A composite lipid nanoparticle and methods of making and using the same

Abstract

The application discloses a kind of composite lipid nanoparticles and preparation method and application thereof.The composite lipid nanoparticles include biotinylated LNP, biotinylated antibody protein and streptavidin for the connection of two, the ratio between biotinylated LNP, streptavidin and biotinylated antibody protein is 1:1:0.2 molar ratio.The preparation method is stable in process, coupling efficiency is high, biotin and streptavidin are mildly reacted, can greatly protect antibody activity, one streptavidin has four binding sites with biotin, can be combined multiple biotinylated LNP and biotinylated antibody protein, realize plug and play, signal amplification effect, has good application prospect in biomedical detection, drug delivery and other fields.

Description

Technical Field

[0001] This invention relates to the field of lipid nanoparticle biotechnology, and in particular to a composite lipid nanoparticle, its preparation method, and its application. Background Technology

[0002] Lipid nanoparticle (LNP) encapsulation technology for mRNA delivery has demonstrated profound application value and development potential in fields such as vaccine development and gene therapy. However, during LNP circulation in vivo, serum proteins rapidly adsorb onto its surface, forming a "biomolecular crown," which directly determines the organ-selective targeting characteristics of LNP. Among them, apolipoprotein E (ApoE) is an important component of the biomolecular crown, mainly synthesized by hepatocytes and most abundant in the liver, making it highly effective for LNP to target liver tissue after systemic administration.

[0003] Currently, in clinical applications, LNPs require precise targeted delivery to non-hepatic tissues, primarily through two technical approaches: First, optimizing LNP formulations by screening specific components and adjusting their proportions to achieve selective organ targeting. For example, the reported Selective Organ Targeting (SORT) method achieves tissue-specific mRNA delivery by adding a fifth component. Second, modifying LNP surface properties by conjugating antibodies or other targeting molecules to the LNP surface to enhance its targeting ability. The maleimide-thiol chemical conjugation "post-insertion" method is a classic antibody conjugation technique. Imbuing LNPs with precise targeting capabilities, enabling them to specifically reach target tissues, organs, or cells in vivo, is crucial for the development of in situ cell therapy. Unmodified LNPs, during circulation, are mostly cleared by immune cells in the liver and spleen, making it difficult to meet the needs of active targeted therapy.

[0004] To address this issue, LNPs need to be conjugated to antibodies with high specificity and high affinity to achieve maximum enrichment of LNPs in target organs or cells, thereby reducing the dosage and minimizing adverse reactions.

[0005] Currently, extensive research and development have been conducted on methods for linking LNPs to antibodies. Conventional chemical conjugation methods include thiol-maleimide reactions, carbodiimide reactions, and disulfide bond exchange reactions. These methods are relatively mature and low-cost, but they have significant drawbacks: they require antibody engineering, the chemical reactions are random, there are many side reactions, they have stringent reaction conditions, and their stability is poor. Meanwhile, some novel linking technologies are in the early stages of research. For example, linking to the antibody Fc fragment holds promise for precise multi-target delivery, but this technology relies on antibodies with high binding affinity, and targeting the Fc antibody fragment carries potential immunogenicity risks. Summary of the Invention

[0006] The present invention addresses the aforementioned problems by providing a composite lipid nanoparticle, its preparation method, and its application.

[0007] The present invention adopts the following technical solution: a composite lipid nanoparticle, wherein the composite lipid nanoparticle comprises biotinylated LNP, biotinylated antibody protein, and streptavidin for the connection of the two; The biotinylated LNP includes ionizable cationic lipids, cholesterol, distearylphosphatidylcholine, and distearylphosphatidylethanolamine-polyethylene glycol-biotin; The biotinylated antibody protein is a biotinylated anti-human CD5 antibody.

[0008] Furthermore, the preparation method of biotinylated LNP is as follows: (1) Preparation of organic phase: Dissolve ionizable cationic lipids, cholesterol, distearate phosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol-biotin in anhydrous ethanol at a mass ratio of 50:37.5:10:2.5 to obtain organic phase; (2) Aqueous phase preparation: Calculate the required amount of mRNA according to the molar ratio of amino N to phosphate P of 8:1, dissolve the mRNA in sodium citrate solution to obtain the aqueous phase; (3) LNP preparation: The organic phase and aqueous phase were mixed at a volume ratio of 1:3 and a flow rate of 1.5 min. -1 LNPs were prepared by mixing using a microfluidic device. (4) Purification and preservation: Disperse the LNP obtained in step (3) in sodium citrate solution, gradually replace it with TBS solution through a 100 kDa ultrafiltration tube and concentrate it for preservation.

