Lipid nanoparticle, application thereof and drug delivery carrier

Abstract

The invention belongs to the field of biological medicine, and particularly relates to lipid nanoparticles, application thereof and a drug delivery carrier. The lipid nanoparticle comprises an ionizable lipid, cholesterol, phospholipid, a pegylated lipid and a sterol substitute, and is characterized in that the ionizable lipid is Lipid 5, the phospholipid is DSPC, the pegylated lipid is 18: 0 mPEG2000 PE, and the sterol substitute is one or more of beta-sitosterol, stigmasterol or stigmasterol or a derivative thereof; the molar ratio of the ionizable lipid to the cholesterol to the phospholipid to the pegylated lipid to the sterol substitute is (40-60): (5-38.5): (5-30): (1-2): (5-38.5). According to the invention, the LNP with unique physicochemical properties is constructed through high synergy of the components, the technical problems of low delivery efficiency and poor specificity of hematopoietic stem progenitor cells in the prior art are solved, and high-efficiency and high-specificity drug delivery of HSPCs in vivo and in vitro is realized. The invention also provides a drug delivery carrier containing the lipid nanoparticles and a preparation method thereof.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and particularly to a drug delivery carrier of lipid nanoparticles, especially to drug delivery in vivo and in vitro to hematopoietic stem cells. Background Technology

[0002] Hematopoietic stem and progenitor cells (HSPCs) proliferate and differentiate to produce blood cells and the vast majority of immune cells in the human body, playing a vital role in maintaining essential physiological functions such as promoting hematopoiesis, immune regulation, and tissue repair and regeneration. Disorders of the hematopoietic system can lead to various pathological conditions, including sickle cell disease, β-thalassemia, primary immunodeficiency, and bone marrow and blood-related tumors. These diseases typically originate from HSPC gene mutations, which, as they proliferate and differentiate, affect normal physiological functions. However, with the continuous development of gene therapy and gene editing technologies, new hope has emerged for treating hematopoietic system-related gene defects such as sickle cell disease. For example, in vivo delivery of deoxyribonucleic acid (DNA) or messenger RNA (mRNA) can achieve long-term or transient expression of specific genes, compensating for related defects; delivery of small interfering RNA (siRNA) can specifically mediate the degradation of mRNA of specific genes, thereby inhibiting their abnormal expression or overexpression; co-delivery of Cas9 protein mRNA and unidirectional guide RNA (sgRNA) of specific genes can precisely edit their sequences, achieving DNA-level repair of defective genes. By introducing the above-mentioned exogenous nucleic acid drugs in vivo, precise regulation of defective genes can be achieved through different strategies, effectively treating hematopoietic system-related gene diseases while avoiding or reducing the use of high-side-effect therapies such as chemical drugs.

[0003] However, the negative charge and large molecular size of nucleic acid drugs make it difficult for them to overcome physiological barriers such as cell membranes, intercellular spaces, and tissue barriers to enter the cytoplasm and exert their effects. Simultaneously, their exogenous nature makes them easily recognized and cleared by the immune system, inducing inflammation. Nucleic acid drugs, especially RNA drugs, also suffer from poor stability, requiring stringent storage conditions and prone to inactivation or degradation before use. Therefore, in actual therapeutic production, to address these issues, nanoparticle carriers are generally used to encapsulate and protect nucleic acid drugs, while simultaneously assisting in delivery and transfection. Nucleic acid nanoparticle carriers are generally divided into viral and non-viral types. Viral delivery carriers, such as lentiviruses (LV) and adeno-associated viruses (AAV), pose risks in terms of immunogenicity and mutagenicity, and are limited in delivery efficiency, transformation, and production. Non-viral carriers, especially synthetic carriers, are more suitable for use in nucleic acid-based gene editing therapies due to their relatively lower biosafety risks, biodegradability, ease of functionalization, and large-scale production. Lipid nanoparticles (LNPs) are a representative example of non-viral nucleic acid delivery carriers. LNPs are generally composed of both biologically derived and artificially produced lipids, ensuring both low immunogenicity and the potential for functional modification, and they have a relatively mature large-scale production process. Since their clinical approval as a delivery vector for RNA vaccines during the COVID-19 pandemic, the application prospects of LNPs in the biomedical field have attracted widespread attention.

[0004] To further improve the delivery efficiency of LNPs, numerous explorations have been conducted in this field. For example, WO2021026358A1 discloses an LNP composition for enhancing drug delivery, in which the document discloses that the combination of I-301 / I-321 / I-182 / I-49 with P-428, β-sitosterol and cholesterol, and DSPC can enhance the delivery efficiency to hepatocytes and spleen cells. However, although this approach has made progress in enhancing the targeted delivery of LNPs to specific organs such as the liver and spleen, it is not applicable to the specific target of HSPCs. The main challenges in HSPC delivery are: (1) Poor physiological accessibility: For example, the liver is a sinusoidal organ and the spleen is a blood filter, and LNPs are easily enriched in these organs. However, HSPCs are mainly located in the bone marrow and are protected by the "blood-bone marrow barrier," making them difficult for conventional LNPs to reach. Furthermore, HSPCs are located in areas with high concentrations of other bone marrow cells, making accurate identification very difficult. Even when HSPCs are temporarily mobilized into the peripheral blood using mobilizing agents, the complex blood circulation contains many types of blood cells and immune cells, making efficient and specific gene and drug delivery to them extremely difficult.

