Preparation and application of non-immunogenic phospholipid nanodelivery system

Abstract

The application relates to the technical field of medicines, in particular to preparation and application of an immunogenic phospholipid nano delivery system. The nano particle comprises 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG 2k ), cholesterol (Chol) and an immunogenic lipid. The purpose is to solve the inflammatory reaction caused in the process of cationic lipid delivery of genes or drugs, and meanwhile, the hydrophilic lipid 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG 2k ) in the immunogenic lipid composition can successfully shield protein adsorption, and the long circulation effect in the body is realized.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical technology, and more specifically, to the preparation and application of a non-immunogenic phospholipid nanodelivery system. Background Technology

[0002] RNA is rapidly developing as a novel drug target (including proteins, RNA, and the genome) for therapeutic purposes, particularly with gene or drug-based RNA therapies for various diseases attracting the most widespread attention. Currently, two gene or drug-based coronavirus disease (COVID-19) vaccines have received FDA approval, a significant breakthrough that has spurred global interest in gene or drug-based therapies. A gene or drug is a single-stranded RNA that can rapidly produce a transient therapeutic effect after entering cells. Currently, gene or drug-based cancer therapies, genetic disease therapies, protein replacement therapies, and vaccines have entered clinical trial phases.

[0003] Despite the significant advantages of gene or drug delivery, many challenges remain. First, single-stranded genes or drugs are unstable and more easily degraded by nucleases in the body. Second, their large molecular weight (300kDa-5000kDa) and electronegativity make it difficult for them to enter target cells. Third, as foreign molecules, genes or drugs can be recognized by the body's immune system, inducing paraimmune responses. Finally, once inside the target cell, if they cannot reach the cytoplasm promptly, they are quickly degraded by lysosomes. Therefore, overcoming these delivery barriers requires the use of effective gene or drug delivery vectors.

[0004] Traditional gene vectors include viral and non-viral vectors. Although viral vectors for gene delivery have achieved successful clinical results, the effectiveness of these methods can be limited by factors such as innate immunity, virus-induced immunogenicity, harmful genome integration, limitations in gene load, inability to repeat dosing, complications associated with scaling up doses, and high vector production costs. As an alternative to viral vectors, non-viral vectors, such as polymers, liposomes, and lipid nanoparticles (LNPs), have attracted widespread attention from researchers, promoting research into non-viral vector-based delivery systems. While non-viral vectors have achieved some breakthroughs in gene delivery efficiency, many problems remain to be solved. Commonly used polymer gene or drug delivery vectors are mostly cationic polymers; excessively high positive charges can lead to hemolysis and in vivo inflammatory reactions. Furthermore, many polymer backbones are non-degradable carbon chains, which can cause high toxicity in vivo. Currently, clinically approved gene or drug vaccine delivery vectors are still LNPs; however, traditional LNPs deliver genes or drugs using cationic lipid components, which can cause severe in vivo inflammatory reactions. Therefore, the design of gene delivery vectors still faces significant challenges. Therefore, it is essential to develop a non-toxic, non-inflammatory LNP as a gene or drug delivery system, in conjunction with clinically translated LNPs. Summary of the Invention

[0005] This disclosure provides the preparation and application of an immunogenic phospholipid nanodelivery system, aiming to address the inflammatory response caused by cationic lipid delivery of genes or drugs. Simultaneously, the immunogenic lipid component contains the hydrophilic lipid 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG). 2k It can successfully block protein adsorption and achieve the effect of long-term circulation in the body.

[0006] In a first aspect, this disclosure provides an immunogenic phospholipid nanoparticle, said nanoparticle comprising 1,2-dimyristic-rac-glycerol-3-polyethylene glycol (DMG-PEG). 2kThe non-immunogenic lipids are dihexyl((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DHECG), dioctyl((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DOTCG), didecyl((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DDCCG), dilauryl((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DDDCG), difarnesol((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DFACG), dipalmitoyl((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DHXCG), and distearatel((2,3-dihydroxypropyl)aminomethylthioformamide)glutamate (DOCCG).