[0009] A method for preparing composite lipid nanoparticles, comprising mixing biotinylated LNP, streptavidin and biotinylated anti-human CD5 antibody in a molar ratio of 1:1:0.2, incubating overnight and then shaking to obtain composite lipid nanoparticles.

[0010] Furthermore, the incubation temperature is 4°C.

[0011] A method for preparing composite lipid nanoparticles, wherein the method comprises first mixing biotinylated LNP and streptavidin at a 1:1 molar ratio and incubating for the first time, and then adding 1 / 5 molar ratio of biotinylated anti-human CD5 antibody for the second incubation to obtain composite lipid nanoparticles.

[0012] Furthermore, the first incubation temperature of the biotinylated LNP with streptavidin is room temperature and the incubation time is 30 min, and the second incubation temperature is 4 °C and the incubation time is 1 h.

[0013] An RNA vaccine comprising the composite lipid nanoparticles of claim 1.

[0014] A drug comprising the composite lipid nanoparticles of claim 1.

[0015] Beneficial effects of this invention: This invention modifies LNPs and antibodies by biotinylation, and forms an "irreversible" link between LNPs and antibodies through streptavidin. This method has a relatively stable process and extremely high coupling efficiency. Moreover, this binding method effectively solves the shortcomings of insertion instability in insertion methods. At the same time, the reaction between biotin and streptavidin is mild, which can greatly protect the antibody activity. One streptavidin has four binding sites with biotin, which can bind to multiple biotinylated LNPs and antibodies, achieving plug-and-play and signal amplification effects. Attached Figure Description

[0016] Figure 1 This is a scatter plot of flow cytometry data from Example 4 of the present invention. Figure 2 This is a fluorescence intensity histogram of flow cytometry data from an embodiment of the present invention. Detailed Implementation

[0017] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0018] In this embodiment of the invention, the biotinylated anti-human CD5 antibody was purchased from BioLegend; the ionizable cationic lipid SM102 was purchased from MCE.

[0019] In this embodiment of the invention, the T-cell sorting kit was purchased from Stemcell.

[0020] Example 1: Preparation of biotinylated LNP, including the following steps: (1) 1.02 mg SM102, 0.42 mg cholesterol, 0.23 mg DSPC, 0.11 mg DMG-PEG2000 and 0.029 mg DSPE-PEG2000-mal were added sequentially and dissolved in anhydrous ethanol to prepare an organic phase. The amount of mRNA required was calculated based on the ratio of positively charged amino N to negatively charged phosphate P. 60 mg mRNA was dissolved in sodium citrate solution to prepare an aqueous phase. The volume ratio of organic phase to aqueous phase was 1:3 and the flow rate of the two phases was 1.5 min. LNP was prepared using a microfluidic device. (2) The prepared LNP was uniformly dispersed in sodium citrate solution and gradually replaced from sodium citrate solution to a mixed solution of sodium citrate and TBS using a 100 kDa ultrafiltration tube. Finally, it was completely concentrated in TBS solution and stored at 4°C for a short period of time.

[0021] Example 2: A method for preparing CD5-LNP-mRNA, comprising the following steps: (1) Mix biotinylated LNP, streptavidin and biotinylated anti-human CD5 antibody in a molar ratio of 1:1:0.2, incubate overnight at 4°C, and shake to mix.

[0022] (2) All modified LNPs were removed from unbound components using a 100kDa ultrafiltration tube and concentrated to the original sampling volume. They were then stored at 4℃ for a short period to obtain CD5-LNP-mRNA.

[0023] Example 3: A method for preparing CD5-LNP-mRNA, comprising the following steps: (1) Mix biotinylated LNP and streptavidin in a 1:1 molar ratio, incubate at room temperature for 30 minutes, and gently shake to avoid precipitation; (2) Use a 100 kDa ultrafiltration tube to remove streptavidin that is not bound to LNP; (3) Add 1 / 5 molar ratio of biotinylated anti-human CD5 antibody and incubate at 4° for 1 hour. Shake to mix well and prepare CD5-LNP-mRNA.

[0024] The CD5-LNP-mRNAs prepared in Examples 2 and 3 were characterized, and the characterization results are shown in Table 1. Table 1. LNP-mRNA characterization results

[0025] As shown in Table 1, the CD5-LNP-mRNA prepared using the method in Example 3 has a small particle size and a PDI value of less than 0.2, which is more in line with the optimal delivery size of LNP.

[0026] Example 4: The expression rate of CD5-LNP-mRNA transfected into T cells with CAR prepared in Example 3 was detected; the blank group, SM102, SM102-biotinn and IgG-SM102 were used as control groups.