[0005] (2) Differences in phagocytic activity: Hepatocytes highly express receptors such as LDLR and are prone to endocytosis of LNPs. HSPCs are mostly in a metabolically inactive resting phase, and their endocytic capacity is much weaker than that of professional phagocytic cells such as hepatocytes.

[0006] (3) Poor cell tolerance: HSPCs are less tolerant to the toxicity of transfection process and related agents than cells such as hepatocytes.

[0007] Therefore, transfecting HSPCs is like special operations, requiring overcoming their low endocytosis and low translational activity, accurately identifying targets in complex blood circulation or densely packed other bone marrow cells, and safely delivering and efficiently expressing drugs without causing damage to HSPC target cells and maintaining cell viability. This is something that existing universal solutions for delivery to the liver and other organs cannot accomplish. Summary of the Invention

[0008] Therefore, the purpose of this invention is to solve the technical problem of efficient and specific delivery of LNPs to hematopoietic stem and progenitor cells (HSPCs) in the prior art. This invention is not a simple replacement or optimization of existing LNP components, but rather provides a highly synergistic formulation combination of components to construct an LNP with novel physicochemical properties and biological functions, achieving efficient and specific nucleic acid delivery and protein expression to HSPCs.

[0009] The objective of this invention is achieved through the following measures: A lipid nanoparticle (LNP) for hematopoietic stem and progenitor cells comprises ionizable lipids, cholesterol, phospholipids, polyethylene glycol-modified lipids, and sterol substitutes, characterized in that: the ionizable lipid is Lipid 5, the phospholipid is DSPC, the polyethylene glycol-modified lipid is 18:0 mPEG2000 PE, and the sterol substitute is one or more of β-sitosterol, stigmasterol, or stigmasterol or their derivatives; the molar ratio of ionizable lipids: cholesterol: phospholipids: polyethylene glycol-modified lipids: sterol substitutes is 40-60: 5-38.5: 5-30: 1-2: 5-38.5.

[0010] Under electron microscopy, the aforementioned lipid nanoparticles exhibit a bilayer membrane structure on the outer layer, with some parts displaying a polyhedral structure. The core of this invention lies in the specific, synergistic combination of the various components of the LNP, whose overall function is far more than a simple sum of the effects of its individual parts, manifested in: Structural synergy: A specific combination of components—Lipid 5, 18:0 mPEG2000 PE, cholesterol, β-sitosterol / stigmasterol / stigmasterol or their derivatives, and DSPC—is employed, with each component's dosage controlled within a specific ratio. Furthermore, the molar ratio of cholesterol to sterol substitutes is maintained at a specific ratio (preferably 3:1), thereby constructing a LNP with a unique physical morphology (as observed under an electron microscope as a polyhedral structure). This forms the physical basis for achieving subsequent synergistic biological functions.

[0011] Biological synergy: This invention discovers that the LNP with the unique structure described above can form a novel biological complex upon binding with apolipoprotein E (ApoE). This complex can be efficiently recognized and internalized by certain specific receptors on the surface of HSPCs, and it also facilitates the efficient expression of mRNA in HSPCs.

[0012] The molecular formula of the above-mentioned Lipid 5 is C 44 H 87 NO5, CAS No. 2089251-33-0. The above DSPC is 1,2-Distearoyl-sn-glycero-3-phosphocholine, with the Chinese name 1,2-distearateyl-sn-propyltriyl-3-phosphocholine. The above 18:0 mPEG2000 PE is a polyethylene glycol-modified lipid with the molecular formula (C2H4O)nC. 43 H 84 NO 10 P·H3N, CAS No.: 474922-77-5. The above-mentioned derivatives refer to derived sterols formed by attaching an electron acceptor / donor (such as a carboxylic acid, sulfonic acid, secondary amine, tertiary amine group, etc.), a lipid chain (such as a simple linear lipid chain such as stearate or palmitate, or a more complex lipid structure with branched structures, degradable ester bonds, ionizable amino groups, etc.), or a functional polysaccharide / peptide (such as hyaluronic acid, chitosan, or other targeted peptides) to the C-3 hydroxyl group common to β-sitosterol, stigmasterol, or stigmasterol.

[0013] Preferably, the molar ratio of the sterol substitute in this invention is 3:1. More preferably, the molar ratio of Lipid 5:cholesterol:DSPC:18:0 mPEG2000 PE:sterol substitute is 50:28.8:10:1.5:9.6.

[0014] Another object of the present invention is to provide a drug delivery carrier specifically for HSPCs and a method for encapsulating the same.

[0015] The hematopoietic stem cell drug delivery carrier contains the aforementioned lipid nanoparticles. This carrier fully utilizes the high efficiency and specificity of the aforementioned LNPs for HSPCs in vitro and in vivo.

[0016] The preparation method of the above-mentioned hematopoietic stem cell drug delivery carrier includes: dispersing the lipid component of lipid nanoparticles and the mRNA drug in anhydrous ethanol organic phase and in an aqueous phase of pH 4.0 citrate-sodium citrate buffer solution, respectively.

[0017] Preferably, the organic phase concentration is 1.45 mM, and the molar ratio of ionizable lipid (Lipid 5) to RNA in the lipid nanoparticles is 6:1.

[0018] Preferably, lipid nanoparticles encapsulating mRNA are synthesized by microfluidic mixing of organic and aqueous phases, with an organic phase to aqueous phase volume ratio of 1:3 and a total flow rate of 12 mL / min.

[0019] Preferably, the lipid nanoparticles were diluted with 20 times the synthesis volume of 1×PBS buffer solution, and then subjected to solvent exchange and volume concentration in Amicon Ultra 30kDa ultrafiltration centrifuge tubes. After completion, the nanoparticles were stored at 4°C.