[0007] The components of the non-immunogenic lipid nanoparticles function as follows: 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG) 2k PEG acts as a hydrophilic lipid component. After assembling into lipid nanoparticles, PEG is dispersed in water, which is beneficial for resisting protein adsorption and prolonging blood circulation time. Non-ionic lipids, due to the thiourea structure at the end, can encapsulate genes or drugs through hydrogen bonds and hydrophobic interactions. Cholesterol is used to improve the structural stability of lipid nanoparticles.

[0008] Preferably, the non-immunogenic lipid component consists of a hydrophobic carbon chain and a hydrophilic thiourea terminus, wherein the 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG) 2k The PEG acts as a hydrophilic lipid component, and after assembling into lipid nanoparticles, the PEG is dispersed in water. The non-immunogenic lipid nanocarrier serves as a gene or drug delivery carrier, wherein the gene or drug is required to be translated into a specific protein in antigen-presenting cells, tumor cells, or other target cells.

[0009] 1,2-Dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG) in non-immunogenic nanolipids 2k It can enhance the stability of aqueous solutions of nanolipid carriers, reduce the interaction between proteins in plasma and nanocarriers, and prolong the circulation time of nanocarriers in the blood.

[0010] Preferably, the vector can encapsulate genes or drugs through hydrogen bonding between the thiourea terminus and the gene or drug. The vector alters the toxicity and inflammatory effects of traditional cationic lipid gene vectors, enabling successful delivery of genes or drugs to APCs and tumor cells.

[0011] The gene- or drug-loadable components in non-immunogenic nanolipids consist of hydrophobic carbon chains and hydrophilic thiourea, which can bind to genes or drugs through hydrogen bonds to achieve effective gene or drug loading.

[0012] Preferably, the non-immunogenic lipid nanoparticles have a particle size of 70–110 nm, and the gene- or drug-carrying nanomicelles can exist in the form of an aqueous solution or lyophilized powder.

[0013] Cholesterol in non-immunogenic nanolipids can improve the stability of nanolipids, thereby effectively preventing the leakage of genes or drugs.

[0014] Preferably, the immunogenic lipid nanoparticles have a potential of -15 to 0.5 mV, and the 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG) 2k The PEG present in the sample has a molecular weight of 2000.

[0015] Preferably, the non-immunogenic lipid nanoparticles and the gene- or drug-carrying nanoparticles are prepared into corresponding nanoparticle aqueous solutions by microfluidic technology and dialysis, and the nanolipid aqueous solutions are freeze-dried to obtain nanomicelle lyophilized powder.

[0016] Preferably, the loaded gene or drug may be a gene or drug that translates a specific cytokine, major histocompatibility complex (MHC) or a specific protein.

[0017] None of the components contain ionic components; gene or drug encapsulation primarily occurs through hydrogen bonding at the thiourea terminus of the non-immunogenic lipid components. Among these, the hydrophilic lipid DMG-PEG... 2k Chol is used to improve the water solubility, stability, and anti-protein adsorption of lipid nanocarriers, prolonging the circulation time of drug-loaded lipid nanoparticles in the blood. Non-immunogenic lipids enhance the loading capacity of genes or drugs, improving their ability to escape from early intracellular endosomes. The non-immunogenic lipid components include a hydrophilic head-thiourea structure, a hydrophobic tail comprising a saturated or unsaturated alkyl chain, and are bonded to glutamic acid.

[0018] Secondly, this disclosure provides an immunogenic phospholipid nanomicelle aqueous solution system, wherein the immunogenic phospholipid nanomicelle aqueous solution system is formed by the association of hydrophobic chains in each component, with cholesterol interspersed in the hydrophobic region and the hydrophilic part extended in the aqueous phase, forming a nanovesicle structure, and the concentration of the nanolipid is 1-2 mg / mL.

[0019] The series of non-immunogenic lipid nanoparticles and their gene-loaded nanoparticles exhibit redispersibility in aqueous solutions or lyophilized powders. Their gene or drug loading mechanism involves hydrogen bonding between the thiourea structure at the terminal end of the non-immunogenic lipid component and the gene or drug. This gene or drug loading method replaces the traditional method in LNPs where cationic lipid components load genes or drugs through positive and negative charge interactions, thus avoiding the in vivo inflammatory response caused by cationic lipid components.

