Cancer cell membrane-encapsulated lipid nanoparticle for nucleic acid drug delivery, preparation method therefor and use thereof

Abstract

The present invention relates to a cancer cell membrane-encapsulated lipid nanoparticle for nucleic acid drug delivery, a preparation method therefor and the use thereof. The solution comprises encapsulating the outer surface of LNP with a layer of a cancer cell membrane to endow same with a biological activity similar to that of tumor cells, so that the LNP can effectively reside at a lesion for a long time after being injected into the tumor tissue of a patient. Moreover, experimental data shows that CLNPs encapsulated with the cell membrane of cancer cell HepG2 have a generally improved transfection efficiency compared with that of LNPs, and for three different liposome formulations (LNP1, LNP2 and LNP3), the transfection efficiency is improved to varying degrees in three different cell lines. In an in vitro transfection experiment in MC38, the maximum increase of 7.5-fold is achieved.

Description

A cancer cell membrane-encapsulated lipid nanoparticle for nucleic acid drug delivery, its preparation method and application Technical Field

[0001] This invention belongs to the field of nucleic acid drug delivery carriers, specifically relating to a cancer cell membrane-encapsulated lipid nanoparticle for nucleic acid drug delivery, its preparation method and application. Background Technology

[0002] The tremendous success of messenger RNA (mRNA) technology in vaccine development has directly accelerated vaccine research and development and resolved some of the technical bottlenecks in applying mRNA technology to vaccine development. The application of nucleic acid-based drugs in the development of novel therapeutic and preventative cancer vaccines has also brought new opportunities for cancer treatment.

[0003] Current technologies for delivering nucleic acid drugs based on lipid nanoparticles (LNPs) have the following shortcomings:

[0004] 1) The polyethylene glycol-modified lipids (PEG lipids) in its formula can activate anti-PEG antibodies, causing hypersensitivity reactions, accelerating blood clearance, and causing systemic immunogenicity after repeated administration, resulting in rapid drug clearance and failure to produce sufficient efficacy.

[0005] 2) LNPs adsorb onto serum proteins and accumulate in the liver, producing local toxicity. Furthermore, they have poor targeting to tumor tissues, and even after intratumoral injection, they can still spread to adjacent tissues and organs.

[0006] Therefore, based on this, the technical solution of the present invention is proposed.

[0007] Summary of the Invention

[0008] To address the problems of existing technologies, this invention encapsulates lipid nanoparticles with a bioactive cancer cell membrane. After injection into the tumor lesion, the lipid nanoparticles coated with the cancer cell membrane can interact with the cancer cells and remain effectively within the lesion. Furthermore, the cancer cell membrane on the surface of the lipid nanoparticles also possesses an immune adjuvant effect, enhancing the body's immune response.

[0009] This invention provides a method for preparing lipid nanoparticles encapsulated in cancer cell membranes for nucleic acid drug delivery, the method comprising the following steps:

[0010] (1) Phase A and Phase B are mixed and homogenized to obtain lipid nanoparticles (LNPs); wherein:

[0011] Phase A includes cationic lipids, ionizable lipids, phospholipids (DSPC), cholesterol (CHO), 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol (DMG-PEG2000), and solvents;

[0012] Phase B includes nucleic acid drugs and acidic buffer solution;

[0013] (2) The cancer cell sample (derived from the patient's own cancer cells) was diluted using phase C, then centrifuged and the supernatant was discarded to obtain a precipitate; wherein:

[0014] Phase C is HEP buffer;

[0015] (3) The precipitate was resuspended using phase D, frozen, thawed, and then centrifuged and resuspended sequentially to obtain the purified cancer cell membrane; wherein:

[0016] Phase D is a lysis buffer solution;

[0017] (4) The lipid nanoparticles and the purified cancer cell membrane are mixed and homogenized to obtain the cancer cell membrane-encapsulated lipid nanoparticles (CLNP) for nucleic acid drug delivery.