[0020] Specifically, the encapsulation method for the aforementioned drug delivery carrier includes the following steps: Step 1: Dissolve the above lipid nanoparticles in anhydrous ethanol organic phase, and dissolve the mRNA drug in a citrate-sodium citrate buffer solution aqueous phase at pH 4.0; Step 2: With an organic phase concentration of 1.45 mM (total lipid molar concentration), the lipid nanoparticles and mRNA drug were mixed at a ratio of 6:1 (molar ratio of Lipid 5 to mRNA); Step 3: Lipid nanoparticles encapsulating mRNA were synthesized by microfluidic mixing of organic and aqueous phases, with a lipid-to-aqueous phase volume ratio of 1:3 and a total flow rate of 12 mL / min. Step 4: Dilute the lipid nanoparticles with 20 times the synthesis volume of 1×PBS buffer solution, and perform solvent exchange and volume concentration in Amicon Ultra 30kDa ultrafiltration centrifuge tubes. Store at 4°C after completion.

[0021] In some preferred embodiments, the drug delivery carrier further includes apolipoprotein E (ApoE), which can form protein-nucleic acid nanoassemblies. Co-incubating the LNP of the present invention with ApoE for in vitro application can significantly and synergistically enhance the targeted delivery efficiency and specificity to HSPCs.

[0022] In other preferred embodiments, particularly for in vivo application, the lipid nanoparticles are also coupled with targeting ligands, such as antibodies coupled via DSPE-PEG-Mal that target surface markers of HSPCs (e.g., c-Kit / CD117). This active targeting strategy, synergistic with the aforementioned intrinsic biological properties of LNPs, overcomes complex in vivo physiological barriers, enabling precise navigation and efficient delivery of HSPCs to the bone marrow.

[0023] The aforementioned drug delivery vectors primarily deliver nucleic acid drugs, such as mRNAs like enhanced green fluorescent protein mRNA (eGFP mRNA, modified with N1-methylpseudouridine), nucleic acid editing systems (such as Cas9 protein mRNA + sgRNA), small interfering RNA (siRNA), oligonucleotides, etc.

[0024] The present invention has the following beneficial effects: This invention achieves highly efficient and specific delivery to hematopoietic stem and progenitor cells (HSPCs), a target cell type that is extremely difficult to transfect. Existing technologies and commercially available transfection reagents have extremely low transfection efficiency for HSPCs (e.g., ...). Figure 1 and Figure 7 As shown, the percentage is typically below 5%, and at most not exceeding 40%. However, the optimized LNP formulation of this invention, in synergy with ApoE, can increase the in vitro transfection efficiency of human CD34+ HSPCs to nearly 90%. Even more encouragingly, this delivery exhibits extremely high cell specificity, producing almost no transfection in the predominantly CD34- non-target cells in the mixed cell line (e.g., ...). Figure 8 (As shown). This demonstrates that the present invention is a novel delivery carrier capable of intelligently identifying and targeting HSPCs.

[0025] This invention solves the challenge of in vivo delivery of HSPCs. By combining the LNP of this invention with a targeting antibody, it successfully achieves efficient gene delivery of HSPCs deep in the bone marrow in a live mouse model via simple intravenous injection. Figure 9 As shown, this protocol achieved an 88% LNP uptake rate and a 41.9% functional protein expression rate in LSK cells (HSPCs-enriched cell population). Furthermore, this protocol can efficiently transfect various HSPC subsets, including the most primitive long-term hematopoietic stem cells (LT-HSCs) capable of lifelong hematopoiesis. This demonstrates the great potential of this invention to achieve "in vivo gene editing" and radically cure a variety of major hematological and immune diseases, potentially revolutionizing traditional complex and expensive ex vivo cell therapy modalities.

[0026] This invention is not a single-purpose tool, but a universal delivery platform for HSPCs with "plug-and-play" characteristics. The LNP formulation of this invention can be used for efficient in vitro cell modification by binding with ApoE, and can also be used for precise in vivo therapy by conjugating different ligands. This vector can carry a variety of nucleic acid drugs, such as mRNA for gene substitution, the CRISPR-Cas9 system for gene editing, and siRNA for gene regulation. It provides a powerful and flexible tool for tackling a series of major diseases caused by HSPC defects (such as sickle cell disease, β-thalassemia, and immunodeficiency diseases), and has extremely broad clinical application prospects. Attached Figure Description

[0027] Figure 1 The results of in vitro transfection of HEK293 cell line and primary hematopoietic stem and progenitor cells in human umbilical cord blood mononuclear cells (CBMCs) using the basic formulation LNP, LNP+serum and commercial reagent LipoMAX (Thermo) in Example 1 are shown.

[0028] Figure 2 Adding ApoE to Example 1 can enhance the HSPC delivery expression rate of LNPs in the basic formulation for delivering human primary HSPCs.

[0029] Figure 3 The table shows the physicochemical characteristics of different cholesterol substitutes for LNPs in Example 2, including hydration particle size, polydispersity index, encapsulation efficiency, and screening results for delivery effects of transfected human HSPCs.

[0030] Figure 4 Transmission electron microscopy analysis of the β-sitosterol LNP sample with high delivery efficiency for Example 2.

[0031] Figure 5 This study aims to compare and screen the delivery-promoting effects of different plant-derived sterol substitution LNP formulations on transfected human HSPCs in Example 3, and to investigate the influence of plant-derived sterol substitution rate and in vitro transfection drug concentration on the delivery-promoting effect.