[0020] Thirdly, the application of a non-immunogenic phospholipid nanomicelle aqueous solution system, wherein the non-immunogenic phospholipid nanomicelle aqueous solution system is applied to gene or drug encapsulation, endosome escape, long-term circulation in vivo, and in vivo gene or drug vaccines, tumor immunotherapy, or other gene defect diseases based on non-immunogenic lipid gene or drug delivery nanocarriers.

[0021] Preferably, the application of the non-immunogenic phospholipid nanomicelle aqueous solution system is to inject the gene- or drug-carrying lipid nanoparticles into the subcutaneous or bloodstream of mice via subcutaneous or intravenous administration. The nanomicelles can successfully deliver the gene or drug to APC cells in lymph nodes or specific cells in the body, enabling them to express corresponding cytokines or major histocompatibility complex (MHC) or target proteins, activate T cells, or highly express a certain missing protein in specific cells, thereby forming corresponding immune memory.

[0022] In summary, this application has the following beneficial effects:

[0023] 1. Because the components of the non-immunogenic lipid nanoparticles in this application function as 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG) 2k PEG acts as a hydrophilic lipid component. After assembling into lipid nanoparticles, PEG is dispersed in water, which is beneficial for resisting protein adsorption and prolonging blood circulation time. Non-ionic lipids, due to the thiourea structure at the end, can encapsulate genes or drugs through hydrogen bonds and hydrophobic interactions. Cholesterol is used to improve the structural stability of lipid nanoparticles.

[0024] 2. The immunogenic nanolipids in this application contain 1,2-dimyristoyl-rac-glycerol-3-polyethylene glycol (DMG-PEG). 2k It can enhance the stability of aqueous solutions of nanolipid carriers, reduce the interaction between proteins in plasma and nanocarriers, and prolong the circulation time of nanocarriers in the blood.

[0025] 3. The non-immunogenic lipid nanoparticles and gene-loaded nanoparticles in this application series, whether in aqueous solution or lyophilized powder, all exhibit redispersibility. Their mechanism of gene or drug loading involves the formation of hydrogen bonds between the thiourea structure at the end of the non-immunogenic lipid component and the gene or drug. This gene or drug loading method replaces the traditional method of loading genes or drugs through positive and negative charge interactions using cationic lipid components in LNPs, thus avoiding in vivo inflammatory responses caused by cationic lipid components.

[0026] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit the scope of protection of this disclosure. Attached Figure Description

[0027] 1. Figure 1 This is the nuclear magnetic resonance spectrum of the DHECG prepared in Implementation Example 1 of this application (the positions of the nuclear magnetic peaks of various hydrogen protons in the figure prove the structural composition of the DHECG);

[0028] 2. Figure 2 This is the nuclear magnetic resonance spectrum of DOTCG prepared in Implementation Example 2 of this application (the positions of the nuclear magnetic peaks of various hydrogen protons in the figure prove the structural composition of DOTCG);

[0029] 3. Figure 3 This is the NMR spectrum of DDDCCG prepared in Implementation Case 3 of this application (the NMR peak positions of various hydrogen protons in the figure prove the structural composition of DDDCCG);

[0030] 4. Figure 4 This is the NMR spectrum of DDDCG prepared in Implementation Case 4 of this application (the NMR peak positions of various hydrogen protons in the figure prove the structural composition of DDDCG).

[0031] 5. Figure 5 This is the nuclear magnetic resonance spectrum of the DFACG prepared in Implementation Case 5 of this application (the positions of the nuclear magnetic peaks of various hydrogen protons in the figure prove the structural composition of the DFACG);

[0032] 6. Figure 6 This is the NMR spectrum of DHXCG prepared in Implementation Case 6 of this application (the NMR peak positions of various hydrogen protons in the figure prove the structural composition of DHXCG);

[0033] 7. Figure 7 This is the NMR spectrum of the DOCCG prepared in Implementation Case 7 of this application (the NMR peak positions of various hydrogen protons in the figure prove the structural composition of DOCCG);

[0034] 8. Figure 8 This is a particle size diagram of the non-immunogenic lipid nanoparticles prepared in Implementation Case 8 of this application;

[0035] 9. Figure 9 This is a potential diagram of the non-immunogenic lipid nanoparticles prepared in Implementation Case 8 of this application;

[0036] 10. Figure 10 This is a test of the ability of non-immunogenic lipid nanoparticles prepared in Implementation Case 9 of this application to carry genes or drugs (the results in the figure prove that all non-immunogenic nanoparticles can effectively encapsulate genes or drugs).