[0027] I. T cell sorting and activation: (1) PBMC cell resuscitation: PBMC cells were taken and resuscitated at 37°C; (2) Centrifugation: Add the resuscitated PBMC cells to EasySep Buffer and mix well. Centrifuge at 500xg speed, 5°C increment / 5°C mode for 8 min, and then count the cells. (3) Cell resuspension and incubation: Resuspend the cells to 5E7 / mL, add Isolation Cocktail to the sample, mix well and incubate at room temperature for 5 min; (4) Magnetic bead separation: Add RapidSpheres to the sample and mix well. Place the test tube in a magnetic rack and incubate at room temperature for 10 min. Carefully transfer the enriched cell suspension to a new centrifuge tube using a pipette. (5) Second round of separation: Remove the test tube from the magnet, add 5 mL of new EasySep Buffer, continue incubation for 10 min, and perform the second round of magnetic bead separation; (6) Activation culture: Add 1640 complete culture medium, centrifuge and count the cells, adjust the cell density to 1E6 / mL; add CD3 / CD28 activation beads (12.5μL per 1E6 cells), place the cells in a CO2 incubator and activate for 48h, then set aside.

[0028] II. CD5-LNP-mRNA transfection and CAR positivity rate detection: (1) LNP pretreatment: CD5-LNP encapsulated with CAR mRNA was quantified. Before transfection, LNP was concentrated using a 100kDa ultrafiltration tube and replaced in 1640 medium. (2) Transfection procedure: The pretreated LNP was added to the activated T cells at a ratio of 0.5 μg / 1E5 T cells; (3) Incubation and detection: After 6 hours of incubation, the CAR positivity rate was detected by flow cytometry.

[0029] The results are as follows Figure 1 and Figure 2 It can be seen that the CD5-LNP-mRNA prepared by the method of the present invention has a CAR positivity rate of 17.6% when transfecting T cells, which is better than the transfection effect of LNP conjugated with isotype control IgG antibody and LNP without antibody conjugation.

[0030] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A composite lipid nanoparticle, characterized in that: The composite lipid nanoparticles include biotinylated LNP, biotinylated antibody protein, and streptavidin for linking the two. The biotinylated LNP includes ionizable cationic lipids, cholesterol, distearylphosphatidylcholine, and distearylphosphatidylethanolamine-polyethylene glycol-biotin; The biotinylated antibody protein is a biotinylated anti-human CD5 antibody.

2. The composite lipid nanoparticles according to claim 1, characterized in that: The preparation method of the biotinylated LNP is as follows: 1) Preparation of organic phase: Dissolve ionizable cationic lipids, cholesterol, distearate phosphatidylcholine, distearate phosphatidylethanolamine-polyethylene glycol-biotin in anhydrous ethanol at a mass ratio of 50:37.5:10:2.5 to obtain the organic phase; (2) Aqueous phase preparation: Calculate the required amount of mRNA according to the molar ratio of amino N to phosphate P of 8:1, dissolve the mRNA in sodium citrate solution to obtain the aqueous phase; (3) LNP preparation: The organic phase and aqueous phase were mixed at a volume ratio of 1:3 and a flow rate of 1.5 min. -1 LNPs were prepared by mixing using a microfluidic device. (4) Purification and preservation: Disperse the LNP obtained in step (3) in sodium citrate solution, gradually replace it with TBS solution through a 100 kDa ultrafiltration tube and concentrate it for preservation.

3. The method for preparing composite lipid nanoparticles according to claim 1, characterized in that: The preparation method involves mixing biotinylated LNP, streptavidin, and biotinylated anti-human CD5 antibody in a molar ratio of 1:1:0.2, incubating overnight, and then shaking to mix and obtain composite lipid nanoparticles.

4. The method for preparing composite lipid nanoparticles according to claim 3, characterized in that: The incubation temperature is 4°C.

5. The method for preparing composite lipid nanoparticles according to claim 1, characterized in that: The preparation method involves first mixing biotinylated LNP and streptavidin at a 1:1 molar ratio and incubating for the first time, then adding 1 / 5 molar ratio of biotinylated anti-human CD5 antibody and incubating for the second time to obtain composite lipid nanoparticles.

6. The method for preparing composite lipid nanoparticles according to claim 5, characterized in that: The first incubation of biotinylated LNP with streptavidin was at room temperature for 30 minutes, and the second incubation was at 4°C for 1 hour.

7. An RNA vaccine, characterized in that: The RNA vaccine comprises the composite lipid nanoparticles as described in claim 1.

8. A drug, characterized in that: The drug comprises the composite lipid nanoparticles as described in claim 1.

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