Technology Co., Ltd.) and DMG-mPEG 2K (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.), the molar ratio is 50:38.5:10:1.5. The new lipid DMG-PEG was introduced into the liposome system2K -NH2 (purchased from Xi'an Ruixi Biotechnology Co., Ltd.), composed of two containing DLin-MC3-DMA, cholesterol, DSPC, DMG-PEG 2K -NH2 and DMG-mPEG 2K The molar ratios of the mixed lipids were 50:28.5:10:10:1.5 and 50:18.5:10:20:1.5, respectively. In order to fluorescently label and track the liposomes, 2% (w / w) DiR dye was added to the lipid components.

[0030] The preparation method of liposome nanoparticles is as follows:

[0031] (1) Lipids (DLin-MC3-DMA, cholesterol, DSPC, DMG-PEG 2K -NH2 and DMG-mPEG 2K A mixed lipid composition with a molar ratio of 50:28.5:10:10:1.5 and 50:18.5:10:20:1.5 was dissolved in ethanol to prepare a solution with a total lipid concentration of 12 mM.

[0032] (2) Prepare liposome nanoparticles using microfluidic technology, that is, a lipid solution is sucked into the ethanol phase syringe of the microfluidic device, and PBS is sucked into another water phase syringe of the microfluidic device, and the extrusion rate of the ethanol phase is set to 3 mL / min, and the extrusion rate of the water phase is set to 9 mL / min. Start the microfluidic device, after the ethanol phase and the water phase are fully mixed, receive the mixed liposome solution from the discharge port of the microfluidic device.

[0033] (3) After extrusion is completed, the collected mixed liposome solution is transferred to a dialysis bag with a molecular weight cutoff of 20 kDa and dialyzed with PBS 100 times larger in volume than the mixed liposome solution. After dialysis for 1 day, liposome nanoparticles can be obtained and collected and stored at 4°C for later use.

[0034] In order to distinguish, DMG-PEG 2K -NH2 component liposome nanoparticles, denoted as commercial LNP; containing 10% molar ratio of DMG-PEG 2K -NH2 component liposome nanoparticles, denoted as LNP-10% NH2; containing 20% ​​molar ratio of DMG-PEG 2K -NH2 component liposome nanoparticles are denoted as LNP-20% NH2.

[0035] Example 2 In vivo tumor targeting validation of tumor-targeted liposome nanoparticles

[0036] (1) About 10,000 mouse breast cancer cells (4T1 cells) were injected subcutaneously into Balb / c mice. Subcutaneous tumors formed within one week, thus establishing a tumor-bearing mouse model.

[0037] (2) DiR fluorescently labeled commercial LNP, LNP-10% NH2, and LNP-20% NH2 solutions were injected into tumor-bearing mice in equal amounts by tail vein injection. Five days later, the mice were killed, and the heart, liver, spleen, lung, kidney, and subcutaneous tumor tissues were removed and the fluorescence intensity of each tissue was detected.

[0038] (3) Fluorescence intensity test results of each tissue Figure 1 As shown. It can be seen that in the LNP-20% NH2 treated group, the fluorescence intensity of the tumor site was second only to the liver, and higher than other tissues such as the heart, spleen, kidney, and lung. Moreover, the fluorescence intensity of the tumor site in the LNP-20% NH2 treated group was also stronger than that in the commercial LNP and LNP-10% NH2 treated groups. This shows that LNP-20% NH2 nanoparticles exhibited better tumor targeting performance.

[0039] (4) In order to further quantify the fluorescence intensity of each tissue, the fluorescence intensity ratio of each tissue was analyzed, as shown in the attached figure. Figure 2 As shown. It can be seen that compared with the commercial LNP treatment group, the proportion of fluorescence intensity in the liver of the LNP-20% NH2 treatment group decreased, while the proportion of fluorescence intensity in the tumor tissue increased. This also further verifies the tumor targeting performance of LNP-20% NH2 nanoparticles.