[0037] 11. Figure 11 The gene-carrying, non-immunogenic lipid nanoparticles prepared in Implementation Case 10 of this application can achieve the effect of intracellular gene or drug delivery in DC2.4 cells and exhibit a certain gene expression ability. Detailed Implementation

[0038] The following detailed description of this application is provided in conjunction with the embodiments. It should be noted that: unless otherwise specified, the conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following embodiments are all from commercially available sources.

[0039] Example

[0040] Example 1

[0041] Synthesis of DHECG

[0042] N-Boc-glutamic acid (2 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol) to the mixture. The mixture was stirred for 1 h until the solution became clear. Hexanol (1.653 g, 16.18 mmol) was then added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0043] Intermediate 1 (3.011 g, 7.25 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.5 mL, 10.821 mmol) and CS2 (0.90 mL, 14.50 mmol). The mixture was stirred at room temperature for 3 h, and p-toluenesulfonyl chloride (1.7 g, 8.70 mmol) DCM solution (5 mL) was slowly added dropwise under ice-water bath conditions. The mixture was allowed to react completely for 48 h, washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (1.32 g, 14.50 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DHECG (2.5 g, 77% yield).

[0044] Example 2

[0045] Synthesis of DOTCG

[0046] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol), and the mixture was stirred for 1 h until the solution became clear. Then, n-octanol (2.107 g, 16.18 mmol) was added and the reaction mixture was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0047] Intermediate 1 (2.531 g, 5.37 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.1 mL, 7.936 mmol) and CS2 (0.70 mL, 10.74 mmol). The mixture was stirred at room temperature for 3 h, and p-toluenesulfonyl chloride (1.22 g, 8.70 mmol) DCM solution (5 mL) was slowly added dropwise under ice-water bath conditions. The mixture was allowed to react completely for 48 h, washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (0.98 g, 10.74 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DHECG (2.1 g, 77% yield).

[0048] Example 3

[0049] DDCCG Synthesis

[0050] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol) to the mixture. The mixture was stirred for 1 h until the solution became clear. Then, n-decanol (2.561 g, 16.18 mmol) was added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0051] Intermediate 1 (3.823 g, 7.25 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.52 mL, 10.966 mmol) and CS2 (0.88 mL, 14.50 mmol), and stirred at room temperature for 3 h. Under ice-water bath conditions, p-toluenesulfonyl chloride (1.66 g, 8.70 mmol) DCM solution (5 mL) was slowly added dropwise, and the reaction was allowed to proceed for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (1.328 g, 14.5 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DHECG (3.77 g, 92% yield).

[0052] Example 4

[0053] DDDCG Synthesis

[0054] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol) to the mixture. The mixture was stirred for 1 h until the solution became clear. Lauryl alcohol (3.015 g, 16.18 mmol) was then added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0055] Intermediate 1 (4.188 g, 7.18 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.51 mL, 10.893 mmol) and CS2 (0.88 mL, 14.50 mmol), and stirred at room temperature for 3 h. Under ice-water bath conditions, p-toluenesulfonyl chloride (1.66 g, 8.70 mmol) DCM solution (5 mL) was slowly added dropwise, and the reaction was allowed to proceed for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (1.31 g, 14.36 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DDDCG (5.8 g, 76% yield).