[0032] Figure 6 The effect of adding apolipoprotein during the transfection process on the transfection efficiency of LNP human HSPCs in vitro, as described in Example 3.

[0033] Figure 7 The LNP formulation optimized for Example 3 and the apolipoprotein transfection method were used to assess the transfection effect on human HSPCs in vitro.

[0034] Figure 8 The nucleic acid delivery method optimized for Example 3 demonstrates the in vitro transfection specificity of different primary HSPCs in human CBMCs.

[0035] Figure 9 For the in vivo delivery application study of the optimized LNP in a mouse model in Example 4, n=5 [A. Flow cytometry results of mouse bone marrow LSK cells (containing hematopoietic stem cells (HSCs) and pluripotent progenitor cells (MPPs)); B. DiR and GFP positivity rates of different cells]. Detailed Implementation

[0036] The following experimental examples are further illustrative of the present invention and are not intended to limit the invention. Specific experimental conditions and methods are not specified in the following examples. Experimental methods not specified in the following examples are performed according to conventional methods and conditions, or as selected according to the product instructions. In this patent, "h" represents hours and "min" represents minutes.

[0037] Example 1: Optimization of LNP Formulation LNP transfection cell efficiency assay Experimental materials: The basic LNP formula (common LNPs represented by Pfizer's vaccine LNP formula) consists of the following components: ALC-0315 (GLPBIO): Cholesterol (Averestol): DSPC (Averestol): ALC-0159 (Averestol) = 50:38.5:10:1.5 (molar ratio).

[0038] Commercial reagent LipoMAX.

[0039] The mRNA is eGFP mRNA (N1-methylpseuuridine modified, MCE, GenScript).

[0040] The experimental groups are as follows: Basic formulation LNP group: LNP + eGFP mRNA. ALC0315: mRNA molar ratio of 6, total lipid:mRNA molar ratio of 12. Dosage in HEK293 cells: 150 ng mRNA / well, concentration of 300 ng mRNA / mL; Dosage in human CBMCs: 2 μg mRNA / well, concentration of 10 μg mRNA / mL.

[0041] Basic formulation LNP+serum group: LNP+eGFP mRNA+serum; serum 10v / v%, other dosages are the same as basic formulation LNP group.

[0042] LipoMAX group: LipoMAX+ eGFP mRNA; dosage same as the basic formulation LNP group.

[0043] Before cell transfection, mRNA-LipoMax transfection reagent was synthesized according to the instructions of the Lipofectamine Messenger Max kit.

[0044] Dosage: LipoMAX (uL): mRNA (ug) = 3:2.

[0045] Dilute the mRNA in 100uL of Opti-MEM (Gibco) medium according to the dosage (synthesis dosage = transfection dosage).

[0046] Dilute the LipoMAX transfection reagent in 100 μL of Opti-MEM medium and vortex for 2-3 seconds.

[0047] Add the mRNA dilution buffer to LipoMAX dilution buffer (1:1 volume ratio), mix well and incubate for 5 minutes, then load the sample onto the cells.

[0048] The experimental groups were transfected in vitro with HEK293 cell line and primary hematopoietic stem and progenitor cells from human CBMC, and the transfection results were detected.

[0049] In vitro transfection methods: LNP transfected human CBMC samples The cell culture medium used for transfection was SFEM-II medium, with additional additions of appropriate amounts of human cytokines SCF, TPO, and FLT-3.

[0050] Transfection procedure: Gently pipette CBMC cells, collect cell suspension using culture medium, centrifuge at 500g for 5 minutes at room temperature (adjust speed to the slowest setting), resuspend and count, then centrifuge at 5×10⁻⁶. 5 Cells were seeded per well in a 96-well plate. Then, appropriate components were added sequentially according to the experimental group, such as LNP samples (e.g., 2 μg mRNA / well LNP, the amount of mRNA encapsulated in the LNP was detected in the encapsulation efficiency assay), with a final culture medium volume of 200 μL per well. After incubation in a CO2 incubator for 48 h, flow cytometry was used to detect cell viability, as well as the eGFP expression rate and intensity in different cell types.

[0051] • LNP transfection of HEK293 cells The transfection medium used was DMEM medium, with an additional 10 v / v% fetal bovine serum (FBS) and 2% penicillin antibody (PS).

[0052] Gently pipette the cell sample, collect the cell suspension using culture medium, centrifuge at 800g for five minutes at room temperature (adjust the speed to the slowest setting), resuspend, count, and then centrifuge at 1×10⁻⁶. 5 Seed cells per well in a 24-well plate and place in a CO2 incubator. Observe cell proliferation and adhesion after 24 hours. Once cells have covered 70-90% of the well area and adhered well, proceed with transfection.

[0053] Basic formulation LNP group: Mix the encapsulated basic formulation LNP with the culture medium at a concentration of 500 ng mRNA / mL, remove the old culture medium from the wells, and add 500 uL of culture medium containing LNP to each well (LNP dosage is 150 ng mRNA / well).

[0054] LipoMAX group: As a control, LipoMAX was prepared with eGFP mRNA according to the kit instructions to form a transfection reagent, and transfection was performed at the same concentration and with the same steps as the LNP group with the same basic formula.

[0055] Basic formulation LNP + serum group: When LNP is mixed with culture medium, 10% (v / v) of serum is added to the serum group.

[0056] After incubation in a carbon dioxide incubator for 24 hours, the expression rate and intensity of eGFP in the cells were detected by flow cytometry.