[0040] (5) To further analyze the tumor targeting performance of nanoparticles, the fluorescence intensity in tumor tissue was normalized per unit mass. Figure 3 As shown. It can be seen that compared with the commercial LNP treatment group, the fluorescence intensity per unit mass of the tumor in the LNP-10% NH2 and LNP-20% NH2 treatment groups of mice increased, and with the increase of DMG-PEG 2K The data also further demonstrated that LNP-10% NH2 and LNP-20% NH2 nanoparticles have enhanced tumor targeting performance.

[0041] Example 3 Preparation of tumor-targeted liposome nanoparticles loaded with nucleic acid (piR-2158)

[0042] (1) Lipids (DLin-MC3-DMA, cholesterol, DSPC, DMG-PEG 2K -NH2 and DMG-mPEG 2KA mixed lipid composition (with a molar ratio of 50:18.5:10:20:1.5) was dissolved in ethanol to prepare a solution with a total lipid concentration of 12 mM; in order to fluorescently label and track the liposomes, 2% (w / w) DiR dye was added to the lipid component.

[0043] (2) piR-2158 (purchased from Shanghai Jierui Biotechnology Co., Ltd.) was dissolved in pH 4.0 citrate buffer (purchased from Shanghai Yuanye Biotechnology Co., Ltd.) to prepare a nucleic acid solution with a concentration of 80 ng / μL.

[0044] (3) Prepare liposome nanoparticles using microfluidic technology, that is, a lipid solution is sucked into an ethanol phase syringe of a microfluidic device, and a nucleic acid solution is sucked into another water phase syringe of the microfluidic device, and the extrusion rate of the ethanol phase is set to 3 mL / min and the extrusion rate of the water phase is set to 9 mL / min. Start the microfluidic device, and after the ethanol phase and the water phase are fully mixed, receive the mixed liposome solution from the discharge port of the microfluidic device.

[0045] (4) After extrusion, the collected mixed liposome solution was transferred to a dialysis bag with a molecular weight cutoff of 20 kDa and dialyzed with PBS 100 times larger than the volume of the mixed liposome solution. After dialysis for 1 day, tumor-targeted liposome nanoparticles loaded with piR-2158 were obtained, recorded as LNP-20% NH2@piR-2158, and collected and stored at 4°C for later use.

[0046] (5) The prepared LNP-20% NH2@piR-2158 was subjected to dynamic light scattering (DLS) test, and its particle size distribution was measured as shown in the attached figure. Figure 4 As shown in A, its average diameter is 166.5±3.3nm and PDI is 0.372.

[0047] Example 4 Tumor-targeted liposome nanoparticles are further modified with hyaluronic acid (HA) and loaded with piR-2158

[0048] (1) First, as shown in Example 3, tumor-targeted liposome nanoparticles loaded with piR-2158 were prepared.

[0049] (2) Hyaluronic acid (HA) with a molecular weight of 100-200 kDa (purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was weighed and dissolved in deionized water to prepare a solution with a concentration of 2 mg / mL, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added to the solution, wherein the mass ratio of HA:EDC·HCl:NHS was 4:1:1. After adjusting the pH of the solution to 4.0, the solution was placed at 37°C for activation for 2 hours.

[0050] (3) Add the HA solution to the liposome solution prepared in step (1) and stir the reaction at room temperature for 24 hours. Then place the solution in a dialysis bag with a molecular weight cutoff of 300 kDa and dialyze with PBS 100 times larger than the volume of the mixed liposome solution. After 2 days of dialysis, the obtained liposome nanoparticles are recorded as LNP-20% NH2-HA@piR-2158 and collected and stored at 4°C for later use.

[0051] (5) The prepared LNP-20% NH2-HA@piR-2158 was subjected to dynamic light scattering (DLS) test, and its particle size distribution was measured as shown in the attached figure. Figure 4 As shown in Figure B, the average diameter is 210.1±15.3 nm and the PDI is 0.456. Its particle size is slightly larger than that of LNP-20%NH2@piR-2158 particles without HA modification.