[0018] To facilitate understanding of this invention, the relevant principles are explained as follows:

[0019] To improve the biocompatibility and targeting function of lipid nanoparticles, cell membranes obtained after lysing and purifying cancer cells were coated with the LNPs. The main structure of these cell membranes was a lipid-supported structure of phospholipids and cholesterol, incorporating various membrane proteins and transmembrane proteins. This preserved their biological activity while removing intracellular substances that might trigger an immune response. Subsequently, LNPs were mixed with purified cancer cell membranes in a certain proportion, and the cell membranes were coated onto the outer surface of the LNPs using microfluidic, membrane extrusion, or ultrasonic treatment methods, forming a core-shell structure (the core being the LNP and the shell being the cancer cell membrane), which is known as CLNP (Cancer cell membrane coated Lipid Nano Particle).

[0020] Preferably, phase A comprises the following raw materials in parts by weight: 15-45 parts cationic lipids, 15-45 parts ionizable lipids, 5-20 parts phospholipids, 20-40 parts cholesterol, and 0.5-1.5 parts 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol;

[0021] And / or, in phase B, the concentration of the nucleic acid drug is 50-500 μg / mL (more preferably 200-300 μg / mL), and the acidic buffer is a citrate-sodium citrate buffer (5-15 mM) with pH=4;

[0022] And / or, the volume ratio of phase A to phase B is 1:3 to 5.

[0023] Preferably, the cationic lipid is one of (2,3-dioleoxypropyl)trimethylammonium chloride (DOTAP), 1,2-bisoctadecenoxy-3-methylammonium propane (DOTMA), and DC-cholesterol;

[0024] And / or, the ionizable lipid is one of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), methyl 4-(N,N-dimethylamino)butyrate (dilinyl) ester (DLin-MC3-DMA), or 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate (SM-102);

[0025] And / or, the solvent is ethanol;

[0026] And / or, the nucleic acid drug is one of mRNA, siRNA, ASO, etc.

[0027] Preferably, in step (1), the mixing and homogenization is one of microfluidic technology, membrane extrusion technology, and ultrasonic treatment technology; wherein:

[0028] In the microfluidic process, the flow rate of phase A is 3 to 7 mL / min, and the flow rate of phase B is 3 to 5 times that of phase A.

[0029] In the membrane extrusion process, filter membranes with pore sizes of 400 nm and 200 nm are used sequentially, and phase A and phase B are mixed and extruded repeatedly for 9 to 11 cycles.

[0030] In the ultrasonic treatment process, after mixing phase A and phase B, ultrasonic treatment is performed for 5 to 15 minutes using an ultrasonic water bath device with a power of 200 to 1000 kW.

[0031] Preferably, in step (2), the cancer cells are one of liver cancer cells, lung cancer cells, or breast cancer cells;

[0032] And / or, each 500 mL of the HEP buffer contains 4.091 g NaCl, 0.101 g KCl, 0.453 g HEPES, and 0.546 g EGTA, and then the pH of the HEP buffer is adjusted to 7.0–7.4 using hydrochloric acid or sodium hydroxide;

[0033] And / or, the dilution to a cell density of 0.1–0.2 × 10⁻⁶ 8 cells / mL

[0034] And / or, the centrifugation is performed at 500g for 10 min.

[0035] Preferably, in step (3), the lysis buffer comprises 100 mM EGTA, and the pH of the lysis buffer is adjusted to 7.2 using hydrochloric acid or sodium hydroxide;

[0036] And / or, the cell density after resuspension is 0.5–1 × 10⁻⁶. 8 per mL.

[0037] More specifically: The precipitate was resuspended using lysis buffer, and the resuspended cell density was 0.5–1.0 × 10⁻⁶. 8 Cells / mL were collected and transferred to a -80°C freezer for 20 minutes; then thawed in a 37°C water bath for 5–10 minutes until substantially thawed (repeat this operation 4 times); after that, the thawed cell suspension was centrifuged at 10,000g for 15 minutes, the supernatant was discarded and the suspension was resuspended with the same volume of lysis buffer; then, the resuspended cell suspension was centrifuged at 20,000–40,000g for 15 minutes, the supernatant was discarded and the suspension was resuspended with lysis buffer and stored at -80°C for later use.