[0057] Experimental results: such as Figure 1 The results showed that both the basic LNP formulation and the commercial LipoMAX transfection reagent could effectively transfect cell lines such as HEK293, but in HSPCs in CBMC, the transfection rate and protein expression intensity were much lower than those of cell line transfection.

[0058] In addition, the addition of serum has the effect of promoting LNP transfection of cell lines in vitro, but this conclusion is inconsistent in HSPCs.

[0059] Introducing additives to enhance LNP transfection efficiency 1) The effect of adding ApoE (concentration 100ug / mL) to the basic formulation LNP on the in vitro delivery of primary HSPCs in human CBMC.

[0060] Figure 2 The results showed that introducing ApoE alone into LNP could enhance the delivery and expression rate of LNP HSPCs, but the effect was still limited and could not achieve effective delivery (less than 40%). 2) Active cholesterol analogs partially or completely replace the cholesterol in the original LNP formula. To improve delivery efficiency, a strategy was adopted to partially or completely replace the cholesterol in the original LNP formulation with an active cholesterol analog to further optimize the in vitro primary HSPCs delivery efficiency of LNP.

[0061] Common components of LNP: Lipid 5 (MCE, GLPBIO), 18:0 mPEG2000 PE (MCE), cholesterol (Avita), DSPC (Avita).

[0062] The groups are as follows: The eight LNP treatment groups were divided into a cholesterol LNP group as a control and seven experimental groups in which cholesterol was partially replaced by seven different cholesterol substitutes at a replacement rate of 25 mol%.

[0063] Cholesterol LNP group (control group): The composition was Lipid 5: cholesterol: DSPC (phospholipid): 18:0 mPEG2000 PE = 50:38.5:10:1.5 (molar ratio), and the molar ratio of Lipid 5 to eGFP mRNA (N / P ratio) was 6.

[0064] The 7-group LNP group with sterol substitutes has the following composition: Lipid 5: Cholesterol:DSPC (phospholipid): 18:0 mPEG2000 PE: Sterol substitute = 50:28.8:10:1.5:9.6, with a molar ratio (N / P ratio) of 6 for Lipid 5 to eGFP mRNA. Based on the different sterol substitutes, they are divided into HCho-LNP (7α-hydroxycholesterol substitute), SS-LNP (β-sitosterol substitute), SSta-LNP (stigmasterol substitute), FS-LNP (fucosterol substitute), UA-LNP (ursolic acid substitute), GA-LNP (sodium glycochenodeoxycholate substitute), and TA-LNP (taurine deoxycholic acid substitute).

[0065] 4 μL of ApoE was added to each well in all groups.

[0066] like Figure 3 As shown, the LNP particles synthesized from the active ingredients selected in the above experimental groups all had a particle size of 75-150 nm, a PDI of less than 0.2, an encapsulation rate of nearly 100%, and no obvious cytotoxicity.

[0067] pass Figure 4 Transmission electron microscopy analysis revealed a characteristic double membrane structure on the outer layer of LNPs formed by the partial substitution of cholesterol by highly expressed β-sitosterol, stigmasterol, or stigmasterol, and the LNPs partially exhibited a polyhedral rather than a circular structure.

[0068] The selected preferred LNP formulation is an LNP formed by partially replacing cholesterol with β-sitosterol, stigmasterol, or stigmasterol, with the composition being Lipid 5: Cholesterol: DSPC: 18:0 mPEG2000 PE: Sterol substitute = 50:28.8:10:1.5:9.6 (molar ratio), and the molar ratio of Lipid 5 to eGFP mRNA (N / P ratio) is 6.

[0069] Transfection efficiency of plant-derived sterol alternatives to LNP formulations A comparative screening of the effects of different plant-derived sterols on promoting the delivery of LNP-transfected human HSPCs was conducted, and LNP formulations were optimized by replacing cholesterol with three plant-derived sterols: β-sitosterol (SS), stigmasterol (SSte), and stigmasterol (SSta).

[0070] Phytosterol LNP: The composition is Lipid 5: Cholesterol: DSPC: 18:0 mPEG2000 PE: Phytosterol = 50:28.8:10:1.5:9.6, with a Lipid 5 to eGFP mRNA molar ratio (N / P ratio) of 6. Depending on the specific phytosterol used, it is classified as SS-LNP (β-sitosterol), SSte-LNP (stigmasterol), and SSta-LNP (stigmasterol).

[0071] The results showed that all three phytosterols could effectively and specifically transfect HSPCs, with expression rates close to 80%. Based on the fluorescence intensity (MFI) of eGFP, β-sitosterol, which had the highest expression rate among the three, not only effectively increased the protein expression rate but also had a more outstanding ability to increase its expression intensity.

[0072] Considering both expression rate and expression intensity, β-sitosterol substitution was chosen as the main formulation optimization strategy.

[0073] Optimize replacement rate like Figure 5 As shown, while the delivery efficiency remained stable, the expression efficiency decreased with increasing phytosterol substitution ratio for cholesterol. A 25 mol% substitution rate was chosen for subsequent studies. We found that complete substitution was not the best approach; a 25 mol% substitution ratio was optimal.

[0074] The LNP with 25 mol% cholesterol replaced by β-sitosterol, and with the addition of ApoE during transfection, showed the best transfection efficiency. The LNP formulation was Lipid 5:cholesterol:DSPC (phospholipid):18:0 mPEG2000 PE:β-sitosterol = 50:28.8:10:1.5:9.6, with a molar ratio of Lipid 5 to eGFP mRNA (N / P ratio) of 6, and 4 μL of ApoE added per well. In CBMC samples, the transfection rate of LNPs with CD34+ hematopoietic stem and progenitor cells reached nearly 90%, while the transfection rate of LNPs without cholesterol replacement and / or without ApoE was no more than 40%, and as low as ~1%. The fluorescence intensity produced by eGFP expression reached 1.8 × 10⁻⁶. 6 The maximum LNP without substituted sterols and / or without added ApoE is 3 × 10⁻⁶. 4 The minimum is ~3000.