[0052] Example 5 Validation of in vivo tumor-targeted delivery of piR-2158 by LNP-20%NH2@piR-2158 and LNP-20%NH2-HA@piR-2158 nanoparticles

[0053] (1) DiR fluorescently labeled LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158 solutions were injected into tumor-bearing (breast cancer) mice by tail vein injection. Three days later, the mice were killed and subcutaneous tumor tissues were obtained to verify the fluorescence intensity and intratumoral piR-2158 expression.

[0054] (2) Attachment Figure 5 The fluorescence intensity of mouse tumors after injection of LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158 particles. It can be seen that the fluorescence intensity of the tumor in the LNP-20% NH2-HA@piR-2158 treatment group is significantly stronger than that of LNP-20% NH2@piR-2158, indicating that HA further enhances the tumor targeting performance of liposome nanoparticles.

[0055] (3) To further analyze the tumor targeting performance of nanoparticles, the fluorescence intensity in tumor tissue was standardized per unit mass. Figure 6 As shown. It can be seen that the fluorescence intensity per unit mass of the tumor of mice in the LNP-20% NH2-HA@piR-2158-treated group was higher than that in the LNP-20% NH2@piR-2158-treated group, indicating that the enrichment degree of LNP-20% NH2-HA@piR-2158 nanoparticles in the tumor site was higher.

[0056] (4) Attachment Figure 7The expression of piR-2158 in mouse tumor tissue after treatment with LNP-20% NH2@piR-2158 and LNP-20% NH2-HA@piR-2158. It can be seen that compared with the tumor tissue (control group) that has not been treated in any way, LNP-20% NH2 liposome nanoparticles can deliver piR-2158 to tumor tissue, increasing the expression of piR-2158 in tumor tissue by nearly 3 times, while HA-modified LNP-20% NH2-HA liposome nanoparticles further increased the expression of piR-2158 in the tumor site by about 9 times. This shows that HA modification on LNP-20% NH2 liposome nanoparticles further enhances the enrichment of nucleic acid drugs in tumor tissues.

Claims

1. Use of amino-containing polyethylene glycol 1,2-dimyristyl glyceride in the preparation of a carrier for targeted delivery of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 5-50:50-95.

3. The use according to claim 2, characterized in that: The carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 10-20:80-90.

4. The use according to claim 2 or 3, characterized in that: The lipid is any one of ionizable cationic lipids, cationic lipids, anionic lipids, cholesterol, phospholipids, and pegylated lipids, or a combination of two or more thereof.

5. The use according to any one of claims 1 to 4, characterized in that: The carrier is composed of DLin-MC3-DMA, cholesterol, distearoylphosphatidylcholine, amino-containing polyethylene glycol 1,2-dimyristyl glyceride and DMG-mPEG. 2K The composition has a molar ratio of 50:28.5:10:10:1.5 or 50:18.5:10:20:1.

5.

6. A tumor-targeted liposome nanoparticle, characterized in that: The invention is composed of PEGylated 1,2-dimyristyl glyceride with amino group and lipid, wherein the lipid is any one of ionizable cationic lipid, cationic lipid, anionic lipid, cholesterol, phospholipid and PEGylated lipid or a combination of two or more thereof.

7. The tumor-targeting liposome nanoparticle according to claim 6, characterized in that: The carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 5-50:50-95.

8. The tumor-targeting liposome nanoparticle according to claim 7, characterized in that: The carrier is composed of amino-containing polyethylene glycol 1,2-dimyristyl glyceride and lipid, and the molar ratio of the amino-containing polyethylene glycol 1,2-dimyristyl glyceride to the lipid is 10-20:80-90.

9. A tumor-targeting liposome nanoparticle according to any one of claims 6 to 8, characterized in that: The molecule was composed of DLin-MC3-DMA, cholesterol, distearoylphosphatidylcholine, amino-PEGylated 1,2-dimyristylglycerol and DMG-mPEG. 2K The composition has a molar ratio of 50:28.5:10:10:1.5 or 50:18.5:10:20:1.5.

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