[0038] Preferably, in step (4), the ratio of the total lipid concentration in the lipid nanoparticles to the protein concentration in the purified cancer cell membrane is 10:1 to 2:1.

[0039] And / or, the mixing and homogenization is one of the following processes: microfluidic process, membrane extrusion process, and ultrasonic treatment process.

[0040] More specifically: When using membrane extrusion process, after pretreatment with a membrane with a pore size of 400nm, a 100nm or 200nm filter membrane is used to extrude the liquid back and forth for 9 or 11 cycles.

[0041] Based on the same technical concept, another aspect of the present invention is to provide cancer cell membrane-encapsulated lipid nanoparticles for nucleic acid drug delivery obtained by the above preparation method.

[0042] Based on the same technical concept, another aspect of the present invention is to provide the application of cancer cell membrane-encapsulated lipid nanoparticles for nucleic acid drug delivery in the preparation of cancer treatment drugs.

[0043] Preferably, the drug is an injectable preparation.

[0044] The beneficial effects of this invention are as follows:

[0045] The CLNP described in this invention maintains the biological activity of cancer cells, resulting in better biocompatibility, delivery efficiency, and lesion targeting. Experimental data shows that CLNP coated with the HepG2 cancer cell membrane generally improves transfection efficiency compared to LNP. For three different liposome formulations (LNP1, LNP2, and LNP3), transfection efficiency was improved to varying degrees in three different cell lines. The largest improvement, 7.5-fold, was observed in the in vitro transfection experiment with MC38 cells. The increased luciferase signal intensity indicates improved mRNA transfection efficiency. Related data show that coating the HepG2 cancer cell membrane can effectively improve transfection efficiency for specific cell types. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] [Corrected according to Rule 91 06.08.2024] Figure 1 is the particle size distribution of LNP1 in Table 2 (the three curves represent three tests, the same below).

[0048] Figure 2 shows the particle size distribution of CLNP1 in Table 2.

[0049] Figure 3 shows the particle size distribution of LNP2 in Table 2.

[0050] Figure 4 shows the particle size distribution of CLNP2 in Table 2.

[0051] Figure 5 shows the particle size distribution of LNP3 in Table 2.

[0052] Figure 6 shows the particle size distribution of CLNP3 in Table 2.

[0053] Figure 7 shows the luciferase signal intensity data for each group in the HEK-293T cell line.

[0054] Figure 8 shows the luciferase signal intensity data for each group in the HeLa cell line.

[0055] Figure 9 shows the luciferase signal intensity data for each group in the MC38 cell line. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0057] Example

[0058] Step 1 (Preparation of lipid solution): The total lipid concentration was 5 mg / mL. 0.75 mL was prepared for each of the three examples, as shown in Table 1.

[0059] Table 1 Lipid Solution Formulation

[0060] Step 2 (mRNA solution preparation): Dissolve 202 μg of mRNA encoding firefly luciferase in 2.25 mL of 10 mM citric acid solution (pH 4.0).

[0061] Step 3 (Preparation of lipid nanoparticles): 0.75 mL of lipid solution (flow rate set to 7 mL / min) and 2.25 mL of mRNA solution (flow rate set to 21 mL / min) were mixed using a microfluidic device to prepare lipid nanoparticles. The particle size, mRNA concentration, PdI, and encapsulation efficiency were then measured. The lipid nanoparticles obtained in Example 1 were named LNP1, those obtained in Example 2 were named LNP2, and those obtained in Example 3 were named LNP3.

[0062] Step 4 (HepG2 Cancer Cell Membrane Purification): Collect adherent HepG2 cells using a cell scraper, dilute in culture medium, and centrifuge at 500g, 10℃ for 10 min, discarding the supernatant. Resuspend the cells in HEP ​​buffer and centrifuge again at 500g, 10℃ for 10 min, discarding the supernatant. After resuspending the cells in freeze-thaw buffer, freeze at -80℃ for 20 min, then thaw in a 37℃ water bath. Repeat the freeze-thaw cycle 5 times to lyse the cells. After the last thaw, centrifuge at 500g, 10℃ for 10 min, discard the pellet, and retain the supernatant. Then centrifuge the supernatant at 10000g, 10℃ for 15 min, discarding the supernatant; resuspend in freeze-thaw buffer, centrifuge at 21100g, 10℃ for 15 min, discarding the supernatant. The precipitate was resuspended in purified water, sonicated for 15 minutes, and then centrifuged at 10,000g for 5 minutes. The supernatant contained the purified HepG2 cell membrane.