[0075] Investigating the effect of LNP in vitro administration concentration (ug mRNA / well) on expression. In vitro transfection: LNP transfection of human CBMC samples The culture medium used for transfection was SFEM-II medium, with the addition of appropriate amounts of human cytokines SCF, TPO, and FLT-3.

[0076] Gently pipette the cell sample, collect the cell suspension using culture medium, centrifuge at 500g for five minutes at room temperature (adjust the speed to the slowest setting), resuspend, count, and centrifuge at 5 × 10⁻⁶. 5 Cells were seeded at 1000 mRNA / well in 96-well plates, and recombinant human ApoE (and / or ApoA1) and LNP samples (2 μg, 4 μg, and 8 μg mRNA / well, respectively) were added sequentially according to experimental requirements, with a final culture medium volume of approximately 200 μL per well. After incubation in a CO2 incubator for 48 h, flow cytometry was used to detect cell viability and the expression rate and intensity of eGFP in different cell types.

[0077] Result: As Figure 5 As shown, within the dosage range of 2-8 μg mRNA / well, the drug concentration was positively correlated with the expression rate and expression intensity.

[0078] Example 2: LNP-encapsulated mRNA drug A method for preparing lipid nanoparticle-encapsulated mRNA drugs, comprising the following steps: Step 1: The lipid components of LNP were dissolved in anhydrous ethanol organic phase, with a total molar concentration of 1.45 mM. Specifically, the LNP contained Lipid 5:cholesterol:DSPC (phospholipid):18:0 mPEG2000 PE:β-sitosterol = 50:28.8:10:1.5:9.6. The mRNA was dissolved at a concentration of 13.3 mg / L in a citrate-sodium citrate buffer solution at pH 4.0.

[0079] Step 2: Mix the LNP organic phase and the mRNA drug aqueous phase at a ratio of 6:1. Based on the 6:1 N / P (molar ratio) between ionizable lipid (Lipid 5) and mRNA in the LNP formulation, prepare the aqueous and organic phases according to the amount of mRNA to be used. (For a 20ug mRNA dosage, the aqueous phase volume is 1.5mL, and the calculated lipid-organic phase volume is 0.5mL.) Step 3: Using a microfluidic device (Microfluidic LNP-S1), organic and aqueous phases were mixed to encapsulate and synthesize LNPs containing mRNA. The total flow rate was 12 mL / min, and the volume ratio and flow rate ratio of organic and aqueous phases were both 1:3. Step 4: Dilute the LNPs containing the mRNA with 1×PBS buffer (20 times the volume of the LNPs from Step 3), and perform solvent exchange and volume concentration in an Amicon Ultra 30kDa ultrafiltration centrifuge tube. Store at 4°C after completion.

[0080] Step 5: After encapsulation, the hydration particle size and polydispersity index of LNPs were determined using a DLS particle size analyzer (Beckman Delsa Nan°C), and the amount of nucleic acid inside and outside LNPs was detected using the Quant-iT RiboGreen kit to calculate the encapsulation efficiency.

[0081] Experimental results: After testing, such as Figure 3 As shown, the LNP samples encapsulating mRNA have a water content and particle size both below 150 nm, demonstrating their typical nanoscale size and good permeability in various biological samples, indicating potential for effective delivery. The PDI values ​​are all below 0.2, demonstrating the stability and homogeneity of the nanosystem and its good condition. The encapsulation efficiency is close to 98%, proving that the mRNA is bound to lipids and tightly encapsulated within the LNP, demonstrating the effectiveness of the LNP in protecting and delivering the load.

[0082] Nucleic acids are generally believed to bind to a portion of ionizable lipids (Lipid 5 in this patent), and are mostly encapsulated within the LNP. This is because, in encapsulation efficiency characterization, ~98% of the nucleic acids in untreated LNPs did not produce fluorescence after treatment with nucleic acid dyes, while fluorescence was observed after demulsification with Triton X-100. This demonstrates that the vast majority of nucleic acids are encapsulated within the lipid portion of the LNP in a stable LNP system. The component ratio of LNP to nucleic acid is mainly quantified by the nitrogen-to-phosphorus ratio (N / P), defined as the molar ratio of ionizable lipids (Lipid 5 in this patent, accounting for 50% mol of total lipids) to nucleic acids. In this study, N / P = 6 was generally used, meaning the molar ratio of total lipids to nucleic acids was 12. Under transmission electron microscopy, LNPs are generally round or elliptical. Partial bilayer membrane structures and suspected polygonal structures were found on the exterior of highly efficient SS (β-sitosterol) and SSta (stigmasterol) LNPs, which are considered to be characteristic structures of highly efficient LNPs. Figure 4 ).

[0083] Example 3: LNP-loaded mRNA delivery to hematopoietic stem and progenitor cells 1. Based on the optimized LNP formulation and procedures of Examples 1 and 2, the effects of additives on the in vitro delivery of primary HSPCs were investigated in depth. ApoE and ApoA1, two apolipoproteins associated with LNP endocytosis and hematopoietic stem and progenitor cells, were selected as additives for in vitro LNP transfection of hematopoietic stem and progenitor cells to verify their effects on delivery efficiency.