[0056] Example 5

[0057] Synthesis of DFACG

[0058] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol), and the mixture was stirred for 1 h until the solution became clear. Farnesol (3.598 g, 16.18 mmol) was then added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0059] Intermediate 1 (3.291 g, 5.02 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.1 mL, 7.936 mmol) and CS2 (0.60 mL, 10.04 mmol), and stirred at room temperature for 3 h. Under ice-water bath conditions, p-toluenesulfonyl chloride (1.15 g, 6.024 mmol) DCM solution (5 mL) was slowly added dropwise, and the reaction was allowed to proceed for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (0.92 g, 10.04 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DFACG (3.43 g, 97% yield).

[0060] Example 6

[0061] Synthesis of DHXCG

[0062] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol) to the mixture. The mixture was stirred for 1 h until the solution became clear. Then, n-hexadecyl alcohol (3.923 g, 16.18 mmol) was added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0063] Intermediate 1 (5.050 g, 7.26 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.51 mL, 10.89 mmol) and CS2 (0.88 mL, 14.52 mmol). The mixture was stirred at room temperature for 3 h, and p-toluenesulfonyl chloride (1.66 g, 8.712 mmol) DCM solution (5 mL) was slowly added dropwise under ice-water bath conditions. The mixture was allowed to react completely for 48 h, washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (1.32 g, 14.52 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DHXCG (5.29 g, yield 87%).

[0064] Example 7

[0065] DOCCG Synthesis

[0066] N-Boc-glutamic acid (2.00 g, 8.09 mmol) was dissolved in dichloromethane (100 mL), followed by the addition of carbonyl diimidazole (CDI, 2.624 g, 16.18 mmol) to the mixture. The mixture was stirred for 1 h until the solution became clear. Stearyl alcohol (4.377 g, 16.18 mmol) was then added and the reaction was stirred for 12 h. The mixture was washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous magnesium sulfate, and rotary evaporated to give intermediate 1.

[0067] Intermediate 1 (3.40 g, 5.22 mmol) was dissolved in DCM / TFA (10 mL, V / V = 1 / 1), deprotected at room temperature for 6 h, solvent was removed by rotary evaporation, DCM (5 mL) was added again, followed by TEA (1.20 mL, 7.83 mmol) and CS2 (0.67 mL, 10.44 mmol), and stirred at room temperature for 3 h. Under ice-water bath conditions, p-toluenesulfonyl chloride (1.20 g, 6.264 mmol) DCM solution (5 mL) was slowly added dropwise, and the reaction was allowed to proceed for 48 h. The mixture was washed three times with saturated brine, dried over anhydrous magnesium sulfate, and rotary evaporated to obtain intermediate 2. Subsequently, product 2 was dissolved in DMF (10 mL), and 3-amino-1,2-propanediol (0.95 g, 10.44 mmol) was added. The mixture was reacted at room temperature for 2 h, then at 80 °C for 24 h. DCM was added, and the mixture was washed with saturated brine to remove DMF and unreacted 3-amino-1,2-propanediol. The mixture was dried over anhydrous magnesium sulfate and rotary evaporated to obtain the final product DOCCG (2.72 g, yield 67%).

[0068] Example 8

[0069] Preparation and characterization of immunogenic lipid nanoparticles

[0070] Non-immunogenic lipids, cholesterol, and DMG-PEG 2k Dissolved in anhydrous ethanol, the mixture was extruded into a centrifuge tube along with a certain volume of deionized water using a microfluidic device. The ethanol was removed by dialysis, and the volume was adjusted to obtain non-immunogenic lipid nanoparticles with a concentration of 1 mg / mL. The nanoparticles were stored at 4°C for later use.

[0071] The non-immunogenic nanoparticle solution was transferred to a sample cell, and the particle size and potential of DHECG, DOTCG, DDDCG, DDDCG, DFACG, DHXCG and DOCCG nanoparticles were detected by dynamic light scattering particle size analyzer (DLS) at a temperature of 25°C and an angle of 173°.

[0072] like Figure 9 The results shown in the figure demonstrate that all non-immunogenic lipid components are related to DMG-PEG. 2k Both Chol and other technologies can be used to obtain lipid nanoparticles through microfluidic technology, and all non-immunogenic lipid nanoparticles have electronegative or electroneutrally neutral potentials.