[0063] Step 5 (Preparation of Lipid Nanoparticles Coated with Cancer Cell Membranes (CLNPs): The lipid nanoparticles prepared in Step 3 and the cell membrane prepared in Step 4 were mixed at a lipid concentration:protein concentration = 5:1. The mixed solution was extruded using an extruder. The extrusion process was as follows: first, a filter membrane with a pore size of 0.4 μm was extruded, followed by a filter membrane with a pore size of 0.2 μm. After extrusion, cell membrane-coated lipid nanoparticles were obtained. The particle size, mRNA concentration, PdI, and encapsulation efficiency were measured. The cell membrane-coated lipid nanoparticles obtained in Example 1 were named CLNP1, those obtained in Example 2 were named CLNP2, and those obtained in Example 3 were named CLNP3.

[0064] The sample test results are shown in Table 2.

[0065] Table 2 Sample test results

[0066] The particle size distribution diagrams of LNP1, CLNP1, LNP2, CLNP2, LNP3, and CLNP3 are shown in Figure 1, 2, 3, 4, 5, and 6, respectively.

[0067] Validation example (in vitro transfection experiment)

[0068] Three cell lines (HEK-293T, HeLa, and MC38) were digested with 0.25% trypsin and counted. The cell concentration was adjusted to 3 x 10⁻⁶ cells / year. 5 Add 100 μL of cell culture to each well of a 96-well cell culture plate to achieve a cell count of 3 x 10⁶ cells / mL. 4 Cells / well. Place the cell culture plate in an incubator at 37°C and 5% CO2, and incubate overnight (16-18 h). Dilute the LNP and PLNP samples with culture medium to a mRNA concentration of 200 ng / mL. Discard the culture medium after overnight culture, and add 100 μL of the diluted sample to the corresponding well of the cell culture plate, i.e., the actual added mRNA is 20 ng. Place the plate in an incubator at 37°C and 5% CO2, and incubate for 24 h. Remove the cell culture plate and equilibrate to room temperature (25°C). Then add 100 μL of luciferase detection reagent to each well, shake for 30 s, let stand for 3 min, and then read the plate using a microplate reader.

[0069] The results, shown in Table 3, indicate that among the three formulations (LNP1, LNP2, and LNP3), CLNP coated with the HepG2 cancer cell membrane exhibited varying degrees of improved transfection efficiency compared to LNP. For formulations LNP1 and LNP2, significant improvements were observed in both HEK-293T and MC38 cells, with a maximum improvement of 7.5-fold. For LNP3, a significant improvement was observed in HEK-293T cells. The increased luciferase signal intensity indicates improved mRNA transfection efficiency, suggesting that coating the HepG2 cancer cell membrane can effectively enhance transfection efficiency in specific cell types.

[0070] Table 3 Summary of in vitro transfection experiment results (luciferase signal intensity, unit: AU)

[0071] Note: The calculation method for "*" is: Signal strength enhancement ratio = CLNP i / LNP i i = 1, 2 or 3.

[0072] For example, when i=1, in the HEK-293T cell line, the signal intensity enhancement ratio is 235243.0 / 60131.5 = 3.9.

[0073] In addition, the corresponding HEK-293T, Hela and MC38 data graphs are shown in Figures 7 to 9 respectively.