[0084] In vitro transfection: LNP transfected human CBMC, 2ug mRNA / well LNP (96-well plate).

[0085] ApoE and ApoA1 are grouped as follows: 1) No addition; 2) 1uL ApoE / hole; 3) 2uL ApoE / hole; 4) 4uL ApoE / hole; 5) 2uL ApoA1 / hole; 6) 1uL ApoE + 1uL ApoA1 / hole.

[0086] LNP transfected human CBMC samples The cell culture medium used for transfection was SFEM-II medium, with the addition of appropriate amounts of human cytokines SCF, TPO, and FLT-3. Transfection procedure: Gently pipette CBMC cells, collect cell suspension using culture medium, centrifuge at 500g for 5 minutes at room temperature (adjust speed to the slowest setting), resuspend and count, then centrifuge at 5×10⁻⁶. 5 Cells were seeded per well in a 96-well plate. ApoE and 2 μg mRNA / well LNP (the amount of mRNA encapsulated in the LNP was detected in the encapsulation efficiency assay) were added to the culture medium sequentially according to the experimental group. After incubation for 5 min, the cells were transferred to the 96-well plate, with a final culture medium volume of 200 μL per well. After incubation in a CO2 incubator for 48 h, cell viability and eGFP expression were assessed.

[0087] 1) Results are as follows Figure 6 As shown, the addition of ApoE played a crucial role in the effectiveness of transfection, and its promoting effect on transfection was positively correlated with the amount added. In contrast, the addition of ApoA1 did not promote transfection, and its co-addition with ApoE inhibited its expression intensity.

[0088] 2) Based on a comparison of the basic LNP formulation and the optimized LNP, the effects of not adding or adding apolipoprotein ApoE during transfection on the in vitro delivery of primary HSPCs were investigated.

[0089] The results are as follows Figure 7 As shown, the basic formulation of LNP is not effective for in vitro transfection of primary HSPCs in CBMCs. Optimization strategies involving the addition of ApoE or partial substitution of phytosterols alone have limited effect on improving transfection efficiency and overall effectiveness. Only by employing both strategies simultaneously can effective in vitro transfection of human primary HSPCs be achieved.

[0090] 2. In vitro transfection specificity of LNP-delivered mRNA An optimized in vitro delivery strategy involving LNP and the addition of ApoE during transfection was employed to transfect different human CBMCs (CD34+ and CD34- cells). The procedures were the same as described above. Results ( Figure 8 The results showed a clear preference for delivery to HSPCs (CD34+ cells), while the transfection rate was almost zero for the majority of non-target cells (CD34-), suggesting that the method has transfection specificity for CD34+ HSPCs.

[0091] In research and clinical settings (such as bone marrow transplantation and umbilical cord blood transplantation), CD34 is the most practical and commonly used biomarker for identifying and isolating HSPCs. CD34+ cell populations are widely recognized as containing hematopoietic stem and progenitor cells capable of reconstructing the hematopoietic system. Experimental results demonstrate that this LNP system possesses an intrinsic predisposition to be efficiently taken up by CD34+ cells, a capability not shared by most other mature blood cells (CD34-). This LNP system can precisely locate this therapeutically valuable HSPC population from target cells mixed with various cell types within a mixed bone marrow or umbilical cord blood cell population, solving the challenge of specific delivery in complex cellular mixtures.

[0092] Example 4: In vivo delivery application of optimized LNP in a mouse model In vivo transfection: LNP transfection of C57 mice First, the LNP samples were modified with conjugated antibodies: The optimized formulation is the LNP encapsulated with mRNA prepared in Example 2. Lipid 5: cholesterol: DSPC: 18:0mPEG2000 PE: β-sitosterol = 50:28.8:10:1.5:9.6, and the molar ratio (N / P ratio) of Lipid 5 to eGFP mRNA is 6.

[0093] By adding 0.5 wt% DSPE-PEG-Mal (manufacturer MCE) to the optimized LNP formulation, Mal-LNP with thiol coupling ability was synthesized according to the steps of Example 2.

[0094] DSPE-PEG-MAL (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Polyethylene glycol-Maleimide) is an amphiphilic block copolymer composed of DSPE (distearylphosphatidylethanolamine), PEG (polyethylene glycol), and MAL (maleimide).

[0095] The mouse CD-117 antibody (c-Kit) was modified by thiolation. The antibody was mixed with Traut reagent (manufacturer McLean) in PBS and reacted at room temperature for 1 h. The antibody was then purified by Zeba desalting centrifuge column (Thermo).

[0096] The thiolized antibody was mixed with Mal-LNP (based on the molar amount of DSPE-PEG-Mal used in synthesis) (Mal:antibody = 20:1 molar ratio) in 1×PBS, and the mixture was reacted with gentle shaking on a shaker for 1 h, followed by overnight incubation at 4°C. The LNP conjugated with c-Kit was obtained after purification using a cytiva column.

[0097] For four days prior to LNP transfection, mice were intraperitoneally injected with G-CSF (250 μg / kg) daily. On the day of transfection, plerixafor (5 mg / kg) was injected intraperitoneally one hour prior to transfection to induce bone marrow mobilization. Subsequently, mice were administered LNP via tail vein injection at a dose of 1 mg mRNA / kg. After 36 hours, major organs, femurs, and tibias of the mice were harvested for imaging analysis of LNP expression in vivo. Bone marrow from the femurs and tibias was then collected, and eGFP expression in different cells was detected using flow cytometry.