[0073] Example 9

[0074] Detection of the ability of non-immunogenic nanoparticles to encapsulate genes or drugs

[0075] Gene or drug aqueous solution and non-immunogenic lipid ethanol solution were extruded into centrifuge tubes via a microfluidic device, dialyzed with DEPC water, and brought to a final volume to obtain a certain concentration of gene or drug non-immunogenic lipid nanoparticles, which were then stored at 4°C. A certain volume of the gene or drug non-immunogenic lipid nanoparticle aqueous solution was mixed thoroughly with loading buffer and transferred to the sample wells of an agarose gel plate. The agarose gel plate was then transferred to an electrophoresis tank for gel chromatography, and the gene or drug loading capacity was detected by UV light.

[0076] like Figure 10 The results shown in the figure demonstrate that all non-immunogenic nanoparticles can effectively encapsulate genes or drugs.

[0077] Example 10

[0078] Detection of GFP expression in in vitro DC2.4 cells

[0079] DC2.4 cells (1.0*10) 5 The cells were spread in 24-well plates overnight and then co-incubated with non-immunogenic lipid nanoparticles loaded with mEGFP for 4 h under serum-containing and serum-free conditions, respectively. The culture medium was then replaced with new complete culture medium and cultured for another 20 h. The expression of GFP was observed by confocal fluorescence microscopy.

[0080] The above description is merely an exemplary embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. An immunogenically inert phospholipid nanoparticle, characterized in that, The nanoparticles comprise DMG-PEG 2k cholesterol and an immunologically unreactive lipid, the immunologically unreactive lipid being one of dihexyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DHECG), dioctyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DOTCG), didecyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DDCCG), didodecyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DDDCG), difarnesyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DFACG), dipalmitoyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DHXCG), and distearyl ((2,3-dihydroxypropyl)thiocarbamoyl) glutarate (DOCCG).

2. The non-immunogenic phospholipid nanoparticle of claim 1, wherein, The non-immunogenic lipid component consists of a hydrophobic carbon chain and a hydrophilic thiourea head group, the DMG-PEG 2k serves as a hydrophilic lipid component, DMG-PEG 2k dispersed in water, assembling into non-immunogenic phospholipid nanoparticles that serve as gene or drug delivery vehicles, the genes or drugs being translated into specific proteins within antigen presenting cells, tumor cells or other cells of interest.

3. The non-immunogenic phospholipid nanoparticle of claim 2, wherein, The carrier can encapsulate the gene or the drug through hydrogen bonding of thiourea end with the gene or the drug.

4. The non-immunogenic phospholipid nanoparticle of claim 3, wherein, The non-immunogenic phospholipid nanoparticle has a particle size of 70-110 nm.

5. The non-immunogenic phospholipid nanoparticle of claim 1, wherein, The non-immunogenic phospholipid nanoparticle has a potential of -15-0.5 mV.

6. The non-immunogenic phospholipid nanoparticle of claim 3, wherein, The non-immunogenic phospholipid nanoparticle and the encapsulated gene or drug nanoparticle are prepared into corresponding aqueous nanoparticle solution through microfluidic technology or dialysis, and the aqueous nanoparticle solution is freeze-dried to obtain nanoparticle micelles freeze-dried powder.

7. An aqueous solution system of non-immunogenic phospholipid nanomicelles characterized in that, The non-immunogenic phospholipid nanoparticle micelles aqueous solution system comprises the non-immunogenic phospholipid nanoparticle micelles, and the non-immunogenic nanoparticle micelles aqueous solution system is a nano-vesicle structure in which hydrophobic chains of each component are associated, cholesterol is intercalated in the hydrophobic region, and hydrophilic parts are stretched in the water phase.

8. Use of a non-immunogenic phospholipid nanomicellar aqueous solution system according to claim 7, characterized in that, The non-immunogenic phospholipid nanoparticle micelles aqueous solution system is used for preparing a non-immunogenic lipid-based gene drug.

9. Use of a non-immunogenic phospholipid nanomicellar aqueous solution system according to claim 8, characterized in that, The non-immunogenic phospholipid nanoparticle micelles aqueous solution system is applied through subcutaneous administration or intravenous administration.

Images