[0074] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing lipid nanoparticles encapsulated in cancer cell membranes for nucleic acid drug delivery, characterized in that, The preparation method includes the following steps: (1) Phase A and Phase B are mixed and homogenized to obtain lipid nanoparticles; wherein: Phase A includes cationic lipids, ionizable lipids, phospholipids, cholesterol, 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol, and solvents; Phase B includes nucleic acid drugs and acidic buffer solution; (2) The cancer cell sample was diluted using phase C, then centrifuged and the supernatant was discarded to obtain a precipitate; wherein: Phase C is HEP buffer; (3) The precipitate was resuspended using phase D, frozen, thawed, and then centrifuged and resuspended sequentially to obtain the purified cancer cell membrane; wherein: Phase D is a lysis buffer solution; (4) The lipid nanoparticles and the purified cancer cell membrane are mixed and homogenized to obtain the cancer cell membrane-encapsulated lipid nanoparticles for nucleic acid drug delivery.

2. The preparation method according to claim 1, characterized in that, In step (1), phase A comprises the following raw materials in parts by weight: 15-45 parts cationic lipids, 15-45 parts ionizable lipids, 5-20 parts phospholipids, 20-40 parts cholesterol, and 0.5-1.5 parts 1,2-dimyristic-rac-glycerol-3-methoxy polyethylene glycol. And / or, in phase B, the concentration of the nucleic acid drug is 50-500 μg / mL, and the acidic buffer is a citrate-sodium citrate buffer with pH=4; And / or, the volume ratio of phase A to phase B is 1:3 to 5.

3. The preparation method according to claim 1 or 2, characterized in that, The cationic lipid is one of (2,3-dioleoxypropyl)trimethylammonium chloride, 1,2-bisoctadecenoxy-3-methylammonium propane, and DC-cholesterol. And / or, the ionizable lipid is one of ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 4-(N,N-dimethylamino)butyrate (dilinoleyl)methyl ester, and 1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate; And / or, the solvent is ethanol; And / or, the nucleic acid drug is one of mRNA, siRNA, and ASO.

4. The preparation method according to claim 1, characterized in that, In step (1), the mixing and homogenization is one of the following: microfluidic process, membrane extrusion process, and ultrasonic treatment process; wherein: In the microfluidic process, the flow rate of phase A is 3 to 7 mL / min, and the flow rate of phase B is 3 to 5 times that of phase A. In the membrane extrusion process, filter membranes with pore sizes of 400 nm and 200 nm are used sequentially, and phase A and phase B are mixed and extruded repeatedly for 9 to 11 cycles. In the ultrasonic treatment process, after mixing phase A and phase B, ultrasonic treatment is performed for 5 to 15 minutes using an ultrasonic water bath device with a power of 200 to 1000 kW.

5. The preparation method according to claim 1, characterized in that, In step (2), the cancer cells are one of liver cancer cells, lung cancer cells, or breast cancer cells; And / or, each 500 mL of the HEP buffer contains 4.091 g NaCl, 0.101 g KCl, 0.453 g HEPES, and 0.546 g EGTA, and then the pH of the HEP buffer is adjusted to 7.0–7.4 using hydrochloric acid or sodium hydroxide; And / or, the dilution to a cell density of 0.1–0.2 × 10⁻⁶ 8 cells / mL And / or, the centrifugation is performed at 500g for 10 min.

6. The preparation method according to claim 1, characterized in that, In step (3), the lysis buffer contains 100 mM EGTA, and the pH of the lysis buffer is adjusted to 7.2 using hydrochloric acid or sodium hydroxide; And / or, the cell density after resuspension is 0.5–1 × 10⁻⁶. 8 per mL.

7. The preparation method according to claim 1, characterized in that, In step (4), the ratio of the total lipid concentration in the lipid nanoparticles to the protein concentration in the purified cancer cell membrane is 10:1 to 2:

1. And / or, the mixing and homogenization is one of the following processes: microfluidic process, membrane extrusion process, and ultrasonic treatment process.

8. Cancer cell membrane-encapsulated lipid nanoparticles for nucleic acid drug delivery obtained by the preparation method according to any one of claims 1 to 7.

9. The use of the cancer cell membrane-encapsulated lipid nanoparticles for nucleic acid drug delivery as described in claim 8 in the preparation of cancer therapeutic drugs.

10. The application according to claim 9, characterized in that, The drug is an injectable form.

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