[0098] Experimental results are as follows Figure 9 As shown, the optimized LNP (Ab-LNP) after conjugation with antibody can effectively transfect hematopoietic stem and progenitor cells in mouse bone marrow via intravenous injection. The cell delivery efficiency in LSK cells (hematopoietic stem and progenitor cells, including HSCs and MPPs) is close to 90%, and the eGFP expression rate reaches 41.9%, which is significantly higher than that in total bone marrow cells. This indicates that all aspects of the entire delivery process, including targeting, endocytosis, and endosome escape of HSCs, have been highly optimized and coordinated, demonstrating a high level of technological maturity.

[0099] It is almost not expressed in Lin+ cells, which represent mature, differentiated blood cells (such as lymphocytes, granulocytes, monocytes, etc.) and constitute the "majority" of bone marrow. Their lack of expression can maximize the restriction of gene manipulation to the target HSPCs, thus eliminating the safety risks caused by off-target effects. This echoes the specificity of "sterol replacement + ApoE" in in vitro experiments. In the more complex in vivo environment, this targeting enhances this specificity to a level that ensures the width of the therapeutic window.

[0100] The LNP exhibits effective expression at 20-40%+ in various HSPCs, including pluripotent progenitor cells (MPP), short-term hematopoietic stem cells (ST-HSC), and long-term hematopoietic stem cells (LT-HSC). Transfecting LT-HSC is a recognized extreme challenge in the field, as they are the "seed" cells that maintain lifelong hematopoiesis. The results of this experiment show that this LNP can not only transfect relatively active progenitor cells, but also reach and modify the most primitive and core long-term hematopoietic stem cells, penetrating the entire HSPC hierarchy to achieve deep, durable, and even lifelong gene therapy effects on the hematopoietic system.

[0101] Figure 9This delivery system demonstrates that it simultaneously achieves three often contradictory goals: high efficiency (90% delivery in LSK), high specificity (no expression in Lin-), and depth of action (reaching LT-HSC). The system solves the key technical challenge of efficient and safe gene drug delivery to hematopoietic stem cells in vivo.

[0102] In summary, this invention provides an LNP formulation and a vector and method for delivering mRNA to hematopoietic stem and progenitor cells. The mRNA drug is encapsulated at approximately 98% concentration. When transfecting umbilical cord blood mononuclear cells in vitro, the addition of apolipoprotein ApoE achieves a high specific transfection rate for CD34+ hematopoietic stem and progenitor cells (nearly 90% transfection efficiency in CD34+ hematopoietic stem and progenitor cells, while almost no expression is observed in CD34- cells). After antibody conjugation, LNP can also achieve specific and efficient transfection of in vivo bone marrow hematopoietic stem and progenitor cells in the C57 mouse model (90% transfection rate, eGFP expression rate of 41.9%, significantly higher than that of total bone marrow cells).

Claims

1. A lipid nanoparticle for hematopoietic stem progenitor cells comprising an ionizable lipid, a cholesterol, a phospholipid, a pegylated lipid, a solid sterol substitute, characterized in that: The ionizable lipid is Lipid 5, the phospholipid is DSPC, the PEGylated lipid is 18:0 mPEG2000 PE, and the sterol substitute is one or more of beta-sitosterol, stigmasterol, or stigmastanol, or derivatives thereof; the molar ratio of ionizable lipid:cholesterol:phospholipid:PEGylated lipid:sterol substitute is 40-60:5-38.5:5-30:1-2:5-38.

5.

2. The lipid nanoparticle for hematopoietic stem progenitor cells according to claim 1, wherein: The molar ratio of cholesterol to sterol substitute is 3:

1.

3. The lipid nanoparticle for hematopoietic stem progenitor cells according to claim 1, wherein: The molar ratio of Lipid 5:cholesterol:DSPC:18:0 mPEG2000 PE:sterol substitute is 50:28.8:10:1.5:9.

6.

4. Use of the lipid nanoparticle of any one of claims 1-3 in the manufacture of a product for delivering a drug to hematopoietic stem progenitor cells.

5. A drug delivery vehicle for hematopoietic stem progenitor cells, characterized by: The product comprises the lipid nanoparticle of any one of claims 1-3.

6. The method for preparing a drug delivery carrier for hematopoietic stem progenitor cells comprising a lipid encapsulated mRNA according to claim 5, characterized in that: The encapsulation comprises the following steps: Step 1: dissolve lipids in anhydrous ethanol organic phase, and dissolve mRNA drug in citric acid-sodium citrate buffer solution with pH 4.0 aqueous phase; Step 2: the total molar concentration of lipids in the organic phase is 1.45 mM, and the lipids nanoparticles and mRNA drug are mixed according to the molar ratio of Lipid 5 to mRNA of 6:1; Step 3: use microfluidic mixing to synthesize mRNA-encapsulated lipid nanoparticles by mixing the organic phase and the aqueous phase, the volume ratio of the organic phase to the aqueous phase is 1:3, and the total flow rate is 12 mL / min; Step 4: dilute the lipid nanoparticles with 1x PBS buffer solution with a volume 20 times that of the mRNA-encapsulated lipid nanoparticles in step 3, and perform solvent exchange and volume concentration in an ultrafiltration centrifuge tube.

7. The drug delivery vehicle for hematopoietic stem progenitor cells according to claim 5, wherein: The lipid nanoparticle comprises apolipoprotein E.

8. The drug delivery vehicle for hematopoietic stem progenitor cells according to claim 5, wherein: The lipid nanoparticle is coupled with a targeting ligand, and the targeting ligand is DSPE-PEG-Mal coupled antibody.

Images